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ARP/ wARP User Guide

1. e Click on Run and choose Run now There are a number of options that can be added A brief description is given below e Required parameters ARP REFMAC refinement cycles By default 20 cycles will be carried out However the job may finish earlier if converged Please monitor R factor R free for convergence Free R flag It may be advantageous to use R free flag for solvent building Should you choose to use R free this will cause additional options to ap pear within the section REFMAC parameters The default is not to use R free e ARP wARP flow parameters Add atoms This is followed by two numbers defining the threshold in sigmas of the density above the mean for addition and removal of solvent atoms The defaults are 3 4 and 1 0 respectively which should work for most cases Disable Wilson plot statistics check The current Wilson plot checking rou tine is probably too stringent You may disable the check and the warn ings if you are sure that the X ray data is of high quality However we recommend to not disable the check and in case of warnings inspect the plot and only then proceed e REFMAC parameters Cycles of refinement in each REFMAC run REFMAC is invoked to refine the model before the density maps are computed The default is 1 cycle CHAPTER 3 USING ARP WARP 46 Matrix weight for Xray Geometry The default is automatic weighting This proved to work well Scaling model The default is to use s
2. it will also launch the job The log files and additional output files as well as the building results can be found in the directory created CHAPTER 3 USING ARP WARP 50 3 7 ARP wARP molecular graphics ARP Navigator The graphical front end to ARP wARP Version 7 5 is an OpenGL X window based graphics application that can be launched by pressing the ARP Navigator button in the CCP4 GUI The program can also be started from the command line by typing arpnavigator Figure 3 8 ARP Navigator 3 7 1 Main Menu The main menu sits at the top of the ARP Navigator screen e ArpNavigator CHAPTER 3 USING ARP WARP 51 About This item contains the information of the program Quit ArpNavigator To exit the program using the mouse e Files Open MTZ File Open an MTZ file that contains structure factor amplitudes and phases to feed into fft The first part will be an intuitive file browser then you are asked to select labels resolution range and colour for the map to be displayed Working default values are provided Open MAP File Open a map Use the file browser then go with defaults or choose different values for colour contour level etc Open PDB File Open a coordinate file Open SDF File Open a coordinate file in SDF format typically a ligand molecule Open SMILES File Open a ligand structure specified by a SMILES string in the provided file A three dimensional energy minimised molecule is automatically prepared and sh
3. GLY ALA SER VAL are built and refined Loop building This is invoked if the sequence is available and if the tracing score is above 0 85 It is also invoked after the last building cycle CHAPTER 3 USING ARP WARP 22 R factor after REFMAC during the iterations The value of the R factor typically oscillates At the end of the procedure it should reach a value typical for a restrained refinement Sequence coverage If the sequence is provided the ratio of the number of residues for which the side chains are built to the total number of traced residues is printed A value higher than 0 8 is deemed as good convergence All free atoms are then removed from the file and the task is directed into a few cy cles of restrained refinement with solvent search If however the value of sequence coverage is lower than 0 8 the free atoms DUM are left in the file You can inspect the density maps modify the model on the graphics or submit another model building task using the output of this job Job termination The statement Task completed successfully indicates that the job is finished with no error An error statement QUITTING ARP wARP module stopped with an error message name_of_the_program indicates that one of the modules of the task has terminated with an error message Please refer to the specified log file CPU requirements Automated protein model building may be time consuming Using a standard protocol of 10 building cycles inter
