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FITTED Suite 3.6 User Guide

1. Binding Site Cav BindSite filename Name of the file where to output the binding site cavity If this keyword is not present ProCESs will not create a binding site cavity file Interaction Sites IS filename Name of the file where to output the interaction sites definition file If this keyword is not present ProCEss will not create an interaction sites definition file Ligand lt ligands gt ligand1l mol2 ligand2 mol2 Ligand file s in MOL2 format used to define the active site and its center It should be in the same frame as the protein 21 FITTED Suite 3 5 User Guide V 2 Reading the input files and preparing the output protein files AutoFind Site YIN This function allows the user to have PROCESS automatically finding the flexible residues binding site The default is Y Ligand ligandfile mol2 Ligand file in MOL2 format used to define the active site and its center It should be in the same frame as the protein Ligand Cutoff lt ligand_cutoff gt Protein residues within this cutoff in are considered part of the binding site The default is 6 0 Binding Site lt i Elex pos duces flex residue 1 name flex residue 2 name Manually defines the active site The active site can be automatically defined by providing a ligand see below On the same line following this keyword specify the number of flexible residues This list should be as
2. FITTED Suite 3 5 User Guide V 3 Parameters for the binding Cavity file 22 V 4 Parameters for the Interaction Sites file 23 V 5 A simple Process keyword file for rigid protein docking setup ceeeeeeeeeneeeeeeetteeeeetnneeeetees 25 V 6 A simple Process keyword file for flexible protein docking setup 25 V 7 An advanced Process keyword le 25 VI Preparing a keyword file for SMART cccsccesssceseeeeeeeeeeeeeeeseaeseseeeeneeeessaeseseaesaseeeseeeseseaesaneeeenseeeees 26 VET Inputoutp t fes une accede beet debs a re nt da td ni deli nina ere 26 VI 2 Parameters for the preparation of the ligand file 26 VI 3 A simple typical SMART keyword le 28 VI 4 An advanced typical SMART keyword file 0 ccccceeeeseceeeeeeeeeeeeeeeseeeeeseaeeesaaeseeeeeseaeeesaeeseaeeeeaes 28 VII Preparing a keyword file for FITTED ccceseeeceseeeeeeeseeeeeseseeeeeseseeneeseseeneesaseeneeseseeneeseseenenseseeenens 29 VE Inpuvoutput TTT 29 VIk2 Run parameters tahidct atone E 30 VILS Conjugate gradient parameters usines 31 VIL4 Energy parameters sus antenne tant te nain niet ta nent a nn 32 VIS Scoring e IEN 34 VII 6 Initial population parameters 34 VILT Evolution parameters restreinte tasse a ETA nee date de ATN 35 VII 8 Docking of Covalent inhibitors ss 37 VII 9 Output convergence parameters iii 38 VI1 10 A simple FITTED keyword file for rigid protein docking eeeeseceeeenneeeeeeneeeeeenaeeeeee
3. FITTED Suite 3 6 User Guide MOLECULAR D McGill N Moitessier C Corbeil P Englebienne E Therrien Department of Chemistry Molecular Forecaster Inc McGill University Laval Qu bec Canada Montr al Qu bec Canada 24 08 2012 FITTED Suite 3 5 User Guide Table of Contents 1 1 Conventions used in this guide sisi 5 ee eu nn tn anne t are ere telnet ete A bts ae nt et dent ot tu e 5 Il Before using the FITTED Suite cccsscccsseecsseeeeeseeesneeenseeeeneeeesnaeseseeeenseaessaeseseaesaseaeeneeeeseesasseaeeseneeas 6 Il 1 Recent changes from previous versions ececeeeeeeeeeeenee cece ences ee eaeeeeeeaaeeeeetaaeeeeeeaeeeeeeaeeeeneaa 6 11 2 What s included in the Suite ss 6 IS 1nStallation sise ii t ns etes ce ne er A M neve hi ae 6 UE a tee 6 1922 nu Sede hit he ME Ane ta hed Lae de ie ied MP ee aed Dats 7 SRC Esl Me eege ege ee 7 lA Minimum Requirement iii 7 IS ALICE MSG Eelere ageseent 8 1 5 1 Generating a machine_id fitted le 0n noneneeeeeeeneeenesenesenssrnsstnssrnssrenstnnstnnsnnnntnnntnnntnnntnnnnnnnt 8 1 5 2 License and version tools ins 8 ll Getting started With FITTED 222 e ee a e oe ie non en ete este nen in tr en donne anne 10 111 1 Overview of the programs of the FITTED Suite ccccccceeeeeeeeeeeeeeeeeeeeeaeeeeeeeseeeeeseaeeeeaeeseeeeee 10 III 2 Setting up the system manually iii 10 III 3 Setting up the system with PREPARE iii 12 IE Running th
4. Linux and Mac OS X In a terminal navigate to the installation folder and execute the following command get information about the license status and programs version lt path to the_executable gt Forecaster v license fitted lt path to the executable gt fitted v license fitted FITTED Suite 3 5 User Guide i e CSR C Windows system32 cmd exe St pu lt EE c fitted_exe2 gt Forecaster exe v license fitted License Information for Forecaster version 1 6 License file license fitted fitted 3 6 is valid until 2612 82 28 prepare 1 5 is valid until 2612 62 28 smart 3 6 is valid until 2612 62 28 process 3 6 is valid until 2612 62 28 2dto3d 1 6 is not part of your current license select 1 8 is not part of your current license filter 1 6 is not part of your current license reactor 1 6 is not part of your current license c i fitted_exe2 gt _ FITTED Suite 3 5 User Guide lll Getting started with FITTED This section describes how to prepare and start a docking run with the FITTED suite of programs All the examples and the corresponding files can be found in the example folder 111 1 Overview of the programs of the FITTED Suite The FITTED Suite is a collection of four different actions programs 1 PREPARE adds the hydrogens mutates the truncated residues and optionally optimizes the tautomers and the water molecules according the user defined number of iterations
5. Creation time Thu Feb 15 13 38 45 2007 4 y coordinate i lt TRIPOS gt MOLECULE 5 Z coordinate leZk_protein mol2 6 Atom type 1044 354 o o SMALL 7 Group number USER_CHARGES lt TRIPOS gt ATOM 8 Group name 1 N 5 9870 0 0100 8 6680 n 10 ASP55 0 5200 zeen j 2 CA 5 8090 1 3320 9 2360 chu 10 ASPS5 0 2460 zones 9 Partial charge 3 C 5 0120 2 2540 8 3380 e 10 ASPSS 0 5260 r 10 Misc Information 4 O 4 7670 1 9500 7 1690 o 10 ASPSS 0 5000 zones 5 CB 5 1480 1 2350 10 6450 c2u 10 ASP55 0 2080 6 CG 5 6930 2 2220 11 6570 c 10 ASPSS 0 6200 7 oD1 5 9000 3 4030 11 3160 o 10 ASPSS 0 7060 zeen 8 OD2 5 9770 1 8420 12 8160 o 10 ASPSS 0 7060 9 H 5 1940 0 5390 8 2780 hn 10 ASP55 0 2480 Trtar 10 N 4 2830 6 6210 7 0410 n 12 PROS 0 2570 zen ii CA 4 9940 7 8450 6 8220 chu 12 PROS 0 1120 ttes 12 C 6 3650 7 6260 6 1760 c 12 PROS 0 5260 13 O 6 4870 6 7300 5 3590 o 12 PROS 0 5000 zess 14 CB 4 1380 8 6060 5 7740 c2u 12 PROS 0 0010 zen 15 CG 2 7710 8 0330 5 9870 czu 12 PROS 0 0360 treat 16 CD 2 9980 6 5760 6 3070 c2u 12 PROS 0 0840 t 47 N 7 2700 8 5050 6 5550 n 13 HISES8 0 5200 18 C 8 5950 8 4450 5 8840 chu 13 HISESS 0 2190 tee 19 C 8 4270 8 9230 4 4190 c 13 HISESS 0 5260 trest 20 o 7 4290 9 6060 4 1300 o 13 HISESS 0 5000 zeen 21 CB 9 4960 9 4360 6 5580 c2u 13 HISESS 0 0600 22 CG 9 0530 10 8220 6 8200 ce 13 HISESS 0 1120 ere 23 ND1 8 2050 11 1500 7 8430
6. 0000 1 2 nb 120 0000 60 0000 0 0 i x 0 0000 0 0000 A 5 3 Adding parameters to the torsion list The torsion list starts 2 lines below the fitted torsion parameters title and the end of the list is signaled by having all J K and L atom types as FITTED also allows for the use of wildcard parameters where and L can represent any atom by using the wildcard character in the respective column Parameters added to the force field including wildcards should be placed at the beginning of the torsion list The parameter added must be in a single line in the following format Units V kcal mol 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Force field file Reference Atom Atom Atom Atom V1 dor V2 bo V3 dos Va doa version number number type of type ofJ typeofK type of L E SUM n 1 4 Vn m 1 cos n Phi PhiOd n m multiplicity or total number of torsions centered on the same bond 0 i CG Si 0 0000 0 00 1 2000 180 00 0 0000 0 00 0 0000 0 00 0 CG cl K 0 0000 0 00 0 0000 180 00 0 0000 0 00 0 0000 0 00 0 c cg X 0 0000 0 00 0 0000 180 00 0 000 0 00 0 0000 0 00 0 5 C ch 0 0000 0 00 0 0000 180 00 0 0000 0 00 0 0000 0 00 1 0 c c2 0 0000 0 00 8 7000 180 00 0 0000 0 00 0 0000 0 00 iv A 0 he c3 c3 f 0 1900 0 00 0 0000 0 00 0 0000 0 00 0 0000 0 00 0 he c3 c3 cl 0 2500 0 00 0 0000 0 00 0 0000 0 00 0 0000 0 00 1 0 he es SR
7. 12 PROS O 0229 EEE Oo 100 0 1 2500 8 2124 105 6190773347802700 49 2764967413624500 0 0121513620285930 24 HG2 2 2810 8 5380 6 8440 he 12 PROS 0 0213 veer Oo 100 0 1 2500 8 2109 104 910077542993 6900 66 4703050300625340 0 0192292562929697 25 HD1 3 0580 5 9960 5 3770 he 12 PROS 0 0391 2222 0 100 0 1 2500 8 2167 108 7273966888356400 70 1289098247763580 0 0107572283317070 26 HD2 2 1580 6 1730 6 9000 he 12 PROS 0 0391 12722 0 100 0 1 2500 8 2132 109 4069126544251300 68 5571661820853960 0 0128497050025733 27 N 7 2700 8 5050 6 5550 n 13 HISESS 0 4157 ANSE 0 3 0 1 6400 16 2699 74 1304080266949090 67 7942263593668600 0 0396200694251138 I 2A ra a sasn A_4aasn 5 Anan ES 13 HTSFEA N NSAI seer n an 1 a250 14 834 en Demi aaen AN SA Q33NKNI7777R1270 n nraznagients7 ng i rm For Help press F1 NUM 47 FITTED Suite 3 5 User Guide A 1 3 The XXXX_site txt file The XXXX_site txt file is output by Process and contains the binding site residue list The first line of the file must start with Site followed by the number of residues The following lines 1 per line list the names of the binding site residues B Site 48 ARGE LEU23 ASP25 THR26 GLY27 ALA28 ASP29 ASP30 THR31 VAL32 ILE47 GLY48 GLY49 ILE5O GLY51 GLY 52 PHE53 LEU76 THR80 PROSL VALB2 ASN83 ILE84 GLY86 ARG87 ARGLOBS LEU123 ASHL25 THR126 GLY127 ALAL28 ASP129 ASP130 THR131 VAL132 ILE147 GLY148 GLY149 ILE150 GLY152 ILE15
8. 44 3590 21 C21 45 5420 22 C22 46 6870 23 C23 45 6000 24 H24 48 7158 25 H25 47 8108 26 H26 48 6998 27 H27 51 0182 28 H28 50 4042 29 H29 50 5786 30 H30 51 8889 31 H31 47 6514 32 H32 46 6308 33 H33 45 0360 34 H34 45 1612 lt TRIPOS gt BOND a 22 31 al 2 10 22 ar 3 15 10 1 4 16 15 1 5 12 16 1 6 18 12 a 6 18 al 8 T 6 d 9 29 18 BE 10 30 18 1 11 13 12 1 3 4173 00000010000 FITTED 1173471157 PWNHEENOOFONNORFORFOO 1205 2864 0431 8004 9475 2290 0410 2610 4590 0480 1630 1970 1100 6110 5190 6400 1530 2750 4750 0470 4210 8440 3980 4985 9042 6578 8741 8147 2480 1331 0827 6941 2973 9442 00000000 4 2 BPREBBBBBBBBBBEBBBEBEBEBEBEBEBEBEBBEBEBEBEE UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK UNK GL p Pot 020000000000 Lt 0000000 L 0000000000000000 2094 2098 2259 0600 0319 3944 3911 3843 4079 1975 1860 0257 0660 0040 0782 0003 0364 0525 3246 1930 0224 0501 0437 0319 0645 0337 0276 0276 0563 0563 1010 0296 0296 0296 0 6 0 wuuwu PELLE wuiven Wee HH whe nett wt Het weet nee Kt He ELLE ELLE EH ELLE PELLE TELL kikik Hunii DELLE DLL LI vetevete wee Hee wt Het ELLE ETC ELLE ELLE ELLE w et ck DELLE CLLLLI
9. E VdWScale Pro lt e_ vdwscale pro gt Scaling factor for the ligand protein van der Waals interactions The default is 1 0 E VdWScale Wat lt e vdwscale wat gt Scaling factor for the ligand water van der Waals interactions The default is set the value as the same as E_vdWScale Pro Elec 1 411 5 Select whether 1 4 and or 1 5 and greater electrostatic interactions should be considered 1 4 Used to consider 1 4 interactions and above This is the default setting 1 5 Used to consider 1 5 interactions and above ElecScale 1 4 lt elecscale 1 4 gt Scaling factor for the 1 4 electrostatic interactions The default is 1 0 ElecScale 1 5 lt elecscale 1 5 gt Scaling factor for the 1 5 electrostatic interactions The default is 1 0 E ElecScale Pro lt e_elecscale pro gt Scaling factor for the ligand protein electrostatic interactions The default is 1 0 E ElecScale Wat lt e elecscale wat gt Scaling factor for the ligand water electrostatic interactions The default value is set the same as E ElecScale Pro HBond YIN Selects whether or not hydrogen bonds are included in the energy calculation The default is Y E HbondScale Pro lt e_hbondscale pro gt Scaling factor for the ligand protein hydrogen bond interactions The default is 1 0 E HbondScale Wat lt e hbondscale wat gt Scaling factor for the ligand water hydrogen bond interactions The default value is set the same as E HbondS
10. Generation number for 2nd checkpoint Upper bound score at Max Gen 2 Random number gen seed If 0 Seed is random Resolution for bond rotation during init pop gen For example if a resolution of 120 is selected the bond rotation will occur in multiples of 360 120 or 30 degrees Probability of energy minimiz of parents each gen 41 FITTED Suite 3 5 User Guide pCross 0585 Probability of crossover at each gen pMut 0 05 Probability of mutation at each gen pMutRot 0 30 Probability of mutation of ligand orient each gen pMutWat 0 35 Max rate of mutation of water at Max Gen generations pElite 0 01 Percentage of best individuals passed to next gen pElite Every X Gen 2 pElite will be used every pElite Every X Gen pElite SSize 10 Number of top indiv to select pElite from pOpt 0 20 Probability of optimization of children ligs each gen Evolution Steady State Type of evolution GA Num of Trials 1000 Max number of successive unsuccessful trials to create children CONVERGENCE CRITERIA Diff Avg Best 1 Min diff btw avg energy of pop n and best indiv Diff N Best 0 5 Min diff btw top and N ranked indiv Diff Number Pop Size N ranked individual for Diff N Best Covalent docking Covalent Residue lt residue name gt Name of reacting prot residue SER54 Only CYS and SER implemented so far Covalent Ligand Only Consider only covalent or both types Proton Moved To lt residue name gt lt atom name gt Proton will mo
11. Lig 1 38 368 32 865 0 50 16 179 Lig 2 38 201 32 838 DS TL L679 Lig 3 38 440 32 755 0 50 16 179 Lig 4 38 434 32 664 0 51 17 026 Lig 5 i 24882300 282 657 0 53 16 179 In addition to this previous table information about the internal energy strain of the ligand can be found as well as the on off state of the water molecules when displaceable waters are used VIIL3 The results file This file named XXXX results txt is a brief summary of the output file and contains only the minimum information about the poses the Best Complexes table VIIL 4 The ligand mol2 file When the docking run is over the best pose for each run is generated as a mol2 file that can be visualized The name is XXXX rank 1 Ligl runl mol2 Vill 5 The sdf file An sdf file is also created XXXX sdf which contains the top pose of each run along with the associated energy score rmsd and mscore as sdf fields This file can be visualized easily in any 43 FITTED Suite 3 5 User Guide chemistry spreadsheet program that supports chemical structures or any chemical database programs VIIL6 The protein mol2 file flexible protein mode only Once a flexible protein docking run is performed a mol2 file of the composite protein structure is generated for visualization This file contains only the binding site flexible residues The name is XXXX rank 1 Protl runl mol2 Vill 7 The protein pd