4. Labels and chose appropriate ones Protein model without ligand Provide the PDB file with coordinates of the protein only If the file contains solvent atoms free atoms or fragments of other ligands please make sure that their location is not overlapping with the supposed location of the ligand or have them removed prior to running ligand building Ligand molecule Choose whether to input the ligand as a PDB file or using a 3 letter PDB ligand code Stereochemical information about the ligand to be built is normally read from the provided PDB file if input The file should contain the ligand molecule only The molecule can be in any CHAPTER 3 USING ARP WARP 38 conformation but the interatomic distances bonding angles and chirality if present should be sensible and correspond to the target stereochem istry of the ligand to be built so that the automated recognition of ligand topology works Please also check that there is atom bonded connectivity throughout the whole target ligand molecule i e you do not accidentally have several unconnected clusters of atoms and that there are no atoms that are too close to each other distance lt 0 6 A Otherwise the restraints defined in the REFMAC ligand library file are used if a 3 letter ligand code is input e Click on Run and choose Run now There are a number of options that can be added either in the main GUI panel scrolling bar Build the ligand or under the Parameters section You n
5. The script auto_ligand sh in the warpbin directory allows you to run the ligand building as a single line command without the use of the GUI The use of auto_ ligand sh is fairly simple The script prints out help information if it is invoked without arguments Usage auto_ligand sh datafile either mtzfile or mapfile protein starting_PDB_file_without_ligand ligand PDB_file_with_ligand_to_fit ligandcode 3 letter code of a ligand molecule the code must be present in the REFMAC library workdir FULLPATH_WORKING_DIRECTORY ligandfileout output_PDB_file fp fp_label sigfp sigfp_label freer freer_label nligandcycles number_of_ligandbuild_cycles default is 2 search_model PDB_file_with_model_at_expected_ligand_site search_position X Y Z search_radius radius_in_angstroms reflist textfile_with_FULLPATHnames_of_fitted_ligands_for_comparison extralibrary user_defined_library_for_REFMAC5 additionally if this library contains data for the ligand to be built then these paramters are used to derive ligand topology to the highest level or priority ahead of REFMAC cif or coordinate dervied topology partial 0 for modelling the whole ligand and 4 or higher number to model partially occupied ligand giving 4 would mean to consider 4 atoms as the smallest ligand fragment keepwaters 1 for keeping them before computing the difference map parfile parfilename_if_only_parfile_is_to
6. amplitudes and only one column for their stan dard deviations these will be taken If you wish FOM not to be used please input fom none For test purposes the constructed helices strands can be compared to known reference models hand or pre fitted The required keyword is compareto followed by the full path name of a PDB file You can also enable disable the construction of strands using the keyword nostrands the default is 0 build the strands If auto_albe sh is called with an option parfile the script will create a CHAPTER 3 USING ARP WARP 27 parameter file and a directory in the workdir whose name will be printed The job can subsequently be launched by warpbin warp_albe sh NAME_OF_PARFILE If auto_albe sh is called without an option parfile it will also launch the job The log files and additional output files as well as the building results can be found in the directory created CHAPTER 3 USING ARP WARP 28 3 3 Automated Loop Building 3 3 1 Running loop building from the GUI ARP wARP Loops 200 _ ARP wARP Version 7 4 0 Loop Building _Help Job title For building loops use phases from MTZ file to calculate map Mode for loop building Single loop MTZin _ PROJECT Tn Browse vew Fmap PHimap Protein model for loop building PROJECT se E Use pir file to define the loop sequence W Sequence fle PROJECT a roe Mew Edit list Add input PIR file New loo
7. be shared by multiple users is by doing a command line install The user who is doing the installation should have both write permission to the installation directory and write permission to the CCP4 installation directory gunzip arp_warp_7 5 tar gz tar xvf arp_warp_7 5 tar cd arp_warp_7 5 install sh At the end of the installation the CCP4 startup files will be updated with an addition similar to following lines for c shell Line below added by emph ARP wARP 7 5 installer r destination ccp4 bin arpwarp source csh amp amp source destination ccp4 bin arpwarp source csh where destination ccp4 is the CCP4 directory The file destination ccp4 bin arpwarp source csh will be created and will contain the following instruction r destination arpwarp arpwarp_setup csh amp amp source destination arpwarp arpwarp_setup csh where destination arpwarp is the location where ARP wARP was installed 2 3 Installation of ARP wARP CCP4 as a bundle ARP wARP can be installed together with CCP4 by downloading the joint bundle directly from the CCP4 web site Both packages can be obtained and installed with a single mouse click 3 Using ARP wARP 3 1 Automated Model Building 3 1 1 Running protein model building from the GUI ARP wARP Classic automated model building starting from experimental phases Job title Run ARPAWARP for automated model building starting from experimental