12. PELLE TILL 12111 weitere PELLE zz ananananannn IIIIIIIIIIIWNNNNWWWWWWWZZNNWWIIIII 0000 D0000000000WOD0DONN N OOOOOHHO0O0O 50 FITTED Suite 3 5 User Guide A 3 The binding site cavity file The binding site cavity file is used to determine the empty space within the protein via a collection of spheres of different radius It resembles a MOL2 formatted file with the following changes lt TRIPOS gt MOLECUL E section o onthe second line the number of spheres is specified as the first field fields 2 5 are 0 lt TRIPOS gt ATOM section o column 6 Sybyl atom type is unnecessary therefore it is replaced by a dash o column 9 partial charges is replaced by the radius of the sphere Column Discription Point number Point name x coordinate y coordinate z coordinate Point Type Group number Group name Radius 0 Misc information J O O1 P D St active_site_rigid WordPad amx File Edit View Insert Format Help Del 64 eo B Creating user name FITTED GRID Creation time Thu Feb 15 13 17 06 2007 lt TRIPOS gt MOLECULE output grid rigid 239 D D D SMALL USER_CHARGES B lt TRIPOS gt ATOM 1 PL 1 3810 2 1423 13 3608 1 GRID 2 1602 r 2 P2 19 3810 6 6423 2 8608 1 GRID FEASTS ORR ORR 3 P3 11 8810 9 6423 10 3608 1 GRID S 0GR7 store 4 P4 10 3810 8 1423 13 3608 1 GRID SISO Ree 5 PS 17 8810 5 1423 4 3608 1 GR
13. Site e Keyword options constant width font italic face Examples 1a46 mol2 Docking Yes Please note that the formatting is for clarity of the manual only as it is not possible to format an ASCII file with different typefaces 1 2 Acknowledgements Over the last years the development of FITTED has been funded by ViroChem Pharma research grants and the Canadian Institutes for Health Research CIHR Operating grants while the development of ACE has been funded by the Natural Science and Engineering Research Council NSERC discovery grant These partners are warmly acknowledged More recently the Ministere du D veloppement Economique de l Innovation et de l Exportation du Qu bec has recognized the potential of our drug discovery platform and granted us funding for further development and commercialization as part of a program called Soutien a la maturation technologique Jeremy Schwartzentruber code optimization and Devin Lee comparative study are also acknowledged FITTED Suite 3 5 User Guide ll Before using the FITTED Suite II Recent changes from previous versions The major changes that happened since the version 3 0 of the FITTED Suite are the merger of all the accessory programs in a single program called Forecaster New actions programs are available either trough the FITTED or the Forecaster Suite The keyword files have two new keywords that are required in order to specify the mode and program to use
14. Therefore keyword files from the previous version will not work without the addition of these two new keywords Automatic alignment and superposition of many proteins is now implemented in the Prepare action This new action is essential when performing flexible protein docking with FITTED Since the version 2 6 but were in the version 3 0 changes that were made are the reorganization of the folder architecture the usage of a keyword file for SMART action and the addition of a new action PREPARE Other changes involve a new improved scoring function RankScores faster execution of FITTED many bugs fixed in PROCESS action and a new charging scheme for atomic partial charges in SMART A new java graphical interface is also provided in this new release A license file is now required to run the programs IL2 What s included in the Suite The FITTED Suite is available for three different platforms Linux Windows and Mac OSX The package contains two executables namely fitted exe and Forecaster exe fitted and Forecaster in Linux and Mac OSX It contains also a forcefield folder with the fitted_ff txt forcefield file and a machine_id exe program for generating the license file Finally a Java based graphical user interface for easy file manipulation and program execution Example files for protein 1e2k are given with the pre configured keyword files example 1e2k di forcefield n ifitted exe Forecaster exe Front end jar _ k
15. amine is defined as an atom with an Y_ R N atom type of n3 bound to a single hydrogen R 65
16. br 0 5500 0 00 0 0000 0 00 0 0000 0 00 0 0000 0 00 0 0 ij S SS ii 0 0000 0 00 0 0000 0 00 0 0000 0 00 0 0000 0 00 54 FITTED Suite 3 5 User Guide A 5 4 Adding parameters to the out of plane list The angle list starts 2 lines below the itted_oop parameters title and the end of the list is signaled by having all I J L and K atom types as FITTED also allows for the use of wildcard parameters where K and or L can represent any atom by using the wildcard character in the respective column Parameters added to the force field including wildcards should be placed at the end of the angle list The less specific the parameter higher number of wildcards the lower in the list it should be placed The parameter added must be in a single line in the following format Units K kcal mol 1 2 3 4 5 6 7 8 9 Force field file Reference Atom Atom Atom Atom Kchi N Chio version number number type of typeofJ typeofK typeofL Se el EN EE E e EE ei e e el e EN EE e e gies E Kchi 1 cos n Chi ChiO ER RE PP ee E a ee ey a E Ver Ref I J K L Kehi n Chid ia eet ee a ee i or es eo eg dd eS ee ee ee fitted oop parameters es a a a a a See See Ste EE See ue E mn tu one eue 1 0 c c2 c2 C3 000 2 80 0000 L 0 c ca C3 ca 000 2 80 0000 0 e n C3 hn 000 2 80 0000 0 c n G3 o 000 2 80 0000 at e2 na c2 ES 000 2 80 0000 tee 13
17. contain a single molecule or multiple molecules multi mol2 Ref lt i nls lig ref filel mol2 lig ref file2 mol2 Following this keyword is an integer stating how many reference files are used to calculate the root mean square deviation RMSD of the ligand heavy atoms These ligand files should be in the same reference frame as the protein structure The possible symmetric conformations of the ligand are calculated in silico RMSD calculation can only be done when the ligand s bioactive conformation is known e g self docking study 2 reference files may be needed in some instances where the ligand or protein active site is C symmetric n gt 2 On the following line s the reference file s in MOL2 format are listed one per line If this keyword is missing no RMSD values will be computed Output filename 29 FITTED Suite 3 5 User Guide Name of the output file Forcefield ONCE ele le tt Name of the force field file to use If a forcefield other than fitted_ff txt is to be used The format of this force field should be consistent with the required format for Fitted Default value is fitted_ff txt if the keyword is not provided Binding Site Cav XXXX BindSite mol2 Following this keyword is the file defining the empty space present in the active site cavity a set of spheres prepared by Process If this keyword is missing no grid filter will be used it is highly
18. directory the working directory will be automatically updated The keyword file can be edited by clicking the Edit Keyword File button The program associated with the txt file extension will be used to edit the keyword file To prepare a keyword file to be used by FITTED refer to the appropriate section of this guide To run the program simply click on the Run FITTED button The program will output all the docking files results and mol2 files in the output folder will be created automatically if the folder doesn t exist This output folder can be opened by clicking on the Open output folder button ED ron end T LS FITTED Front end aed a se File Help Select Working Directory Browse C Mitted_exe example_1e2k Open PREPARE SMART ProCESS FITTED Dock using FITTED _ Use Template Keyword File Open output folder Select Keyword File C filted_exe example_1e2kikeyword fitted txt Edit Keyword File Run FITTED M OTL ESCU CAR 111 4 2 Running the FITTED suite from the command line To run PREPARE place the pdb file in your working directory and create an appropriate keyword file see example You can run the program by typing the following command in the terminal where lt path to the executable gt is your installation folder lt path to the executable gt Forecaster keyword prepare txt PREPARE will create the XXXX pro mol
19. exhaustive as possible to avoid missing any important residue defining the active site On subsequent lines the residue name numbers according to Find_Residues are specified one per line Truncate Y N auto Determine if the protein will be truncated keeping only residues within Cutoff of the binding site residues The default is auto The protein will be truncated keeping residues within cutoff distance of the ligand and not within cutoff distance from the binding site residues Cutoff Truncate lt cutoff gt Any residue that does not have an atom within this distance in A from an atom of a flexible residue or of the given ligand will be deleted from the protein file that Process will output The default value is 9 Find Residues Name Number f Active Site is used define in which way Process will identify the residues that make up the binding site Name Search residues by group name This is the default Number Search residues by group number V 3 Parameters for the binding cavity file Grid_Center lt grid_center gt 22 FITTED Suite 3 5 User Guide Specifically defines the center of the binding site The default is to automatically find it using the center of a ligand Grid_Size lt size x gt lt size y gt lt size z gt Specifies the size of the box for the binding site The defaultis 15 15 15 Grid_Boundary Soft Hard Soft When converting from the grid to spheres t
20. flexible protein docking setup Protein Output Binding Site Cav Interaction Sites AutoFind Site AutoFind Center Ligand Run Mode Main Mode 2 1e2k pro mol2 le2p pro mol2 tk process tk bindSite tk_ IS yes yes 2 le2k_lig mol2 le2p_lig mol2 process process V 7 An advanced Process keyword file Protein Output Binding Site Cav Interaction Sites AutoFind Site Ligand Ligand Cutoff Truncate Cutoff Num of IS Run Mode Main Mode 1 protein mol2 protein BindSite mol2 IS mol2 yes lig mol2 9 auto 7 50 process process 25 FITTED Suite 3 5 User Guide VI Preparing a keyword file for SMART SMART is the module used to prepare ligand structures in a modified MOL2 format for use by FITTED It can also assign atomic partial charges and prepare ligand structures for use with ACE Asymmetric Catalyst Evaluation The following section lists the keywords their functions and default values Gray shading indicates a required keyword angle brackets lt gt indicate a numeric value plain text indicates a text string Such as a file name square brackets indicate a choice of values the default shown in italics When a default value is assigned to a keyword the latter can be omitted from the keywordfile SMART keywords files are case sensitive Empty lines are allowed and text after a pound sign is considered a comment Although the val
21. for splitting a multiple ligands file into separate files for docking in parallel charge MMFF DGH none input SMART will assign the atomic partial charges based on the selected method none will zero all the partial charges and input will keep the charges as they appear in the input mol2 file The default value is MMFF assign_bond YIN SMART will assign the bond orders The default value is N ionize Y N SMART will ionize thiols to sulfides The default value is N name Field_ID Field containing the name of the molecule to be used in the sdf file Usually this field contains brackets that should be included ex lt Corporate_ID gt 27 FITTED Suite 3 5 User Guide V1 3 A simple typical SMART keyword file Molecule Output Run_Mode Main Mode le2k_lig mol2 le2k_ lig 1 smart smart V1 4 An advanced typical SMART keyword file Molecule Output mode sane outf charge assign_ bond multi ionize name Run_Mode Main_Mode 1e2k lig mol2 le2k lig 1 fitted mol2 fitted DGH yes y y lt corporate id gt smart smart 28 FITTED Suite 3 5 User Guide VII Preparing a keyword file for FITTED The following sections list the common keywords one that are most frequently changed for a complete list for a specific usage please contact us their functions and default values Gray shading indicates a required keyword
22. lt Max_Gen2 gt On GENETIC ALGORITHM PARAMETERS 100 lt anchor_atom gt lt anchor x gt lt anchor y gt lt O1 O1 O1 D Oo 10000 On 10 5 0 lt Max_Gen_1 gt 4 lt Max_Gen_2 gt 29 29 100 120 Scaling factor for lig wat Hbond energy Cutoff dist in A for lig prot non bond Switching dist in A for lig prot non bond Cutoff dist for lig wat non bond Switching dist for lig wat non bond United All atom Prot repr for init pop gen United All atom Prot repr for evolution Gen to switch from United to All atom On IOff calculation of the solvation energy Number of individuals in the population Initial Min MatchScore Minimum value for PharmScore Number of atom to be used as ctre of rot anchor zz x y and z coord of BS ctre Max value in A for translation in x Max value in A for translation in y Max value in A for translation in z Max number of successive unsuccessful trials On Of Toggle matching algorithm Number of top IS points that interaction site triangle must contain at least one of Factor by which triangles are selected The higher Stringent Triangles is set the more the matching algorithm will favour triangles that have not been used Weight factor used in calculation of Min MatchScore The higher this value the stricter Min MatchScore Maximum numebr of generations Generation number for 1st checkpoint Upper bound score at Max Gen 1
23. ne 13 HISESS 0 5270 zen 24 CD2 9 3410 12 0060 6 1990 cau 13 HISESS 0 1220 e 25 CEL 7 9820 12 4650 7 8530 cau 13 HISESS 0 3840 4 26 NE2 8 6710 13 0030 6 8680 na 13 HISESS 0 4440 tarar v For Help press F1 NUM A 1 2 The XXXX_score mol2 file The format of the file output by Process is a standard MOL2 format with the following changes lt TRI POS gt ATOM section o the atom types column 6 are Amber united atom types instead of Sybyl atom types o the group names column 8 include the advanced residue names see Appendix A lt TR POS gt BOND section o all bonds are listed 46 FITTED Suite 3 5 User Guide Column Description Column Description 1 Atom number 11 Scaling factor 2 Atom name 12 Place Holder 3 x coordinate 13 OPLS Atom Type 4 y coordinate 14 Place Holder 5 z coordinate 15 van der Waals 6 Atom type Radii 7 Group number 16 Atom Volume 8 Group name 17 Atomic Solvation 9 Partial charge 18 vdW solvation 10 Misc Information 19 and gt Water solvation te2k_protein_score mol2 WordPad File Edit View Insert Format Help Oe 64 are B Creating user name FLIPD Creation time Wed Oct 17 14 32 45 2007 lt TRIPOS gt MOLECULE 1le2k_protein_score mol2 4659 4702 609 3581 1216 D SMALL USER_CHARGES lt TRIPOS gt ATOM 1 N 5 9870 0 0100 8 6680 n 10 ASPSS 0 5163 22 0 3 0 1 6400 16 2620 74 7847324292