8. fragments will be docked in sequence and side chains built After the last building cycle the fragments will be arranged to form a globu lar structure or for a case of NCS several NCS related structures The remainder of the structure cis prolines poorly ordered loops and terminal residues for each fragment will have to be completed by the user manually Since the output model is refined its accuracy is comparable to that of the refined structure Mis tracing in correct tracing of polypeptide fragments is not impossible but should normally be a small part of the structure An estimate of the correctness of the model is printed after every model building cycle the accuracy of this estimate is about 2 5 Chains 12 Residues 434 Estimated correctness of the model 99 1 Below the use of the module for a start from a density map is described in de tail input in case of molecular replacement model is very similar and should be straightforward to figure out e Launch the ARP wARP Classic window within the CCP4i GUI e Provide required input Run ARP wARP for Choose the application as described above in X ray data in the MTZ format containing structure factor amplitudes their standard deviations phases and figures of merit If pre weighted struc ture factor amplitudes are to be used to construct initial map please check the corresponding box in ARP wARP flow parameters see below Fobs Sigma PHIB FOM If the MTZ column label
9. monitor the log file in your browser window As soon as the job is finished you will be provided with a link to the results that you can then download please keep in mind that once the job is finished your data will be kept for one week only Make sure that you download your data within that time The remote job submission relies on the curl software installed at your site Avail ability of curl is checked while installing ARP wARP and a warning is given if curl is not available 3 1 3 2 Submitting from a web browser Navigate your browser to e http cluster embl hamburg de ARPwARP remote http html or choose model building via the web at e http www arp warp org 1 View the Disclaimer and agree to the ARP wARP and the CCP4 licensing con ditions 2 Proceed with the remote services to Step One 3 Choose the model building protocol start from experimental phases or exist ing model 4 Enter your Email address to which instructions on how to view the results will be send 5 Provide your MTZ file by using the Browse button the file must have an extension mtz 6 Click Proceed to Step Two 7 Enter starting model unless you have chosen a protocol to start from experi mental phases 8 Enter the total number of residues and the number of chemically identical mo lecules in the asymmetric unit Please make sure you enter these two numbers right If for example the asymmetric unit contains a dimer wit
10. parameterisations of the binding region The default value is 2 There is one run of each ligand building algorithm for each starting grid therefore the CPU time required for building is proportional to this number of cycles Assume partial occupancy of ligand Check this box if you wish to model a partially occupied ligand Keep waters By default all water molecules in the provided structure are deleted from the input structure to ensure that the binding site is not oc cupied by inappropriate waters If you are sure that this is not the case water molecules can be kept by ticking this box e REFMAC parameters Cycles of refinement for REFMAC run REFMAC is invoked to refine your pro tein part of the structure before the difference density map is computed The default is 1 cycle for the fast protocol and 3 cycles for the slow proto col see above Matrix weight for Xray Geometry The default is automatic weighting and there is normally no need to change this parameter Input a user defined library file In case your input protein is already a protein ligand complex then REFMAC will have to refine both entities together in order to obtain a difference electron density map If you already have a REFMAC style cif library for ligand s present in the structure you can input it here Otherwise REFMAC will use its own library if it knows the ligand If it does not it will generate a cif file for the ligand and proceed If the user wishes t