24. of RAM 2GB or more recommended Java 1 6 latest version for gui FITTED Suite 3 5 User Guide Mac OS X Leopard 10 6 64 bit architecture only 1GB of RAM 2 GB or more recommended Java 1 6 latest version for gui IL5 License File The execution of the programs is controlled by the license file license fitted This license ensures that the programs are used on the licensed computer only Therefore you need to generate a machine id fitted file by using the machine id program This file needs to be sent by email at license fitted ca and you will receive a license fitted file This license fitted file needs to be located in the same folder as the executables IST Generating a machine_id fitted file Windows Double click on the program machine id exe and a file named machine id fitted will be created Linux and Mac OS X In a terminal navigate to the installation folder and execute the machine id program by typing lt path to the executable gt machine id Email this file to license fitted ca to obtain your license fitted file Repeat this process on each computer you need to run the programs 1 5 2 License and version tools Windows In a dos window navigate to the folder where the executables are installed and execute the following command to get information about the license status and programs version c fitted exe2 gt Forecaster ex v license fitted c fitted exe2 gt fitted exe v license fitied
25. should include the water molecules ions and co factors if any These files ligand and protein should be saved in mol2 format available within most of the programs If running a rigid docking Mode Rigid the protein file is ready to be submitted to Process If Mode is Semiflex Flex Or Flex Water additional steps may be required to ensure that all protein files are identical i e same number of protein atoms same number of water molecules same residue names If discrepancies were found Process will exit with an error message see Appendix C Process If some crystal structures have more water molecules than others waters can be taken copied from the protein structures that have similar conformations The following section lists some of the common errors in PDB files which need to be corrected o In all cases if the error appears close to the binding site the protein structure should not be considered for the study o Mutated residues if the mutation is far from the binding site at least 10 A from the binding site then the residue can be virtually mutated to the desired residue o Incomplete residue In some case parts of very flexible residues are not observed and are not included in PDB files Again if they are far from the active site they can be virtually reconstructed o Missing Residues H they are far from the active site they can be i added where missing or ii removed from the other files o Terminal Res
26. weight lt TRI POS gt MOLECUL E section o onthe second line the number of constraints is specified as the first field fields 2 5 are 0 lt TRI POS gt ATOM section o column 6 Sybyl atom type is unnecessary therefore it is replaced by a dash o column 7 group number is replaced by a point type descriptor o column 9 partial charges is replaced by the radius of the constraint o column 10 specifies the type of the pharmacophoric point HBD HBA HYD ARO or any combination such as HBA HYD o column 11 specifies the weight of the constraint Column Discription J O O1 ND Point number Point name x coordinate y coordinate z coordinate Point type PHARM Radius Pharmacophoric type Weight SI pharm_rigid WordPad Loe File Edit View Insert Format Help DW 64 a Bo B k A Creating user name FITTED GRID Creation time Thu Feb 15 13 38 39 2007 lt TRIPOS gt MOLECULE output pharm rigid 80 D D D 0 SMALL USER_CHARGES lt TRIPOS gt ATOM 1 P1 14 1620 2 5960 5 3140 d PHARM 2 3000 HBD 5 0000 2 P2 11 9072 2 9535 1 4574 1 PHARM 0 7500 HBA 5 0000 3 P 14 7634 2 8117 1 1667 1 PHARM 0 7500 HBA 5 0000 4 P4 11 0390 2 8050 4 3570 1 PHARM 2 3000 HBD 5 0000 5 PS 10 8470 2 0823 6 8052 1 PHARM 0 7500 HBA 5 0000 6 P6 16 7345 0 9861 4 9241 1 PHARM 0 7500 HBD 15 0000 7 P7 14 7230 1 7110 6 5690 PHARM 2 3000 HBD 5 0000 8 P
27. 0 5160 25 7600 C ar 1 UNK 0 0292 7 C7 44 0900 0 5050 27 1740 C ar 1 UNK 0 0584 8 c8 43 0000 0 0000 27 8680 C ar 1 UNK 0 1160 9 B9 40 4450 1 1990 28 1520 Br 1 UNK 0 0512 10 510 40 4120 1 2290 24 8260 Br 1 UNK 0 0440 Ti pI 43 0000 0 0000 23 1510 Br 1 UNK 0 0531 12 B12 45 5900 1 2290 24 8260 Br 1 UNK 0 0440 13 B13 45 5600 1 1990 28 1520 Br 1 UNK 0 0512 lt TRIPOS gt BOND 1 1 21 2 8 1 3 8 3 ar 4 8 7 ar S T t3 i 6 z 6 ar 7 6 Kee 8 6 5 ar 9 5 LEE 10 5 4 ar 11 4 LD 12 4 3 ar 13 3 gi lt TRIPOS gt SUBSTRUCTURE 1 UNK 1 PERM O sett sete D ROOT lt TRIPOS gt SET LIGAND STATIC ATOMS lt user gt ete RR pe D Re D Sm 6 T7 E e HO E Wee LAB A TYPE STATIC ATOMS LABELGROUP SYSTEM a be Dn Wee E Aa e Abe EC E Mu be aen S y For Help press F1 NUM 45 FITTED Suite 3 5 User Guide A 1 The protein files A 1 1 The XXXX_dock mol2 The format of the file output by Process is a standard MOL2 format with the following changes lt TRI POS gt ATOM section o the atom types column 6 are Amber united atom types instead of Sybyl atom types o the group names column 8 include the advanced residue names see Appendix A lt TR POS gt BOND section o only bonds for the flexible residues are listed 7 p B 1e2k_protein WordPad Jon Column Description Fie ER Vew Insert Fomat Rab 1 Atom number DEH ARA Bo 2 Atom name Creating user name FITTED 3 x coordinate
28. 0 8 2139 110 6679102932152400 75 7357817723 646460 0 0093222318744948 13 N 4 2830 6 6210 7 0410 n 12 PROS 20 2548 trette 0 3 0 1 6400 14 0293 68 1840515678402570 52 5544944163839180 0 0203996157236316 14 CA 4 9940 7 8450 6 8220 c3 12 PROS 0 0266 terre 0 14 0 1 9250 14 8352 58 2076335624130370 53 1157540179168070 0 0278611938359424 15 c 6 3650 7 6260 6 1760 e 12 PROS 0 5896 veer 0 10 1 8500 18 5746 64 4847841400968780 65 6445787111220940 0 0267251255440412 16 6 4870 6 7300 5 3590 o 12 PROS 0 5740 SES 0 20 1 4800 13 6971 99 0381985677257860 65 0691600284429510 0 0181425296663499 17 CB 4 1380 8 6060 5 7740 c3 12 PROS 78 0070 9299 0 9 0 1 9000 29 2410 71 0501934690087750 53 9564801073376610 0 0211308100701858 18 CG 2 7710 8 0330 5 9870 c3 12 PROS 0 0199 0 9 0 1 9000 23 5528 72 7421939287598890 49 1476023370383210 0 0161742847035640 19 CD 2 9980 6 5760 6 3070 c3 12 PROS 0 0192 9888 0 15 0 1 9000 23 6243 70 0584460242238550 51 3409665617368920 0 0141615777604204 20 HA 5 0540 8 4130 7 7730 he 12 PROS 0 0642 8448 0 100 0 1 2500 6 2107 108 5075481594027000 65 1407659161451260 0 0412502942777156 21 HB1 4 4480 8 3290 4 7470 he 12 PROS 0 0253 vers Oo 100 0 1 2500 6 2114 105 3908239427586000 76 3983247594625960 0 0162541064369056 22 HB2 4 2130 9 6980 5 8370 he 12 PROS 0 0253 e Oo 100 0 1 2500 8 2174 106 1665744809384800 68 0353107029286690 0 0255285053789857 23 HG1 2 1060 8 1700 5 1140 he
29. 0 c2 c2 e S 10 5000 2 80 0000 L O o e ei 1 1000 2 80 0000 L 0 SS c 10 5000 2 80 0000 1 0 E ca Ge x 7 1000 2 80 0000 0 0 Si 0 0000 2 80 0000 A 5 5 Adding parameters to the van der Waals list The vdW list starts 2 lines below the itted_vdW_parameters title and the end of the list is signaled by atom type as The parameter added must be in a single line in the following format Units R A ESPI kcal mol 1 2 3 4 5 Force field file Reference Atom type Ri ESPI version number number of type r eps combination arithmetic E EPSij Rij Rij 12 2 Rij Rij 6 where EPSij sqrt EPSi EPSj Rij Ri Rj 2 Ver Ref I RIX EPSi fitted vd parameters 55 FITTED Suite 3 5 User Guide Sa a 1 0 h1 2 7740 0 01570 1 0 h2 2 5740 0 01570 1 0 h3 2 3740 0 01570 1 0 h4 2 8180 0 01500 1 0 h5 2 7180 0 01500 tsa Dee no 3 7360 0 00277 122 k 5 3160 0 000328 E zn2 2 2000 0 0125 0 0 0 0000 0 00000 A 5 6 Adding parameters to the hydrogen bond list The Hbond list starts 2 lines below the itted_Hbond_parameters title and the end of the list is signaled by having both the and J atom types as The parameter added must be ina single line in the following format Units A B kcal mol 1 2 3 4 5 6 Force field file Reference Atom Atom A B version number number type of type o
30. 2 and RankScore5 Please contact us if you need to change the keywords VIL 6 Initial population parameters Pop Size lt pop_size gt Population size for the genetic algorithm conformational search When 10000 is given as value automatic determination based on the ligand s number of torsions is done The default is automatic for rigid docking 200 for flexible docking when keyword is omitted Min MatchScore lt min_matchscore gt This keyword is used only if an interaction site file is provided If the Mode is set to Dock Min_MatchScore is automatically calculated Minimum match of the interaction sites The default is 20 34 FITTED Suite 3 5 User Guide Min PharmScore lt min_constraint gt This keyword is used only if a pharmacophore file is provided Minimum percent match of the pharmacophore The default is 100 Anchor Atom lt anchor atom gt Sequence number of the atom to be used as an anchor This is used to identify the center of translation and rotation for the GA f this keyword is not specified the anchor is automatically set to the gravity center of the ligand Anchor Coor lt anchor x gt lt anchor vz lt anchor z gt Following this keyword must be the x y and z coordinates of the protein active site center lf this keyword is not used it is automatically set to the center of the protein active site defined by the active site flexible residues Max Tx lt max_tx gt Ma
31. 2 and XXXX lig mo12 file as well as the XXXX out file that contains all the information about the calculations and errors To run Process place all protein files in your working directory and create an appropriate keyword file see examples keyword files provided If flexibility is desired make sure the Process 15 FITTED Suite 3 5 User Guide keyword file includes multiple protein structure files for consideration of flexibility Run the program by typing lt path to the executable gt Forecaster keyword process txt Process will create all the files XXXX pro dock mol2 XXXX pro score mol2 XXXX pro site txt XXXX pro IS mol2 XXXX bindSite mol2 XXXX IS mol2 in the working directory within minutes as well as XXXX out which will include information about the calculations and errors If running the same keyword again all the files will be overwritten To run SMART place the ligand file previously prepared in the working directory Run SMART by typing lt path_to the executable gt Forecaster keyword smart txt All files will be output to working directory To run FITTED make sure all the input files ligand protein s active site cavity and interaction site files are in the working directory Create the appropriate keyword examples for rigid and flexible docking are included in the example folder and place them in the working directory To run FITTED type lt path to the executable gt fitted keyword fitted t
32. 4 A 5 4 Adding parameters to the out of plane list 55 A 5 5 Adding parameters to the van der Waals list 55 A 5 6 Adding parameters to the hydrogen bond list 56 A 5 7 Adding parameters to the bond charge increment list 56 A 5 8 Adding parameters to the partial bond charge increment formal charge adjustment factor EE 57 Appendix B FITTED errors and Wari ssnnnnnnnnnnennnnennennnnnennnes 58 Appendix C Process errors and WarningS s eeenness 61 Appendix D SMART errors and warnings eenennnnenennnnnnenness 62 Appendix E Functional group definitions nes 63 FITTED Suite 3 5 User Guide Preface 1 1 Conventions used in this guide This guide describes the use of a suite of programs which are usable either from a graphical user interface or via a command line arguments FITTED and Forecaster require a set of commands to be issued in the form of a keyword file a standard ASCII text file with instructions that follow the form Keyword Option usually one per line but in some cases a keyword might span multiple lines In the remainder of the manual different typefaces will be used to symbolize the following e Filenames and command line input constant width font standard face Examples ligand mol2 keyword txt smart keyword smart txt e Keyword names constant width font bold face Examples Protein Mode AutoFind
33. 4 VAL156 LEUL76 THR180 PRO181 VAL182 ILE184 A 2 The Ligand file The format output by SMART is based on the MOL2 format Some modifications were introduced in order to implement the bitstring when using the filter mode describing the presence of functional groups and to aid in checking the chirality atom connectivity and ring perception The changes from the standard MOL2 format are as follows lt TRIPOS gt MOLECULE section 48 FITTED Suite 3 5 User Guide o the second line data associated with the molecule is expanded by a number of fields describing the ligand and the functional groups present bitstring The presence of a particular group is indicated by a 1 on the respective field The order of the fields is as follows E number of ES number of molecular number of number of number of number of number o net charge atoms bonds weight hydrogen bond donors hydrogen bond acceptors rotatable bonds rings number o ionisable groups aromatic group number of number of number of number of number of number of number of number of number of number of number o aldehyde ester lactone amide amide lactame acid nitrile imine nitro Michael acceptor number of number of number o azide isocyanate number of number of number of number of number of number o acyl chloride sulphonamide carbamate ammonium oxi
34. 461090 60 0223136939803580 0 0066900772065744 2 CA 5 8090 1 3320 9 2360 c3 10 ASPSS 0 0381 22229 0 6 0 1 9250 14 8586 63 3771565926831070 56 3920491594106750 0 0096815631719025 3 c 5 0120 2 2540 8 3380 c 10 ASPSS 0 5366 22229 0 10 1 8500 18 5944 64 5919033101811660 57 9635672887487270 0 0101965418318157 4 o 4 7670 1 9500 7 1690 o 10 ASPSS eo 58 eee Oo 20 1 4800 13 6830 98 9836659022793550 71 0465764178361920 0 0079697015457906 5 CB 5 1480 1 2350 10 6450 c3 10 ASPSS 0 0909 Seter d a 160 1 9000 23 6166 70 4355310967099800 56 7160365414643980 0 0102316603914527 6 CG 5 6930 2 2220 711 6570 c 10 ASPSS D 7994 terr 26 17 0 1 8750 19 2154 69 9922232494050860 62 2455661161640260 0 0153128490315614 ei ODi 5 9000 3 4030 11 3160 o 10 ASPSS 0 9014 HAE 2 18 0 1 6000 17 3916 88 1413334297605220 78 6319398476289850 0 0217067577645443 8 OD2 5 9770 1 8420 12 8160 o 10 ASPSS zc BDT H AES 2 18 0 1 6000 17 3825 88 3552556993307690 66 1335116983712230 0 0150745709823387 9 H 5 1940 0 5390 8 2780 hn 10 ASPSS 0 2936 FAR Oo 40 1 2075 7 4260 115 1579955991750000 64 6771086267408040 0 0054463548513047 10 HA 6 7960 1 8310 9 3330 he 10 ASPSS 0 0880 Oo 100 0 1 2500 6 2103 108 1180154214647800 66 2454656039476650 0 0119148826226527 11 HBL 5 3310 0 2350 11 0850 he 10 ASPSS 70 0122 FAN 1 100 0 1 2500 6 2115 110 1132534823030700 75 3860953532208610 0 0083103769715239 12 HB2 4 0480 1 3100 10 6120 he 10 ASPSS biet MALE 1 100 0 1 250