11. template model of the ligand to build also in PDB format or a ligand 3 letter code Options include the possibility to specify the binding site the ability to compare the run result to some reference ligand s and the possibility to build a ligand taken from a list of candidates cocktail In the latter case the coordinates of the ligand candidates should be concatenated into a single PDB file The different ligands must be dis tinguished by their residue names columns 18 20 chain identifiers column 22 or residue sequence numbers columns 23 26 ARP wARP will automatically choose the best matching ligand candidate and will attempt to build it at the binding site either determined automatically or supplied by the user However since this feature is new the specification of the binding site see below is recommended One can also specify that only well resolved parts of a partially occupied ligand should be modelled and indicate the minimum number of atoms present in the bound ligand fragment The default is 4 or more atoms e Launch the ARP wARP Ligands window within the CCP4i GUI e Provide required input MTZ in X ray data in the MTZ format containing structure factor amplitudes and their standard deviations Fobs Sigma If the MTZ column labels for structure factor amplitudes and their standard deviations have obvious names they will be recognised automatically Otherwise please use the scrolling button navigate to List All
12. to apply not apply NCS extension regardless of the resolution of the data Input ncsrestraints 1 0 has similar effect Required keyword is datafile followed by the mtz file name with the full path Optional keywords include residues followed by the number of residues workdir followed by the absolute path to the working directory fp followed by the fp label sigfp followed by the sigfp label freelabin followed by the Rfree label fbest followed by the label for the fom weighted structure factor amplitudes to be used for initial map calculation phibest followed by the best phi label fom followed by the figure of merit label modelin followed by a starting pdb file with the full path seqin followed by a sequence file name with the full path cgr fol lowed by a number of NSC related copies buildingcycles followed by the num ber of building cycles resol followed by the resolution limit albe followed by the flag to enable or not helix strands building similarly for restraints twin and sad There are additional parameters which can be customised and an experienced user should have no problem in figuring out how to do this Alternatively please contact the ARP wARP developers for advice If auto_tracing sh is called with an option parfile the script will create a pa rameter file and a directory in the workdir whose name will be printed The job can subsequently be launched by warpbin warp_tracing
13. to decrease the threshold for atom addition from 3 2 to 3 0 or 2 8 Change the number of atoms to be added and removed The default is 1 no increase and it is not recommended to change this Disable Wilson plot statistics check The current Wilson plot checking rou tine is probably too stringent You may disable the check and the warn ings if you are sure that the X ray data is of high quality However we strongly recommend not to disable the check and in case of warnings inspect the plot and only then proceed e REFMAC parameters Attempt to correct for data collected from a twinned crystal REFMAC will at tempt fully automated twinning This option is incompatible with SAD refinement Cycles of refinement in each REFMAC run REFMAC is invoked to refine the hybrid model before the density maps are computed The default is 1 cy cle if the data extend to a resolution of 2 3 A or better otherwise 3 cycles There is usually no need to change this parameter CHAPTER 3 USING ARP WARP 15 Damp shifts The default is 1 0 for both types of shifts There is usually no need to change these parameters Matrix weight for Xray Geometry The default is automatic weighting This proved to work well Scaling model The default is to use simple scaling of the low angle part of the X ray data You can change this to bulk solvent correction if you are sure that your low angle data below about 8 A resolution are complete and correct Scaling B f
14. 3 SUBUNIT Cycles of autobuilding total cycles The default is 10 building cycles sepa rated with 5 ARP wARP REFMAC atom update a refinement cycles thus making 50 cycles in total In cases of good starting phases the autobuild ing may converge faster in cases of poorer phases more cycles may be required You can always submit warpNtrace for further cycles using the output of the previous tracing protocol automated model building start ing from existing model Protocol for REFMAC5 Rfree The refinement target gives three choices 1 The default is to use maximum likelihood target 2 The second choice allows the user to use the SAD target This func tion is based on REFMAC5 developments by Skubak amp Pannu and allows to refine against the F F data when these are available A prerequisite when this option is activated is to also provide a PDB file with the anomalous scatterers and define the extent of the anoma lous signal either by providing the wavelength or measured f and f values Currently ARP wARP accepts only one type of atom to be defined when f f values are used If you have more than one atom you just choose the wavelength to fetch theoretical values that should in practice work well CHAPTER 3 USING ARP WARP 13 3 The third choice is the Phased ML function which is not recom mended to use with SAD data If MAD or MIRAS data are available you should use Phased ML in conjunction
15. 5 tar The package will unpack under the directory called arp_warp_7 5 that will contain all the required files and subdirectories The installation script install sh is needed to do the installation and to set the appropriate environmental variables Sudo permissions may be required The README will walk you through the installation process 2 Go to the directory arp_warp_7 5 and run there the install sh script by sim ply typing install sh After installation the CCP4 interface ccp4i should have its model building menu updated and will appear as in figure 2 1 ARP Navigator ARPAWARP Classic ARPAVARP Quick Fold ARP wARP Loops ARP WARP DNA RNA ARP WARP Ligands ARP WARP Solvent Figure 2 1 The CCP4i Model Building menu after ARP wARP installation Unless you are already an experienced ARP wARP user you should try to get started with the test files provided in the directory arp_warp_7 5 examples These include data for protein chain tracing also with NCS helix strands search nu cleotides ligand and solvent building README files are included which give more detailed information regarding which data are to be used for what purposes If things do not work as expected please consult your more experienced col leagues system manager or the ARP wARP developers CHAPTER 2 INSTALLING ARP WARP 9 2 2 1 Installing for Multiple users The recommended way to install ARP wARP so that it can
16. ARP wARP User Guide Version 7 5 December 19 2014 Contents Contents 2 he eRe EEE S Sak HGR WGK FO eS ee S 1 2 Major changes in Version 7 5 1 3 Latest News Bug Reports and Troubleshooting 2 Installing ARP wARP 2 1 Standalone Intel Mac OSX Installation 000 7 7 2 2 Standalone Command Line Installation on Mac OSX or Linux 7 9 9 2 2 1 Installing for Multiple users 2 3 Installation of ARP wARP CCP4 as a bundle 3 Using ARPI wARP 3 1 Automated Model Buildin CONTENTS 3 3 4 Automated Building of Poly Nucleotides 4 Additional Remarks 58 4 1 Quality of the X ray Data a a okay echo Soe Gee SE Sw 58 5 Citing ARP wARP 59 6 Acknowledgements 61 6A Third Party Software sos ecra 4 4 At Oa Ee ee ee ee 62 1 General information 1 1 Introduction ARP wARP is a software project for automated protein model building and struc ture refinement ARP wARP combines pattern recognition based interpretation of an electron density its modelling as a hybrid model and a maximum likelihood pa rameter refinement with REFMAC The ARP wARP software is under continuous development Its present release version 7 5 can be used for the following tasks 1 Automated protein chain tracing in the density map and model building GUI module ARP wARP Classic and command line module auto_tracing sh This constructs polypeptide fragments for the cases of MR solutions or MAD M S IR AS ph
17. MAC refinement cycles of the model For REFMACS refinement do not use the Free R flag mm e e a a Figure 3 7 Solvent Building from the CCP4 GUI 44 Within solvent building module restrained reciprocal space refinement is carried out with REFMAC while ARP wARP is performing automatic adjustment of the solvent structure Resolution of the data should be 2 5 A or higher The output is the protein model with the solvent molecules transformed with symmetry operations to lie around the protein The ARP wARP solvent building module requires the X ray data in MTZ for mat and the protein model in PDB format without solvent or with a partial solvent model e Launch the ARP wARP Solvent window within the CCP4i GUI e Provide required input MTZ in X ray data in the MTZ format containing structure factor amplitudes and their standard deviations CHAPTER 3 USING ARP WARP 45 Fobs Sigma If the MTZ column labels for structure factor amplitudes and their standard deviations have obvious names they will be recognised automatically Otherwise please use the scrolling button navigate to List All Labels and chose appropriate ones Starting model in Provide the PDB file with coordinates of the protein only If the file already contains some solvent sites these will be updated during the iterative solvent building Output model Provide the name of the file where output PDB of the protein with the built solvent will be written to