35. 8 Adding parameters to the partial bond charge increment formal charge adjustment factor list As a more general way of describing bcis MMFF includes a partial bci parameter that is assigned to each atom type 15 a bci for a bond can be obtained as the sum of the partial bci corresponding to each atom type involved Additionally the formal charge on some groups is spread among neighbouring atoms this is specified in the formal charge adjustment factor for the central atom type in those functional groups 15 The parameter list starts below the fitted mmff addl charges title and the end of the list is signaled by having both the and J atom types as The parameter added must be in a single line in the following format 1 2 3 4 5 Version number Atom type Partial bci Formal charge adj Comment MMFF Partial Bond Charge Incs and Formal Charge Adj Factors 19 MAY 1994 source J Comp Chem 17 616 1996 type pbci fcadj Origin Comment fitted mmff addl charges 0 1 0 000 0 000 E94 0 2 0 135 0 000 E94 0 3 0 095 0 000 E94 0 4 0 200 0 000 E94 easa 0 96 2 000 0 000 Ionic charge 0 97 1 000 0 000 Ionic charge 0 98 2 000 0 000 Ionic charge 0 99 2 000 0 000 Ionic charge 57 FITTED Suite 3 5 User Guide Appendix B FITTED errors and warnings ERROR Molecule outside maximum number of angles FITTED can only handle molecules with 3 x atoms ang
36. C c1 L 4600 379 800 1 0 CG 2 L 4060 449 900 1 0 3 L 5080 328 300 1 0 c ca L 4870 349 700 Driza 12 ct ss 7700 256 600 1 2 ct nh 3640 449 000 1 2 nt nt 1 3400 450 000 0 0 x e 0 0000 0 000 A 5 2 Adding parameters to the angle list The angle list starts 2 lines below the fitted angle parameters title and the end is signaled by having all J and K atom types columns 3 5 as FITTED also allows for the use of wildcard parameters where and or K can represent any atom by using the wildcard character in the respective column Parameters added to the force field including wildcards should be placed at the end of the angle list The less specific the parameter higher number of wildcards the lower in the list it should be placed The parameter added must be in a single line in the following format Units add units for R K kcal mol rad 53 FITTED Suite 3 5 User Guide 1 2 3 4 5 6 7 Force field file version Reference Atom type Atom type Atom type Ro K2 number number of of J of K Se ES E K2 Theta Theta0 2 CSS EE ee SSS Se Se Se See EE Ver Ref I J K Theta0 K2 Eeer fitted angle parameters SSS Se Se See SS EE oe ER SS See 0 hw ow hw 104 5200 100 0000 0 hw hw ow 127 7400 0 0000 0 br G Dr 113 1000 66 9000 0 br SG Co 110 7400 63 3000 1 0 br e o 121 4600 63 2000 K L 2 n4 hn 109 0000 35 0000 L 2 n4 x 109 5000 60 0000 L x na K 120 0000 60
37. ID Sr EL ewe 6 P6 11 8810 11 1423 8 8608 1 GRID Genre NAN 7 P7 19 3810 3 6423 4 3608 1 GRID E SEA 8 P8 11 8810 8 1423 11 8608 1 GRID He SOON 9 P9 13 3810 11 1423 10 3608 1 GRID 3 6305 r 10 P10 16 3810 6 6423 2 8608 1 GRID Bi BS Eege EL Pili 19 3810 5 1423 5 8608 1 GRID Care ei LS 12 P12 19 3810 2 1423 5 8608 1 GRID RA 13 P13 13 3810 8 1423 8 8608 1 GRID 3 4609 vers 14 Pia 11 8810 11 1423 11 8608 1 GRID 94588 NESE 15 P15 10 3810 9 6423 11 8608 1 GRID 3 4260 ss ee 16 P16 20 8810 8 1423 4 3608 1 GRID Sat eee 17 P17 13 3810 8 1423 10 3608 1 GRID 3 3765 setae 18 P18 10 3810 11 1423 13 3608 1 GRID Valo hea een 19 P19 11 8810 12 6423 11 8608 1 GRID 32619 ver 20 P20 10 3810 9 6423 14 8608 1 GRID CAS CU Merer 21 P21 11 8810 3 6423 7 3608 1 GRID Oreste Sree 22 P22 10 3610 12 6423 11 8608 1 GRID 3 2255 teeter 23 P23 19 3810 3 6423 7 3608 1 GRID GELGGS EAN 24 P24 17 8810 3 6423 2 8608 al GRID 3 1626 rrrs 25 P25 17 8810 3 6423 5 8608 A GRID DELIO T stews 26 P26 17 8810 8 1423 1 3608_ 1 GRID 3 1765 vervs i For Help press F1 NUM 51 FITTED Suite 3 5 User Guide AA The interaction site binding site cavity and XXXX_IS mol2 files The interaction sites and binding site cavity file are used to create conformations that already have good interaction with the protein Again the format resembles mol2 format with the addition of columns for the interaction site type and
38. Program now Closing The protein file given can not be found in the input directory lt Ligand file name gt file not present Program now Closing The ligand file given can not be found in the input directory Side chain lt residue name gt Not found in lt protein file name gt The residue given can not be found in the protein file Unknown residue name lt residue name gt The residue is not known Refer to Tables 1a and 1b for accepted residue names 61 FITTED Suite 3 5 User Guide Appendix D SMART errors and warnings The following is a list of errors and warnings that SMART outputs to the corresponding log file in the output directory Errors indicate serious problems that cause SMART to either skip a molecule or exit Warnings are potential problems that might cause the SMART output to be incorrect critical examination of the output and input structures in these cases is strongly encouraged ERROR File lt filename gt cannot be opened The input file specified could not be read Make sure that the file is located in the input directory Check the spelling and the file permissions ERROR Atom lt atom name gt cannot find element The specified atom has a non standard Sybyl atom type or is not in the range of atomic numbers 1 35 H Br 44 46 Ru Pd 53 I or 78 Pt Without a proper element assignment atom types cannot be assigned In particular look for i P atoms in ph
39. S 14 6206 1 8493 10 5447 1 PHARM 0 7500 HBD 5 0000 9 P9 16 6852 1 9991 13 2224 1 PHARM 1 2000 HBD 5 0000 10 P10 19 0346 1 4487 9 6946 1 PHARM 1 2000 HED 5 0000 Fr P11 18 5152 5 0332 8 2018 d PHARM 1 2000 HBD 5 0000 12 P12 13 6520 42 7965 9 6955 1 PHARM 1 2000 HBD 5 0000 13 P13 16 7522 0 0367 7 7953 1 PHARM 0 7500 HBA 5 0000 14 P14 20 7056 8 2778 12 9660 1 PHARM 1 2000 HBD 5 0000 15 P15 22 0072 14 0719 12 5012 1 PHARM 1 2000 HBD 5 0000 16 P16 16 6747 15 8059 16 8933 1 PHARM 1 2000 HBD 5 0000 17 P17 19 3110 12 2730 17 9280 1 PHARM 2 3000 HBD 5 0000 18 P18 20 3302 12 3488 13 9023 1 PHARM 0 7500 HBA 5 0000 19 P19 17 1223 20 4562 14 1161 PHARM 0 7500 HBD 5 0000 20 P20 20 5044 14 3237 13 0743 1 PHARM 0 7500 HBA 5 0000 21 P21 17 4681 15 8488 19 3465 PHARM 0 7500 HBA 5 0000 22 P22 11 1580 14 3900 8 7800 1 PHARM 2 3000 HBD 5 0000 23 P23 11 8241 13 7743 6 3782 a PHARM 0 7500 HBA 5 0000 24 P24 11 0604 14 9240 19 2151 2 PHARM 1 2000 HBD 5 0000 25 P25 10 1048 11 2484 15 5964 d PHARM 0 7500 HBD 15 0000 _ 26 P26 9 7988 8 9320 16 2352 1 PHARM 0 7500 HBA 5 0000 Je For Help press F1 NUM 52 FITTED Suite 3 5 User Guide A 5 The force field file The force field file is where all the parameters for the FITTED force field are kept Additionally SMART uses the force field file to assign MMFF charges to molecules if so requested The force field is a modified GAFF force field with MMFF cha
40. al Dock Normal docking run This is the default VS This mode is now deprecated in this new version When selecting this mode it automatically switches to the Dock mode Currently not working bug to fix Scores the ligand input structure in the provided orientation against all input proteins 30 FITTED Suite 3 5 User Guide Local Performs a local search on the ligand input structure The provided orientation translation conformation is used as a starting point and only slight modifications to the ligand conformation orientation and translation are carried out SAR Performs a local search on the ligand input structure The provided orientation translation conformation is used as a starting point and only slight modification to the ligand orientation and translation are carried out while a complete search of conformations is done Flex Type Rigid Semiflex Flex water Flex Rigid The ligand is docked onto one protein structure This is the default if only one protein structure is used Semiflex The ligand is docked onto multiple protein structures requires Protein 2 2 Proteins can be exchanged during the evolution but not the genes corresponding to side chains or water molecules a more complete description of this mode is given in reference 1 This is the default if more than one protein structure is used Flex water The ligand is docked into multiple protein structures requires P
41. angle brackets lt gt indicate a numeric value plain text indicates a text string such as a file name square brackets choicel choice2 indicate a choice of values the default shown in italics When a default value is assigned to a keyword the latter can be omitted from the keywordfile Note that keyword files are case sensitive Empty lines are allowed and text after a pound sign is considered a comment Although the value of many keywords can be altered default values should be used unless a specific system requires different settings These keywords are essentially used by the developers for optimization and evaluation of the program In general modification of a specific value does not significantly improve or affect the accuracy but may result in longer or quicker docking runs At the end of this section a typical keyword file can be found Vil 1 Input output files Protein lt of files gt ci input file 1 input file 2 Following this keyword is the number of protein structure files used as input same protein different conformation These protein files should be prepared using Process prior to the actual docking On the following lines are the protein file names one per line without the file extension mol2 Ligand tem ica kemmert Name of the ligand file to be docked in MOL2 format This ligand files should be prepared using SMART prior to the actual docking The ligand file can
42. b file flexible protein mode only In addition to the protein mol2 file generated when flexible protein docking is performed the complete composite protein structure is generated as a pdb file for the best pose of each run The name is XXXX rank 1 Protl runl pdb 44 FITTED Suite 3 5 User Guide Appendix A FITTED Input File Formats The following sections outline the file formats used for FITTED FITTED uses a modified version of the Sybyl MOL2 format for all of its input files For more information on the original Sybyl MOL2 format visit http www tripos com data support mol2 pdf The following is an example of a standard MOL2 formatted file 1e4h_tig mol2 WordPad og Column Description Fie Edit View Insert Format Help 1 Atom number D SR A BR D 2 Atom name k k Name ie4h tripos lig 2 3 x coordinate Creating user name englebip 4 y coordinate Creation time Fri Sep 22 15 33 22 2006 5 z coordinate Modifying user name englebip 6 Atom type Modification time Fri Sep 22 15 33 22 2006 7 Group number lt TRIPOS gt MOLECULE 8 Group name le4h tripos_lig 9 Partial charge 13 n 1 o 2 SMALL USER_CHARGES INVALID_CHARGES lt TRIPOS gt ATOM a i HI 43 6724 0 6420 29 5009 H 1 UNK 0 2521 2 02 43 0000 0 0000 29 2030 0 3 1 UNK 0 3257 3 CH 41 9070 0 5050 27 1740 C ar 1 UNK 0 0584 4 C4 41 8980 0 5160 25 7600 C ar 1 UNK 0 0292 5 C5 43 0000 0 0000 25 0470 C ar 1 UNK 0 0260 6 C6 44 1000
43. cale Pro Cutdist lt cutdist gt Cutoff distance in A for the non bond interactions with the protein The default value is 9 33 FITTED Suite 3 5 User Guide Switchdist lt switchdist gt Switching distance in A for the non bond interactions with the protein The default value is 7 Cutdist Wat lt cutdist wat gt Cutoff distance for the non bond interactions with the water molecules The default value is 1 20 Switchdist Wat lt switchdist wat gt Switching distance for the non bond interactions with the water molecules The default is 1 75 GI Protein Nbonds United All Atom FITTED will treat protein non bonded interactions with the ligand as either all atom or united for the generation of the initial population The default for this keyword is United GA Protein Nbonds United All Atom FITTED will treat protein non bonded interactions with the ligand as either all atom or united for the evolutional The default for this keyword is United GA Protein Monde lt generation number gt FITTED will switch from united to all atom representation of the non bonded interactions at this generation The defaults is set to Max_Gen2 Solvation On Off Allows the user to turn off the calculation of the solvation energy The default is on VIL5 Scoring parameters The default values for all the keywords are highly recommended as they represent the scaling factors optimized for RankScore
44. change after GI MaxSameEnergy 3 iters is lt GI EnergyBound consider equivalent 40 Maximum number of iterations in evolution 0 02 nitial step size along direction 10 Maximum Step size 0 001 Gradient convergence criteria 0 001 f energy change after GA MaxSameEnergy 3 iters is lt GA_EnergyBound consider equivalent none nonel scorel minimize Scoring initial input 4 VdW interactions to consider 1 4 1 5 0 Scaling factor for 1 4 vdW interactions Da Scaling factor for 1 5 vdW interactions 0 Scaling factor for lig prot vdW energy 0 Scaling factor for lig wat vdW energy 4 Electrostatic interactions to consider 1 4 1 5 0 Scaling factor for 1 4 elec energy 0 Scaling factor for 1 5 elec energy 0 Scaling factor for lig prot elec energy 0 Scaling factor for lig wat elec energy Y YIN Includes HBond in energy calculation 0 Scaling factor for lig prot Hbond energy 40 FITTED Suite 3 5 User Guide E HbondScale Wat Cutdist Switchdist Cutdist Wat Switchdist Wat GI Protein Nbonds GA Protein Nbonds GA Protein Nbonds2 Solvation Pop Size Min MatchScore Min _PharmScore Anchor Atom Anchor Coor Max_Tx Max _Ty Max_Tz GI_Num_of Trials tt tt Matching Algorithm Num of Top IS Stringent Triangles Stringent_MS Evolution Max_Gen_1 CutScore 1 Max_Gen_2 CutScore 2 Seed Resolution pLearn MATCHING ALGORITHM wor 1 20 TvS United United
45. d residues Ligand Exclude lt ligand residues gt Ligand name 1 chain number Ligand name 2 chain number Ligand residue to be excluded from the protein mole file On the same line following this keyword specify the number of ligand residues On subsequent lines the residue name chain and number are specified one per line as it appears in the pdb file ex TMC A 500 Mutate lt residue name gt lt res chain gt lt res number gt lt new_res gt Residue to be automatically mutated to another residue On the same line following this keyword specify the residue name chain number as it appears in the pdb file followed by the new residue name ex TYR A 58 PHE 18 FITTED Suite 3 5 User Guide Delete lt residue name gt ren chain gt lt res number gt Residue to be automatically deleted On the same line following this keyword specify the residue name chain number as it appears in the pdb file ex ASP A 19 IV 3 Additional parameters for the preparation of a mol2 file mode make_mol2 only Mode fitted normal Mode of execution In the fitted mode only a maximum of 20 water molecules within 5 A of the ligand are conserved in the protein mol2 file In the normal mode no water molecule deletion is performed The default is fitted Optimize YIN Optimization of tautomers and water molecules The default is N Iterations lt number gt Number if optimization ite