18. RP wARP Version 7 5 proceeds in three steps first it locates the binding site in the difference density map then builds there a number of putative ligand models and finally selects the best model which is geometrised and real space fit into the density The binding region may be selected automatically by matching ligands shape related properties to the regions of high density For the construction of the ligand set two algorithms are used One exploits the combinatorial assignment of the lig and atom identities to the grid nodes label swap Another algorithm maximises the overlap between the sparse set and the ligand model by a random search in con formational space The output from both algorithms is merged and then undergoes a last stage of real space refinement before the final model is selected The accuracy of ligand building is mainly dependent on ligand size and the res olution of the X ray data As a rough guide about 75 of well ordered ligands of CHAPTER 3 USING ARP WARP 37 a size around 20 to 40 non hydrogen atoms should be built within rm s d of 1 0 A from their correct location Thus the constructed models should be accurate enough for REFMAC5 to straightforwardly refine the protein ligand complex The proce dure can be iterated to locate additional ligands if any are present The ARP wARP ligand building module requires the X ray data in MTZ for mat the built protein without ligands in PDB format and either a
19. _be_created POL MONA OOM POE LO OO AEP OO ME SEE OGL il GE Optional command line arguments are given in square parentheses All input files are assumed to be located in working directory unless they are given with full path If workdir is not given the current directory will be assumed All output files will be written into workdir subdirectory If no ligand is specified then auto identification of the ligand will be attempted provided that a search position is given experimental Required keywords are datafile followed by the mtz file name with the full path or a map file in CCP4 format and protein followed by the pdb file name of the protein model without the ligand with the full path Either the keyword ligand followed by the full path to the pdb file containing the ligand coordinates or ligandcode followed by the 3 letter code of the ligand to be modelled are nor mally provided to indicate the nature of the ligand to be built If they are not spec CHAPTER 3 USING ARP WARP 42 ified automated identification of the ligand is attempted with a database of 84 of the most common ligands in the PDB being screened Note that datafile can also be a density map an option not offered in the CCP4 GUI Optional keywords include workdir followed by the full path to the working directory fp followed by the fp label sigfp followed by the sigfp label The de faults are FP and SIGFP respectively Alternatively if the
20. actor The default is to use anisotropic B factor for scaling the X ray data You can choose isotropic scaling B factor if your data are systemati cally incomplete e g a cone is missing in reciprocal space Free R label This option appears if the free R flag has been chosen for refine ment of the protein part of the model Here you can provide a column label for the free R flag Use of free R reflections This option also appears if the free R flag has been chosen The scaling and calculation of o 4 coefficients by REFMAC can be computed on the basis of the free reflections this is the default or using all reflections Solvent mask correction The default is to use solvent mask correction in REF MAC e Crystal parameters Space group Cell ARP wARP asymmetric unit Wilson B factor and Solvent content are derived automatically from the MTZ file and the total number of residues in the asymmetric unit They are displayed for information only and cannot be changed However you may want to check whether their values conform to your expectations If the solvent content is outside of the expected limits ARP wARP may be able to correct this automati cally during the run Resolution By default all data present in the MTZ file will be used You can check the box and then narrow the range if you are aware of certain defi ciencies of your data e Submit a remote job at the Hamburg Cluster Checking this button will activate remote submi