46. directly and the path will be updated in the gray text area If you already prepared the keyword file or you want to start from a previous keyword file you can select the desired file by clicking the Select Keyword File button IMPORTANT the keyword file and the input files must be in the same directory If you select a keyword file from another directory the working directory will be automatically updated The keyword file can be edited by clicking the Edit Keyword File button The program associated with the txt file extension will be used to edit the keyword file To prepare a keyword file to be used by PROCESS refer to the appropriate section of this guide To run the program simply click on the Run PROCESS button 14 FITTED Suite 3 5 User Guide 1114 1 4 Running FITTED within the gui You can select the FITTED window by clicking on the FITTED tab at the top of the gui FITTED is used dock the desired ligand s onto the desired protein s If you require the use of a template keyword file checking the option Use Template Keyword File will copy a template keyword file to the working directly and the path will be updated in the gray text area If you already prepared the keyword file or you want to start from a previous keyword file you can select the desired file by clicking the Select Keyword File button IMPORTANT the keyword file and the input files must be in the same directory If you select a keyword file from another
47. e FITTED Sutton 225550 delle a t KARAER E esucke cavplecd scbecuedsevuhe cv esuchte cassoeteebedesgeevs 12 IL A 1 Running the FITTED suite from the graphical user interface 12 IL A 1 1 Running PREPARE within the gui ss 13 111 4 1 2 Running SMART within the gui sisi 13 111 4 1 3 Running PROCESS within the gui si 14 111 4 1 4 Running FITTED within the gui sisi 15 IL A 2 Running the FITTED suite from the command line ssssssssessssirssssrnsssirnssrirnssrinnnnrinnnnrnnnnnennn 15 IV Preparing a keyword file for PREPARE ccccesseeceeeeeeeeeeeseeeseenseeeseeeseeeeenseeeeeeseaeeeenseeneeenseeeeeenees 17 Wid INPUT OUTPUTTIOS eren eran Saa RAAS KAAORE eee devpoeate cducuegouvebe cv esuchen dessert ee eia 17 IV 2 Parameters for the preparation of the protein all modes 18 IV 3 Additional parameters for the preparation of a mol2 file mode make_mol2 only 19 IV 4 A simple PREPARE keyword file for make mol mode ssssssssssssssinssssrrsseirrssrirnssinnnsrinnnsnnnnsennn 20 IV 5 A simple PREPARE keyword file for make similar mode c cceeeeeeceeeeeeeeeeeeeeeeeeeteneeeeeeeeeaeens 20 IV 6 An advanced PREPARE keyword file for make_mol2 mode 20 V Preparing a keyword file for PROCESS is isnnnneenneennnmnnennennenennnneennenenenes 21 V1 put output TSS cisco Seca en ar ANDA PACARAN ee ruse rar et tree 21 V 2 Reading the input files and preparing the output protein files eee eee enteeeeeeneeeeeenaeeeeeenaes 22
48. e avg best then the population is considered to be converged The default is 1 Diff N Best lt difference n best gt The absolute difference in energy between the individual with the lowest energy and the individual ranked Diff Number IfDiff Number is defined the default value is 0 4 Diff Number lt number rank gt The number of the indivuals to be used with Diff N Best By default this criteria is not used VIL8 Docking of covalent inhibitors This feature is under validation Covalent Residue lt residue name gt Following this keyword is the name of the residue the covalent inhibitor will react with Only CYS and SER are implemented in the current version e g SER554 37 FITTED Suite 3 5 User Guide Covalent Ligand Only Both Controls the covalent docking FITTED will automatically identify the aldehyde boronate or nitrile groups other groups will eventually be implemented and assign the proper atom types when covalent poses will be considered Only Only covalent poses will be considered This is the default Both Covalent and non covalent poses will be considered concomitantly Proton Moved To lt residue gt lt atom name gt The proton will be moved to atom lt atom name gt of residue lt residue gt VII 9 Output convergence parameters Print Level 0111213 Controls the amount of data output The default value is 1 Print Structures Final Full None Controls
49. e complex from now on The complex may also include ions e g metals water molecules and co factors X ray crystal structures downloaded from the PDB most likely do not contain hydrogen atoms Hydrogens on the ligands are needed for FITTED since the ligand is treated via an all atom representation while the hydrogens on the protein are required to assign advanced residue names according to the protonation states and to compute the solvation parameters Once hydrogens are added their orientation should be optimized for instance performing an energy minimization with the heavy atoms fixed One of the advantages of FITTED is its ability to have mobile and displaceable water molecules However this feature requires the proper setup of waters within the complex Only water molecules 10 FITTED Suite 3 5 User Guide which are perceived as key for the binding of ligands should be kept while all others should be removed maximum of 20 water molecules are allowed Waters are perceived as critical if they interact with both the protein and the ligand bridging interactions and are not exposed to the aqueous medium If the number of key water molecules varies with the protein structure copy the location of the missing waters from the other structure During the docking run FITTED will displace them if necessary At this point the complex es can be split into its their corresponding protein and ligand structure file s The protein file s
50. ename pro mol2 output_filename_lig mol2 and output_filename out will be created Ligand Include lt ligands gt Ligand name 1 chain number Ligand name 2 chain number Manually defines the ligand On the same line following this keyword specify the number of ligand residues On subsequent lines the residue name chain and numbers are specified one per line as it appears in pdb ex TMC B 500 Only one ligand molecule is allowed the number of ligand residues refers to the residues that form the molecule as it appears in the pdb file ONLY one ligand molecule is allowed Forcefield Ee ele files ti Name of the force field file to use If a forcefield other than fitted_ff txt is to be used The format of this force field should be consistent with the required format for Fitted Default value is fitted_ff txt if the keyword is not provided Itis highly non recommended to change this value IV 2 Parameters for the preparation of the protein all modes Protein Include lt protein residues gt Protein name 1 chain number Protein name 2 chain number Residue to be included in the protein mol2 file On the same line following this keyword specify the number of protein residues On subsequent lines the residue name chain and numbers are specified one per line as it appears in pdb ex PTR A 201 Can be used for protein residues that are not recognized automatically by the program as natural amino aci
51. eyword fitted txt _ keyword prepare txt _ keyword process txt _ keyword smart txt machine _id exe IL3 Installation To install the suite of programs simply follow the instructions given below This will install the programs and all the required files in a system folder that should not include white spaces The programs can still be used as a command line with arguments or using the graphical interface Be sure to install the correct version of the suite that corresponds to your system architecture 32 or 64 bits 1 3 1 Windows To install the program on Windows simply unzip the file to the root of the hard drive ex c You can install it anywhere else except that the path to the executables should not contain any white space However the path where you run the calculations i e the working directory can contain white space You can also create a shortcut for the Front end jar gui See below for instruction how to use the gui FITTED Suite 3 5 User Guide 113 2 Linux To install the Linux version open a terminal window and execute the install script with the following command tcsh or bash Fitted Linux install forecasterXX bin where XX 32 or 64 The script will guide you through the installation process The programs can be installed locally user account or in a system folder must be root to run the script don t use sudo In order to be able to run the program from the command line yo
52. f J heu se nee Sie H eel eS ee ne Sectes E Aij r712 Bij r 10 esos i es Se eet ee Eege Ver Ref I J A B Geico oe en Cet Be See ee e eS fitted Hbond parameters Wee leone Soe NS to Soe Sa So eee heey Sa T0 3 hw nb 7557 0000 2385 0000 1 0 2 hw ne 10238 0000 3071 0000 1 0 3 hw o 7557 0000 2385 0000 1 0 3 hw oh 7557 0000 2385 0000 1 0 3 hw os 7557 0000 2385 0000 sl 1 2 3 zn S6 15000 0000 5000 0000 1 2 3 zn ss 15000 0000 5000 0000 1 2 3 zn sh 15000 0000 5000 0000 0 0 3 x x 0 0000 0 0000 A 5 7 Adding parameters to the bond charge increment list The bond charge increment list starts below the fitted charge parameters title and the end of the list is signaled by having both the and J atom types as Each line specifies a bond charge increment for a bond between atoms of type and J bciw such that the resulting charge on J is the bciy while on is bciy The parameter added must be in a single line in the following format 1 2 3 4 5 Version number Atom type of Atom type of J bci Comment 56 FITTED Suite 3 5 User Guide FE EAE TE E EERE REE HEE HEE EERE GL J bond_inc source FE EAE TE E FE AE EERE EERE EE HEE HEHE EERE fitted_charge parameters 0 1 0 0000 C94 0 2 0 1382 C94 0 3 0 0610 C94 0 4 0 2000 x94 as 0 80 81 0 4000 C94 O 101 1 0 0000 empirical O 10 6 0 1900 empirical O 10 37 0 0000 empirical oe A 5
53. f files gt input file 1 input file 2 ligand file mol2 lt _of files gt lig_ref_filel mol2 lig ref file2 mol2 filename fitted Cf Cat bindSite mol2 IS mol2 pharmacophore file mol2 Dock 3 Rigid On off Conjugate gradient parameters Second Number First Second Ligand Number First Second Name o Force Name o Name o by Pro Ha AEE AE HAE EHR RET AE AE RET HR RARE ER RE ER REE ER RR Local Number Type o protei On Of On Of E E AE AE REE E AE REE A AE REE HR EE HR RARE EHR ERE EHR RARE than Dock the default is protein file of prot files used for RMSD ref protein file ref protein file structure file of reference ligand files reference ligand file reference ligand file f the output file field file name f cavity file created by ProCESS f interaction file created CESS Name of Pharmacophore file FEFE AE HE FE AE FE TE HE FE E FE TE FE FE HE REE REE REE HERRERA EERE VS SAR Score Filter Running mode of runs to carry out If using any other mode f docking to be performed If more than one n is used the default is set to Semiflexible f Toggle displaceable waters f Toggle ring conformational search FEE AEE HAE EE AE aR AE AE HR ERE HR EEE AE AE HR RE 40 Maximum number of iters during init pop gen 0 02 nitial step size along direction 10 Maximum Step size 0 001 Gradient convergence criteria 0 001 f energy
54. face gui is only a small front end to help the setup of the docking and the edition of the keyword files To start the gui simply double click on the Front end jar file windows only For Linux and Mac OSX users type java jar Front end jar from a terminal Fitted Linux java jar lt path to the executable gt Front end jar A window with four tabs PREPARE SMART PROCESS and FITTED will open 12 FITTED Suite 3 5 User Guide Select Working Directory Browse PREPARE SMART Process FITTED i Prepares the protein by adding hydrogens and optimizing tautomers and water molecules SS _ Use Template Keyword File pdb code Getpdb Openpdb Select Keyword File Edit Keyword File Run PREPARE M OTL ESC UE AUR The first thing to do when starting to work with the gui is to define the working directory by browsing to the desired folder using the Browse button at the top of the window Once a working directory is selected the path will be shown in the gray box area The working directory can be opened at any time by simply clicking the Open button The location of the executables programs can be changed from the File settings menu 1 4 1 1 Running PREPARE within the gui When you start the gui the PREPARE tab is shown by default If you already have prepared your protein in mol2 format with the hydrogens move to the other tabs If you are connected to the interne
55. file lt TRIPOS gt ATOM is not found in the ligand file preceding the coordinates ti a Kal TD ROR Check water names and atom types The water atom name and atom types are non standard Change to standard names ea D ROR Bonds not found in ligand file lt TRIPOS gt BOND is not found in the ligand file preceding the list of bonds Si x ROR No assignment of Rotatable bonds Please assign rotatable bonds either manually or by using SMART ti ROR Bonds not found in protein file lt TRIPOS gt BOND is not found in the protein file preceding the list of bonds ti a ROR Protein keyword not found in keyword file The keyword Protein is not found within the keyword file Please include this within you keyword file followed by the number of protein files and on the next lines a list of the protein files for the docking virtual screening run ti ROR Can not find lt residue name gt Cannot a residue listed in the keyword file Please check the spelling eal D ROR Flex file lt protein file name gt not found Make sure lt protein file name gt flex txt is in the input directory Error Can not find coordinates in lt Binding Site filename gt is not present in the active site cavity file lt TRIPOS gt MOLECULE is not found in the Binding Site Cav file voy Error Can not find coordinates in lt Binding Site f
56. file The amount of information within this file is controlled by the Print_Level keyword in the keyword file At the beginning of the output file all the parameters used for the docking are printed with their corresponding value Information about the generation of the initial population appears followed by the evolution of the population genetic algorithm At the end when the convergence is reached a table is printed with the information about the top poses of this run When more than one run is performed default is 3 the information is added continuously When more than one ligand is docked within the same docking multi mol2 ligand file the information about the next ligand is added in the same order as in the ligand file The table labeled Best Complexes contains the information used to identify the best pose For each requested top pose of a single run the ligands are ranked by energy To this energy is then associated a score value that can be used to compare with different ligand molecules Therefore to identify the best pose out of the 3 run performed for the same ligand the ligand with the lowest energy should be taken and the score associated to this ligand can then be used for comparison The score is also based on the energy plus additional terms based on the RankScore scoring function therefore the lower the score the better is the pose Best Complexes Ranked by Energy Rank Score Energy rmsd mscore
57. from a pdb structure and outputs file in mol2 format It also superposes and makes similar different pdb structures of the same protein for flexible protein docking with FITTED 2 PROCESS creates the required files from the protein structure to be used for the docking It requires a mol2 format with hydrogens already added 3 SMART is used to prepare the ligand for docking It requires a molecule in 3D mol2 format with hydrogens already added It will add the partial charges assign correct bond orders and correct atom types 4 FITTED is the program used for docking it requires a ligand that is prepared by SMART and a set of files describing the protein as prepared by PROCESS FITTED D gt FITTED Prepare Forecaster E gt Siart ProCESS 111 2 Setting up the system manually In general the structure files downloaded from the Protein Databank PDB require some preparation to ensure optimal results with any docking program For instance the protonation state of some residues may be critical to the binding of a ligand hence to the observed enzymatic activity The accuracy of the docking obtained with the FITTED suite of programs therefore relies on a careful preparation of the input files The following sections give general details on what needs to be done to the protein and ligand structure files before the FITTED suite can be used for optimal results When the ligand and the protein remain as one file they will be referred to as th
58. he boundary of the box will be ignored defined by Grid_Size and spheres can include volume outside of the box This is the default Hard The active site cavity file will be constrained within the box defined by Grid Size Grid_Resolution lt grid_resolution gt o Following this keyword is the resolution A of the grid The default is 1 5 Grid Sphere Size lt grid sphere size gt Specifies the size of a sphere used to trim the sides of the box to make it rounder The default 15 Grid_Clash lt grid clash gt fa protein atom is within this distance of a grid point the point is removed from the grid The default is 1 5 V 4 Parameters for the Interaction Sites file XXX_ Weight lt xxx weight gt This group of keywords xxx being Hydrophobic Metal HBA or HBD specifies the parameters for the assignment of pharmacophoric points xxx weight is used to give weight for favourable xxx type interactions Defaults parameters are highly recommended Hydrophobic Weight lt hydro weight gt Defines the weight for hydrophobic interaction points The default is 1 Metal Weight lt metal weight gt Defines the weight for metal interaction points The default is 50 HBA Weight lt hba_weight gt Defines the weight for hydrogen bond acceptor interaction points The default is 5 HBD Weight lt hbd weight gt lt hbd penalty gt Defines the weight for hydrogen bond donor interaction points The default i
59. higher than 1 Check the bond order assignment in these molecules to make sure it is correct WARNING Missing bond increment Bond lt bond number gt Atoms lt atom namel gt lt atom name2 gt MMFF atom types lt MMFF typel gt and lt MMFF type2 gt Bond increment set to 0 When automatically assigning the MMFF charges charge command line option this message is output for every pair of atoms for which the bond increment is not parameterized Add the bond increment in the forcefield fitted ff txt file WARNING Could not assign charges to molecule When automatically assigning the MMFF charges charge command line option this message is indicative of other problems with the charge assignment Look for warning messages appearing before this one in the log file 62 FITTED Suite 3 5 User Guide Appendix E Functional group definitions Table 1 Definition of functional groups in SMART blue atom type green element Keyword Description Arati ca An aromatic group is present if a ca atom type is within the molecule Aldehyde Y H H An aldehyde is present if there is a c atom type in the molecule bound to a hydrogen C O 3k An ester is present if there is a c atom type Ester A 07 bound to an atom with an os atom type with the c An Ki not bound to an a n atom type with both c and os C OS atoms being acyclic 0 a A lactone is present there
60. idues In some cases terminal residues are not properly described in the PDB or mishandled by the program used to setup the protein e g terminal COO groups are CHO In this case the missing atoms should be added o Missing Waters All proteins files should have the same number of waters If a water molecule is missing one can be virtually added from another protein file FITTED will remove the water if it is not needed o Missing atoms Atom actually missing if it is far away from the active it can be added lf the atom is there make sure the atom name and atom type are the same The atom may be a different part of the protein file If this is the case renumber the atom within your graphical interface and regenerate the protein input file for ProCESS o Nucleic acids The 5 terminus should have a 5 OH and not a phosphate group If necessary remove the phosphate group and protonate the 5 oxygen The residue names need to be corrected before attempting to run Process but after adding hydrogens to the system For this a pair of scripts fix dna awk and fix rna awk are provided on the fitted ca 11 FITTED Suite 3 5 User Guide website This scripts rename the residues according to the names in Table 1b fix rna awk term5 lt 5 term gt term3 lt 3 term gt file mol2 gt file new mol2 lt 5 term gt and lt 3 term gt denote the residue numbers column 7 in the MOL file of the 5 and 3
61. ilename gt lt TRIPOS gt ATOM is not found in the Binding Site Cav file preceding the coordinates Error Can not find coordinates in lt pharmacophore filename gt lt TRIPOS gt ATOM is not found in the Pharmacophore file preceding the coordinates Error Can not find coordinates in lt Interaction Sites filename gt lt TRIPOS gt ATOM is not found in the Interaction Sites file preceding the coordinates Error Ligand can not match minimum pharmacophore Increase value of Min _PharmScore 59 FITTED Suite 3 5 User Guide Error Ligand can not match minimum Interaction Sites Increase value of Min MatchScore ea D ROR Reference file reference filename gt not present The reference file is not located within the input directory E ROR Invalid parameter specified for covalent residue ti a Make sure the residue name is listed in the keyword the same way it is listed in the protein file ROR FITTED cannot find O S and H for the covalent residue Kal TD Format in protein input file may be incorrect In particular make sure that for serine the alcohol atom names are set as OG and HG mi DO ROR Invalid parameter specified for other catalytic residue Make sure the residue and atom name are specified the same in the keyword and protein file ti a ROR The pro
62. ing the keyword specify the main mode to be run Run Mode mode Following the keyword specify the run mode for the program specified in the Mam Mode keyword make similar superposes multiple PDBs then make them similar make mol2 converts a PDB file to a mol2 file alignment provides the sequence alignment of multiple PDB files superpose superposes multiple PDB structures stats collects statistics on the protein side chains pnma reconstructs a protein structure from a complete PDB file and a backbone Protein lt proteins gt protein filel pdb protein file2 pdb On the same line following this keyword specify the number of proteins On subsequent lines the protein filenames pdb files only Next to the pdb filename the chain ID is optional Default is All It can be any combination as desired such as A AB ABC AD or All for everything When Run Mode is make similar or aligment Protein lt i proteins lt chain CO loo considere protein filel pdb A protein file2 pdb A On the same line following this keyword specify the number of proteins On subsequent lines the protein filenames pdb files only Next to the pdb filename the chain ID is optional Default is All It can be any combination as desired such as A AB ABC AD or All for everything 17 FITTED Suite 3 5 User Guide Output output_filename Name of the output file output fil
63. ions The default is 0 01 pElite Every X Gen lt pElite Every X Gen gt pElite will be used every pElite Every X Gen 36 FITTED Suite 3 5 User Guide The default is 2 pElite SSize lt pElite SSize gt The individual to be passed directly onto the next generation will be selected random from the top pElite SSize individuals of the population The default is 10 pOpt lt popt gt Probability of optimization of the ligand at every generation The default is 0 20 Evolution Steady State Metropolis Elite Steady State During the evolution out of a pair of two children and their 2 parents the two best will be saved This is the default Metropolis During the evolution out of a pair of two children and their 2 parents two individuals will be saved following the Metropolis criterion If the children are higher in energy they are checked to see if they have a high probability to exist at room temperature If they do they are saved Elite During the evolution the top pop size individuals of the children and parents will be kept for the next generation GA_Num_of Trials lt ga_num_trials gt Maximum number of successive unsuccessful trials to create children The default is 1000 Diff Avg Best lt difference avg best gt The absolute difference between the average energy of the population and the best individual of the population If the calculated value is below differenc
64. is a c atom type bound N o to an atom with an os atom type with the cnot Lactone bound to an a n atom type with c and os atoms oe involved in a ring O SNE Jk R An amide is present if there is a c atom type Amide UN H bound to an atom with an n atom type with the c RN not bound to an os atom type with both c and n OS C n atoms being acyclic os 9 RW A lactame is present if there is a c atom type N nP bound to an atom with an n atom type with the c Lactame not bound to an a os atom type With both c and n natoms being cyclic o H ke An acid carboxylate is present if an atom with a Acid ars c atom type is bound to two atoms with o atom types C 63 FITTED Suite 3 5 User Guide cl Sen Nitrile nitrile is present if an atom with a c1 atom type is bound to an atom with an n1 atom type C2 vi Z O Animineis present if an atom with a c2 atom Imine Bech type is bound to an atom with an n2 atom type KH n2 both acyclic R cannot be an oxygen atom 0 A nitro is present if there is an atom with an no Ni Na a E DS no atom type within the molecule C om O N e c2 led Michael acceptor is present if an atom with an atom type of c2 is bound to either 1 an atom with Acceptor a catom type which is not a carboxylate or 2 a ci 1 nitrile group The bond between c2 and c c1 must NNA n be acyclic oa Azide N An azide is present if there are three acyclic Ve d nitroge
65. late keyword file to the working directly and the path will be updated in the gray text area If you already prepared the keyword file or you want to start from a previous keyword file you can select the desired file by clicking the Select Keyword File button IMPORTANT the keyword file and the input files must be in the same directory If you select a keyword file from another directory the working directory will be automatically updated The keyword file can be edited by clicking the Edit Keyword File button The program associated with the txt file extension will be used to edit the keyword file To prepare a keyword file to be used by SMART refer to the appropriate section of this guide To run the program simply click on the Run SMART button areal FITTED Front end File Help Select Working Directory Browse C Mitted_exe example_1e2k Open PREPARE SMART ProCESS FITTED Setup the ligand s for docking C Use Template Keyword File Select Keyword File Run SMART _ Edit Keyword File MSOSLIESCEURES AVR Le 111 4 1 3 Running PROCESS within the gui You can select the PROCESS window by clicking on the PROCESS tab at the top of the gui PROCESS is used to setup the protein s for docking with FITTED If you require the use of a template keyword file checking the option Use Template Keyword File will copy a template keyword file to the working
66. le the docking may take longer and be less accurate eal D ROR Reference file lt Reference file name gt not present The reference file could not be located within the input directory ti a ROR Missing Forcefield Parameters FITTED exits because there are missing force field parameters Either add them to the force field file or use Parameters Auto keyword to have FITTED automatically assign parameters WARNING Missing Forcefield parameters assigning parameters automatically List below is the parameter which was assigned automatically If you do not like the automatic assignment add the parameter with your desired value into the force field file ERROR lt keyword filename gt Can not be opened If the keyword file is not found in the keyword directory then an error 1log will be created with this error Please put keyword in keyword directory 58 FITTED Suite 3 5 User Guide eal D ROR Coordinates not found in protein structural file lt TRIPOS gt ATOM is not found in the protein file preceding the coordinates wer ERROR Array size for number of protein atoms and bonds not in Protein mol2 file lt TRIPOS gt MOLECULE is not found in the first protein mol2 file ERROR Array size for number of ligand atoms and bonds not in Ligand mol2 lt TRIPOS gt MOLECULE is not found in the first protein mol2 file ROR Coordinates not found in ligand
67. les If there are more then please contact the developers at nicolas moitessier mcgill ca ERROR Molecule outside maximum number of torsions FITTED can only handle molecules with 6 x atoms torsions If there are more then please contact the developers at nicolas moitessier mcgill ca ti a ROR Forcefield file lt forcefield filename gt not found The force field file listed in the keyword file is not found in the forcefield directory ti a ROR Molecule too big for active site Increase GI_Num_of Trials Increase GI_Initial E Increase GI Minimized E Increase Grid Size in Process to create a larger active site cavity f none of these work the molecule is too big for the active site and cannot be docked eal TD ROR Protein input file lt Protein file name gt not present The protein file could not be found in the input directory ti a ROR Ligand input file lt ligand file name gt not present The ligand file could not be located in the input directory ti a ROR Binding Site Cav file Active site filename gt not present Binding Site Cav file could not be located within the input directory Without an active site file the docking may take longer and be less accurate WARNING Binding Site Cav needed for generation of initial population FITTED issues this warning but will not exit Without an active site fi