21. al command line arguments are given in square parentheses Possible combinations of MTZ labels for map calculation are fp sigfp phib fom or fbest sigfp phib if fbest is already fom weighted In the latter case if fbest is given fom will be ignored All input files are assumed to be located in working directory unless they are given with full path If workdir is not given the current directory will be assumed All output files will be written into workdir subdirectory Required keyword is datafile followed by the mtz file name with the full path In difference to the functionality offered from the CCP4 GUI datafile can also be a density map CHAPTER 3 USING ARP WARP 35 Optional keywords include residues the expected number of residues in the asymmetric unit nucleotides the expected number of nucleotides in the asym metric unit workdir followed by the full path to the working directory fp fol lowed by the fp label sigfp followed by the sigfp label phib followed by phibest label and fom followed by the label to fom The defaults are FP SIGFP PHI and FOM respectively Alternatively if the mtz file contains only one column for struc ture factor amplitudes and only one column for their standard deviations these will be taken If you wish FOM not to be used please fbest You can set resol followed by the resolution limit For test purposes the constructed model can be compared to known referen
22. ases Generally the higher the resolution of the X ray data the more complete and accurate model ARP wARP will deliver Typically X ray data to 2 7 A resolution or better are required although a considerable part of a protein model can sometimes be built at a resolution of 3 0 A or worse 2 Automated building of alpha helical and beta stranded fragments GUI mod ule ARP wARP Quick Fold command line module auto_albe sh This con structs helical and beta stranded polypeptide fragments main chain and CB atoms in low resolution density maps Phased X ray data to 4 5 A resolution or betters are required This module is automatically invoked as part of protein chain tracing 1 above when the resolution of the data is 2 7 A or worse 3 Building poorly defined loops in a protein model GUI module ARP wARP Loops This will generate a set of candidate loops for a short stretch of missing residues given the anchors and the sequence of the missing residues A pro tein model and an X ray data to 3 0 A resolution or higher are required This module is automatically invoked as part of protein chain tracing 1 above provided that the built protein model is sufficiently complete 4 Software for building poly nucleotide fragments DNA or RNA GUI module ARP wARP DNA RNA command line module auto_nuce sh This will pro duce a set of poly nucleotide chains with guessed bases A or C i e large or small the nucleotide sequence is not yet used Phas
23. ber of Ca atoms generated within a shell Ca Ca distance Distance to use between successive Ca atoms Keep Ca atoms with negative density halfway Default for this option is not to keep the atoms e Crystal parameters Space group and Cell are derived automatically from the MTZ and the PDB files displayed for information only and cannot be changed However you may want to check whether their values conform to your expecta tions e Log files of Loopy Message level Choose a value between 0 and 9 the default is 4 Abort level If a message at this level is encountered the module will abort The default value is 8 CHAPTER 3 USING ARP WARP Message file Name for the message file plain text XML output file Name for the XML message file xml format 31 CHAPTER 3 USING ARP WARP 32 3 4 Automated Building of Poly Nucleotides 3 4 1 Running nucleotide building from the GUI ARP wARP DNA RNA Kok ARP wARP Version 7 4 0 DNA RNA Building Help benD peoa e ee ows w Fobs Sigma PHIB FOM Output PDB fle PROJECT aaa Cae J Parameters Number of nucleotides aa and number of residues in the AU Crystal parameters ry Run Save or Restore _ Gose Figure 3 5 Building Poly Nucleotides from the CCP4 GUI Job title DNA and RNA building This module builds fragments of DNA or RNA The input is an MTZ file con taining the phases from which the map
24. best describing the nucleotide region can be computed Thus the map could be a difference map e g after the protein model is completed or a sigma weighted map for the whole asymmetric unit The nu cleotide building procedure within ARP wARP 7 5 proceeds in several steps first it locates putative phosphates in the density map then uses them in a manner analo gous to the CA candidates for protein chain tracing After the nucleotide fragments are obtained a likely base is built and refined in real space The type of the base is currently limited to A large or C small and the nucleotide sequence is not yet used The produced poly nucleotides are quite accurate a typical r m s d for the built backbone atoms is 0 6 A with X ray data extending to around 3 0 A resolution The method is not sensitive to a particular DNA or RNA conformation The module is not very CPU efficient and may take about 10 minutes for a 20 nucleotide structure e Launch the ARP wARP DNA RNA window within the CCP4i GUI e Provide required input CHAPTER 3 USING ARP WARP 33 MTZ in X ray data in the MTZ format containing structure factor amplitudes and their standard deviations Fobs Sigma PHIB FOM If the MTZ column labels for structure factor ampli tudes and their standard deviations have obvious names they will be recognised automatically Otherwise please use the scrolling button nav igate to List All Labels and chose appropriate ones FOM is optional and co