68. me secondary amine primary amine number of number of ketone boronate 49 FITTED Suite 3 5 User Guide number number number number number of lt TRIPOS gt ATOM section oxygens nitrogens sulphurs hetero atoms toxic metals o the Sybyl atom types column 6 are replaced by GAFF atom types the corresponding Sybyl atom types are stored in column 11 o the number of hydrogen atoms attached to an atom is stated on column 12 o the hybridization of an atom is stated on column 13 lt TRIPOS gt BOND section o an additional column specifies the bond as rotatable r or non rotatable nr Column Discription Atom number Atom name x coordinate y coordinate z coordinate Atom type Group number Group name Partial charge Misc Information Sybyl Atom type Number of Hydrogens hybridization OANDARWN a D 4 wo a 1e2k_lig Notepad EX EI File Edit Format View Help Creating user name Creation time lt TRIPOS gt MOLECULE Le2k_lig mol2 34 36 252 27 0 SMALL USER_CHARGES lt TRIPOS gt ATOM 1 H1 50 6487 2 H2 51 2254 3 H3 43 4989 4 H4 49 3923 5 H5 47 7507 6 06 50 3650 7 07 50 7440 8 08 45 4850 9 09 43 1910 10 N10 46 6500 dt N11 44 3790 12 c12 50 1450 13 c13 49 7430 14 C14 48 4730 15 cis 47 9650 16 c16 49 0050 17 cl 50 3270 18 C18 50 8100 19 c19 45 5520 20 C20
69. n_Mode Main Mode e2k pdb e2k fitted TMC B 500 PTR A 201 TMC A 500 Yy 0 make_mol2 prepare protein 20 FITTED Suite 3 5 User Guide V Preparing a keyword file for PROCESS The following section lists the keywords their functions and default values Gray shading indicates a required keyword angle brackets lt gt indicate a numeric value plain text indicates a text string Such as a file name square brackets indicate a choice of values the default shown in italics When a default value is assigned to a keyword the latter can be omitted from the keywordfile PROCESS keywords files are case sensitive Empty lines are allowed and text after a pound sign is considered a comment Although the value of many keywords can be altered default values should be used unless a specific system requires different settings At the end of this section a typical keyword file can be found V 1 Input output files Main Mode process Following the keyword specify the main mode to be run Run Mode process Following the keyword specify the run mode for the program specified in the Mam Mode keyword Protein EE E E protein filel mol2 protein file2 mol12 Following the keyword specify the number of protein structure files to be processed On the following lines specify the protein file names one per line Output output_filename Name of the output file
70. naeeeenenaes 39 VII 11 A simple FITTED keyword file for flexible protein docking 39 VI1 12 A template FITTED keyword file with all the possible keywordS ssssssssseesssessesrrssrsrssren 39 VIII Analysis of a docking run With FITTED 00 cccceseeeeeseeeeeeeseeeeeseeeeeeeseseseeesaseeneesesesneesesesnenenseeenens 42 MUM A The log TG aisn E tn ten nn ET ES rennes Ent teste Rat est tit 42 KA 43 VIES The results tes nn Re EE Te de ea Bt en 43 VIIE4 lt The ligand molz OTT 43 VIIES Th SOP file a sister den Eegeregie Hie 43 VIII 6 The protein mol2 file flexible protein mode only 44 VIIL7 The protein pdb file flexible protein mode only 44 Appendix A FITTED Input File Formats isnsssnnnnnnnennnnennnes 45 Aili TING protein Mess frame nn nm ner da mr AAAF PAAA a a ne ee et 46 E ER eege EE 46 A2 THE XXXXK SCOPE M2 le sees enen d s ei Ries d se soie Sniper deed geet abrite 46 L The XXXX SIS AKT NO EE egen sh ES et arbres an e Ree Ee Aere geed et ei 48 AZ The Ligand TNO nuses ria anaia Mani mnt drama tante Mimet 48 FITTED Suite 3 5 User Guide A 3 The binding site Cavity file sise 51 AA The interaction site binding site cavity and XXXX_IS Mol2 files 52 A 5 The torce field E UE 53 A 5 1 Adding parameters to the bond list 53 A 5 2 Adding parameters to the angle list 53 A 5 3 Adding parameters to the torsion list 5
71. ns in a linear formation SN n2 An isocyanate is present if an atom with an atom G type of c is bound to 2 atoms one with an atom I oms PORTENE E vi 2 type of n2 and another with an atom type of o N ra Ka KE where the c n2 bond is acyclic C o SL x Ck cl An acyl chloride is present if an atom with a atom Acyl Chloride type of cis bound to an atom with an atom type C o of clor br SL Be br 64 FITTED Suite 3 5 User Guide s6 n A sulphonamide is present when an atom with an Sulphonamide an A atom type of s6 is bound to an atom with an atom Vn type of n H d SCH A carbamate is present when an atom with an Carbamate H DIR R atom type of cis bound to an atom with an n N O lt OS n di g atom type and an atom with an os atom type D R SE n ammonium is present if there is an atom with Ammonium N R ZER an n4 atom type O An oxime is present if there is an atom with a c2 Oxime c2 NOR atom type bound to an atom with an n2 atom Deen type which in turn is bound to an oxygen atom p n2 C SO Ketone FAKA A ketone is present if an atom with a c atom type NA cis bound to 2 carbon atoms C c B o e ofp A boronate is present if there is a boron atom oronate B K O R bound to a carbon and two oxygens O n3 A D D if h s H primary amine is present if there is an atom dns ens R N with an atom type of n3 bound to two hydrogens n3 Secondary Amine H A secondary
72. osphates and analogous functional groups the Sybyl atom type for the P atom should be P 3 ii S atoms in sulfoxides sulfones and derivatives the Sybyl atom type for the S atom should be S o or S o2 respectively ERROR could not write to lt filename gt The specified output file could not be written Check permissions on the output and parent directories that there is enough empty space in the volume and that the filename is valid ERROR cannot create Z matrix Does the molecule have a torsion In order to be processed by SMART a molecule must at least have 4 atoms connected sequentially in order to define a torsion If a torsion cannot be defined the molecule is skipped WARNING Sum of partial charges does not equal net charge When assigning MMFF charges the partial charges assigned do not match the predicted formal charge Check atom type assignment and bond connectivity WARNING Cannot assign atomic weight to atom lt atom_number gt lt atom_name gt When generating the bit string the molecular weight is calculated from the sum of atomic weights Currently only atoms of atomic number 1 17 H Cl 34 35 Se Br and 53 I are parameterized WARNING Atom lt atom_name gt has a formal charge of lt formal charge gt When automatically assigning the bond orders assign_bond command line option this message is output to the log file for every atom with a formal charge
73. rations The default is 10 but 5 is also recommended Protonate lt atom_to_protonate gt Atom to be manually protonated by the program On the same line following this keyword specify the residue name chain number and atom name as it appears in the pdb file ex HIS A 58 NE2 Deprotonate lt atom_to_deprotonate gt Atom to be manually deprotonated by the program On the same line following this keyword specify the residue name chain number and atom name as it appears in the pdb file ex RTL A 701 O2 Hybridization lt atom to hybridize gt Atom to manually change the hybridization by the program On the same line following this keyword specify the residue name chain number atom name and hybridization state as it appears in the pdb file ex RTL A 701 C15 sp2 19 FITTED Suite 3 5 User Guide IV 4 A simple PREPARE keyword file for make_mol2 mode Protein Output Ligand_Include Optimize Iterations Run_Mode Main Mode 1e2k pdb le2k 1 TMC A 500 y 5 make_mol2 prepare protein IV 5 A simple PREPARE keyword file for make_similar mode Protein Output Ligand_Include Run_Mode Main Mode 2 le2k pdb A le2p pdb A tk 2 TMC A 500 CCV A 500 make similar prepare protein IV 6 An advanced PREPARE keyword file for make_mol2 mode Protein Output Mode Ligand_Include Protein Include Ligand_Exclude Optimize Iteration Ru
74. recommended to use both Interaction Sites and Binding site cav keywords Interaction Sites XXXX IS mol2 Name of the file containing the interaction site description prepared by Process lf this keyword is missing no interaction site filter will be used It is highly recommended to use both Interaction Sites andBinding site cav Pharmacophore pharmacophore file mol2 Name of the file containing the pharmacophore constraints on the ligands prepared by ProCEss Typically this keyword is used to ensure that the individuals produced match this constraint but it can be softened by setting Min Constraint If this keyword is missing no constraint will be used Protein Ref lt _of files gt SI EC re ile l ext E Et ile 2 ext ES Following this keyword is the number of reference protein structure files used to compute the protein RMSD deviation of the modeled protein structure from the reference structures On the following lines are the protein file names one per line These files will be used in addition to the Protein files listed before to calculate a root mean square deviation RMSD between the protein generated during a fitted docking run and the Protein ref files Additional files can be needed if the protein has a symmetrical structure e g HIV 1 protease If this keyword is missing protein input files will be used as references Vil 2 Run parameters Mode Dock Filter VS Score Loc
75. rge parameters in free format so it can be edited with any text editor Although most of the parameters for drug like molecules are present some may be missing When adding a parameter to the force field file some rules must be followed Each section starts with a title e g itted_bond_parameters followed by the actual parameters i e 1 0 1 c c 1 5500 290 100 and ends with a line with blank parameters designated by stars i e 0 0 1 0 0000 0 000 The title and end lines should not be removed and any line added before the title line and after the end line will be ignored FITTED also allows for the use of wildcard parameters for angles and torsion parameters where I J K or L can represent any atom type by using the wildcard character in the respective column Using wildcards for all the atoms will be read as an end line A 5 1 Adding parameters to the bond list The bond list starts 2 lines following the fitted bond parameters title and the end is signaled by having both the and J atom types columns 3 and 4 as Any parameters added after this last line will be ignored The parameters added must be in a single line in the following format Units R A K kcal mol A 1 2 3 4 5 6 Force field file version Reference number Atom type of Atom type of J Ro K2 number e a a See Ver Ref I J RO K2 Sa ee EE Se Sere eee See Se fitted bond parameters lt 1 0 e 5500 290 100 1 0
76. ro Ligand le2k aligned mutated lig 1 mol2 Output tk flex Parameters Auto Ref 1 le2k aligned mutated lig 1 mol2 Binding Site Cav tk_bindSite mol2 Interaction Sites tk_IS mol2 Mode Dock Flex Type Flex Vil 12 A template FITTED keyword file with all the possible keywords AIS This template file contains all the keywords in use by FITTED For a detailed explanation of their use please see FITTED user guide AIS NPUT OUTPUT FILES FEFE AE HEFE E FE TE HE FE E FE TE FE FE HE FE ST TE FE FE HE FE FE FE FE FE FE E ST Sd HEE FE FE TE HE FE E FE TE FE TE HE FE REE HEE TE FE TE HE FE E FE TE FE TE HE FE E FE TE FE AE EE FE E FE TE ESS RHEE RHEE Protein lt of files gt Number of protein input files input_file_1 First protein file FITTED Suite 3 5 User Guide Protein Ref Ligand Ref Output Forcefield Binding Site Cav nteraction Sites Pharmacophore Run parameters Mode Number of Runs Flex Type Corner Flap GI Max Iter GI StepSize GI MaxStep GI_MaxGrad GI_EnergyBound GI_MaxSameEnergy GA_Max_Iter GA_StepSize GA MaxStep GA MaxGrad GA EnergyBound GA_MaxSameEnergy Energy parameters Score Initial van VdWScale 1 4 VdWScale 1 5 E_VdWScale_ Pro E_VdWScale Wat Elec ElecScale 1 4 ElecScale 1 5 E ElecScale Pro E ElecScale Wat HBond E HbondScale Pro Displaceable Waters input file 2 lt o
77. rotein gt 2 Similar to Semiflex except that each water molecule evolves independently Flex The ligand is docked onto multiple protein structures requires Protein 2 2 The side chains and waters are allowed to be exchanged independently from the protein backbone Number of Runs lt number of runs gt More than one run per ligand can be performed The ligand may be docked several time to ensure a complete search f this keyword is missing the default value is 3 for Dock mode all other modes the default is 1 Displaceable Waters On Off Allows the user to turn off the displaceable waters The default is on which allows displaceable waters Corner Flap On Off Turns the corner flap conformational search for rings on or off By default it is set to Off VII 3 Conjugate gradient parameters The default values for all the keywords described in this section are recommended GA_ or GI There are two sets of the following keywords one for the parameters used during the generation of the initial population GI_ e g GI_MaxInt and another one used during the evolution GA_ e g GA_MaxInt The default values are recommended XX_MaxIter lt maxiter gt 31 FITTED Suite 3 5 User Guide o Maximum number of iterations Once this number is reached the minimization is finished o The default is 20 XX StepSize lt stepsize gt o Initial value of the step taken in the direction of the gradient d
78. s 5 23 FITTED Suite 3 5 User Guide If too many points are found one can reduce this number by using the following keywords Pharm Polar Softness lt pharm polar soft gt Maximum distance in A between two polar points to merge The default is 0 0 Pharm Nonpolar Softness lt pharm nonpolar soft gt Maximum distance in A between two non polar points to merge The default is 0 0 Hydrophobic Level lt hydro_level gt Van der Waals interaction between a probe on the grid point with hydrophobic carbons to be considered hydrophobic H the interaction is found lower than hydro level an hydrophobic point is added at this location For more information see the section on Error Reference ource not found The default is 0 3 Min Weight lt min_weight gt Minimum weight for a pharmacophoric point to be included in the final pharmacophore The defaults are 0 5 0 0 respectively Num_of IS lt num_of spheres gt This determines the maximum number of interaction site spheres in the interaction sites file The default is 75 24 FITTED Suite 3 5 User Guide V 5 A simple Process keyword file for rigid protein docking setup Protein Output Binding Site Cav Interaction Sites AutoFind Site AutoFind Center Ligand Run_Mode Main Mode 1 1e2k pro mol2 le2k 1e2k bindSite 1e2k IS yes yes 1 1e2k lig mol2 process process V A simple Process keyword file for
79. t you can download the pdb file directly by typing the 4 character code in the pdb code box and clicking the Get pdb button Clicking the Open pdb button will open the downloaded pdb file using the program associated with the pdb file extension If you require the use of a template keyword file checking the option Use Template Keyword File will copy a template keyword file to the working directly and the path will be updated in the gray text area If you already prepared the keyword file or you want to start from a previous keyword file you can select the desired file by clicking the Select Keyword File button IMPORTANT the keyword file and the input files must be in the same directory If you select a keyword file from another directory the working directory will be automatically updated The keyword file can be edited by clicking the Edit Keyword File button The program associated with the txt file extension will be used to edit the keyword file To prepare a keyword file to be used by PREPARE refer to the appropriate section of this guide To run the program simply click on the Run PREPARE button 111 4 1 2 Running SMART within the gui You can select the SMART window by clicking on the Smart tab at the top of the gui SMART is used to setup the ligand s for docking with FITTED 13 FITTED Suite 3 5 User Guide If you require the use of a template keyword file checking the option Use Template Keyword File will copy a temp
80. teins do not have the same number of atoms Make sure to run ProCESS with all proteins in one keyword file eal D ROR Problem with creation of z matrix for ligand Make sure there is not a missing bond in the bond list of the ligand mol2 file FITTED cannot handle mol2 with multiple structures ERROR Problem with creation of z matrix for active site residue lt residue name gt A bond is missing from the bond list in one of the protein mol2 files Either add the missing bond s or remove the residue from the XXXX site txt file if it not critical to binding of the ligand 60 FITTED Suite 3 5 User Guide Appendix C Process errors and warnings ua Number of proteins not in keyword file If the number of protein files does not follow Protein_Conformations keyword Coordinates not found in structural file If in either the protein or ligand mol2 file lt TRIPOS gt ATOM does not precede the coordinates of the structure Bonds not found in structural file If in either the protein or ligand mol2 file lt TRIPOS gt BOND does not precede the bond list Ligand file not present now closing f Ligand is not found in the keyword file User wanted automatic finding of active site center Ligand Reference not given Ifthe keyword AutoFind Site is used in the keyword and Ligand is not found in the keyword file lt Protein file name gt file not present
81. terminal residues respectively 111 3 Setting up the system with PREPARE We have developed a new program that automatically prepares the protein from the pdb file and creates two mol2 files the protein and the ligand PREPARE requires the information regarding the residue name and residue number of the ligand as it is used in the pdb file Most common co factor and ions are recognized by the program Two modes are available normal or fitted The latter is different only by the conservation of a maximum of 25 water molecules in the protein file all water molecule beyond 5 A of the ligand are removed PREPARE adds the hydrogens mutates the truncated residues and optionally optimizes the tautomers and the water molecules according the user defined number of iterations All error that might occur is listed in the output file and verification should be made to identify any potential problems with the structure Recently PREPARE was modified to be able to take different pdb structures of the same protein and to superpose them and make them similar based on a sequence alignment Thus this protein ensemble can be used as input in flexible protein docking with FITTED 111 4 Running the FITTED suite All programs work under Windows Mac OSX and Linux All versions are useable from a terminal window as command line or from the graphical user interface 111 4 1 Running the FITTED suite from the graphical user interface The graphical user inter
82. the output of the structures during or at the end of the docking Final Only the final structures will be printed This is the default Full The structures protein and ligand will be printed during the run along with the final structures None No structures will be printed Print Num Structures lt print num structures gt Select how many of the top poses are printed as MOL2 files The default is 1 Number of Best lt number of best gt Select how many individuals to print the score energy and RMSD during the run The default is 10 in Mode Dock and 1 in Mode VS Print Best Every X Gen lt print best every x gen gt How often to print a summary of the run The default is Max Gen 1 Print Energy Full YIN Controls the printout of the detailed energy contributions S Print out a breakdown of the energy bond energy angle energy etc This is the default 38 FITTED Suite 3 5 User Guide N Print out only the total energy Vil 10 A simple FITTED keyword file for rigid protein docking Protein e2k_pro Ligand e2k_lig_1 mol2 Output e2k Forcefield fitted_ff txt Parameters Auto Ref 1 e2k_lig_1 mol2 Binding Site Cav e2k_bindSite mol2 Interaction_Sites e2k_IS mol2 Mode Dock Flex Type Rigid Vil 11 A simple FITTED keyword file for flexible protein docking Protein 4 le2k aligned mutated pro le2p aligned mutated pro 1ki3 aligned mutated pro 2ki5 aligned mutated p
83. tscore 2 gt Upper bound score at Max_Gen_2 to further proceed with the docking run If there is one individual within the top 3 below this CutScore_2 then the program proceeds to Max_Gen_2 The default is 5 5 Max Gen 2 lt max gen 2 gt As for Max Gen L T after Max Gen_1 generations none of the top poses has a score below the one specified by CutScore 2 the program exits Otherwise the program proceeds until it reaches Max_Gen_2 The default is Max Gen Seed lt seed gt Select the starting point within the random number generator If the same run is done with the same seed the exact same result will be obtained If a different seed is used the GA will follow a different path Changing the seed helps the developers to evaluate the convergence of a run The default is 100 pLearn lt plearn gt Probability of energy minimization of the parents at every generation The Default is 0 1 pCross lt pcross gt Probability of crossover at every generation The default is 0 85 pMut lt pmut gt Probability of mutation at every generation The default is 0 05 pMutRot lt pmutrot gt Probability of mutation of the orientation of the ligand at every generation The default is 0 30 pMutWat lt pmutwat gt The maximum rate of mutation of the water at Max Gen generations The default is 0 35 pElite lt pElite gt The percentage of the best of the population to be directly passed on to the next generat
84. u must edit your bashrc file to include the PATH for FITTED To be added to the bashrc file export FITTED your installation path FITTED export PATH you installation path FITTED SPATH The programs can then be executed from any directory by simply typing the name of the program see section III 3 2 and the gui can be launched by typing front end from a terminal window 1 3 3 Mac OS X To install the MAC OSX version open a terminal window and execute the install script with the following command mac install forecasterX bin The script will guide you through the installation process The programs can be installed locally user account or in a system folder must be root to run the script don t use sudo In order to be able to run the program from the command line you need to provide the full absolute path to the executable The programs can then be executed from any directory by typing the full path to the program see section III 3 2 and the gui can be launched by typing the following command from a terminal window mac java jar lt full path to the executable gt Front end jar 11 4 Minimum Requirements Windows Windows XP Windows Vista Windows 7 32 bit and 64 bit architecture 1 GB of RAM 2GB or more recommended Java 1 6 latest version for gui Linux Ubuntu 8 10 CentOS 5 2 32 bit and 64 bit architecture Xterm needs to be installed 1 GB
85. ue of many keywords can be altered default values should be used unless a specific system requires different settings At the end of this section a typical keyword file can be found V1 1 Input output files please notice the dash before some keywords Main Mode smart Following the keyword specify the main mode to be run Run Mode smart Following the keyword specify the run mode for the program specified in the Main Mode keyword Molecule XXXX lig mol2 Name of the ligand file Supported file formats are mol and 3D sdf Files can contain either single or multiple molecules Output output filename Name of the output file Should be different that the input filename If not specified SMART will automatically append _1 to the filename V1 2 Parameters for the preparation of the ligand file mode fitted filter ace metabolism Instructs SMART to write the file in selected format The default is fitted inf mol2 sd sdf fittedl amber File format of the input ligand f not specified SMART will automatically detect the file format from the input file extension outf mol2 std debug File format of the output ligand 26 FITTED Suite 3 5 User Guide The default value is mo12 multi YIN SMART will output a multi mol2 file The default value is Y split number SMART will output multi mol2 files each containing the number of molecules as specified This is used
86. uring minimization o The default is 0 02 XX MaxStep lt maxstep gt o Maximum step size allowed during minimization o The default is 1 XX EnergyBound lt energybound gt o Minimum energy difference between two molecules to be considered similar o The default is 1 0 for GI_EnergyBound and 0 001 for GA_EnergyBound XX MaxSameEnergy lt maxsameenergy gt o Number of times that the same energy defined by EnergyBound can be repeated o The default is 3 XX MaxGrad lt maxgrad gt o Gradient convergence criteria o The default is 0 001 VII 4 Energy parameters Score Initial none score minimize Scoring of the initial ligand binding mode none No scoring of the initial input structure is performed This is the default setting score Only the score of the initial input ligand is output minimize The score of the initial pose and the score of the energy minimized structure will be output Vdw 1 411 5 Selects whether 1 4 and or 1 5 and greater van der Waals interactions should be considered 1 4 Used to consider 1 4 interactions and above This is the default setting 1 5 Used to consider only 1 5 interactions and above VdWScale 1 4 lt vdwscale 1 4 gt Scaling factor for the 1 4 van der Waals interactions The default is 1 0 VdWScale 1 5 lt vdwscale 1 5 gt Scaling factor for the 1 5 van der Waals interactions 32 FITTED Suite 3 5 User Guide The default is 1 0
87. ve to atom lt atom name gt of res lt residue name gt Output Print Level 1 1 4 Controls verbosity Print Structures Final Whether to output structures Print Num Structures 1 Number of structures printed Print Best Every X Gen 5 Print summary of run every X generations Number of Best 10 Number of indivs to print summary during run Print Energy Full no Output detailed energy breakdown VIII Analysis of a docking run with FITTED Once the docking run has completed you will find a new folder called output which contains several files Each file contains different information and they will be explained separately in this section The pose of the ligand are generated as mol2 file and can be visualized within the protein mol2 file When docking in rigid protein mode no protein structure is generated and the input mol2 file of the protein can be used In flexible protein mode structure of the protein is generated in mol2 and pdb files Vill 1 The log file The log file should have the XXXX 1log filename where Xxxx is the value of the outpout keyword in the FITTED keyword file This file contains any error that might occur during the docking 42 FITTED Suite 3 5 User Guide Vill 2 The output file This file is the most important file since it contains a lot of information pertaining to the docking This file is named XXXX out based on the value xxxx of the outpout keyword in the FITTED keyword
88. x Ty lt max_ty gt Max Tz lt max_tz gt Maximum value for translation in A in x y and z respectively The default is 5 for the three values GI_Num_of Trials lt gi_num_trials gt Maximum number of successive unsuccessful trials before exiting The default for Mode Dock is 10 000 and for Mode VS is 1 000 Matching Algorithm On Off Turns on or off the matching algorithm By default it is set to On Num_of Top IS lt num of top IS gt Number of top Interactions sites that the interaction site triangles must contain at least one of The default is 10 Stringent Triangles lt weight of triangles gt Is a factor by which the triangles are selected The higher Stringent Triangles is set the more the matching algorithm will favour triangles that have not been used The default value is 5 Stringent_MS lt stringent MS gt Is a weight factor used in calculation of Min MatchScore The higher this value the stricter Min MatchScore becomes The default value is 4 Vil 7 Evolution parameters Max Gen lt max_gen gt Determine the maximum number of generations for the genetic algorithm 35 FITTED Suite 3 5 User Guide The default is 175 CutScore 1 lt cutscore 1 gt Upper bound score at Max_Gen to further proceed with the docking run If there is one individual within the top 3 below this CutScore_1 then the program proceeds to Max_Gen_1 The default is 4 CutScore 2 lt cu
89. xt All results will be put into a filename out file name specified in the keyword file and all errors warnings in a filename log file same name as output file If structures are output printed these files will be created in the working directory within the output directory H running more than one file sequentially as in virtual screening runs scripts can be used to create keyword files extract data and run FITTED Examples of these scripts are available on the fitted ca website or upon request E fp therriee ROBINSON Modeling ae ae a o gt S 16 FITTED Suite 3 5 User Guide IV Preparing a keyword file for PREPARE The following section lists the keywords their functions and default values Gray shading indicates a required keyword angle brackets lt gt indicate a numeric value plain text indicates a text string Such as a file name square brackets indicate a choice of values the default shown in italics When a default value is assigned to a keyword the latter can be omitted from the keywordfile PREPARE keywords files are case sensitive Empty lines are allowed and text after a pound sign is considered a comment Although the value of many keywords can be altered default values should be used unless a specific system requires different settings At the end of this section typical keyword files can be found IV 1 Input output files Main Mode prepare protein Follow

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