25. button on this box will pull out the mini menu with actions applied to this object only see also Mini menu 3 7 4 Quick Actions When the right mouse button is pressed with no movement then a green button box is displayed that contains functionalities to be applied ad hoc and with no input dialog CHAPTER 3 USING ARP WARP 57 Goto Atom This button launches the goto atom dialog as shift g does The goto atom dialog expects that atoms are specified as e g CA 123 A for the CA atom of residue 123 in chain A Just specifying CA 123 means the first occurrence of CA in residue 123 Specifying 123 means the first atom in residue 123 Typing Z will be interpreted as the first atom of chain Z The program will centre on the atom if found In case the atom cannot be found the dialog gets coloured in pink Real Space Refine Ligand The ligand to be refined is a detached molecule and there is one density map on display The ligand gets refined to that density map lo cally and the initial ligand position must be in the radius of convergence The output will replace the detached model Please note that the refinement is re strained to the ligand stereochemistry which is derived from the input ligand model Thus continuously playing with the ligand by taking it out and then refining it back into its density will successively change the ligand s stereo chemistry Find Ligand Binding Site The ligand to be located is a detached
26. ce model The required keyword is compareto followed by the full path name of a PDB file If auto_nuce sh is called with an option parfile the script will create a param eter file and a directory in the workdir whose name will be printed The job can subsequently be launched by warpbin warp_nuce sh NAME_OF_PARFILE If auto_nuce sh is called without an option parfile it will also launch the job The log files and additional output files as well as the building results can be found in the directory created CHAPTER 3 USING ARP WARP 36 3 5 Automated Ligand Building 3 5 1 Running ligand building from the GUI ARP wARP Ligands eoo ARP wARP Version 7 4 0 Ligand Build _Help Job title automated ligand building Build the ligand in the most likely place of the complete asymmetric unit Mizin PRET WE al Fobs Sigma Protein model without ligand PROJECT SSS Browse a Input ligand as PDB file Ligand molecule coordinates PROJECT T os _vew Ligand output fle PROJECT a e Li Parameters Use with REFMACS5 the Fast protocolanddo notuse the Free R flag 2 Ligand building cycles _ Assume partial occupancy of ligand i Keep waters Retmac parameters Crystal parameters Test and comparison parameters Run Save or Restore Close Figure 3 6 Building Ligands from the CCP4 GUI ee j The ligand building procedure within A
27. e warpbin auto_albe sh datafile mtzfile residues number_of_residues_in_AU workdir FULLPATH_WORKING_DIRECTORY helixfileout foutput_PDB_file jobId desired_job_id_used_for_subdirectory_naming fp fp label sigfp sigfp label phib phi label fom fom label input fom none if no fom is to be used compareto PDB_file_for_comparison nostrands 0 or 1 default 0 parfile parfilename_if_only_parfile_is_to_be_created ZA A A A A A A A AZ Optional command line arguments are given in square parentheses All input files are assumed to be located in working directory unless they are given with full path If workdir is not given the current directory will be assumed All output files will be written into workdir subdirectory Required keyword is datafile followed by the mtz file name with the full path Optional keywords include residues the expected number of residues in the asymmetric unit workdir followed by the full path to the working directory helixfileout the name of the PDB file where the traced both helical and stranded fragments will be output to jobId if you wish that the working sub directory has a particular name fp followed by the fp label sigfp followed by the sigfp label phib followed by phibest label and fom followed by the label to fom The defaults are FP SIGFP PHI and FOM respectively Alternatively if the mtz file contains only one column for structure factor
28. e arguments are given in square parentheses All input files are assumed to be located in working directory unless they are given with full path If workdir is not given the current directory will be assumed All output files will be written into workdir subdirectory Required keywords are datafile followed by the mtz file name with the full path and protein followed by the pdb file name of the protein model with the full path Optional keywords include workdir followed by the full path to the working directory solventfileout followed by the name of the PDB file where the output will be written fp followed by the fp label sigfp followed by the sigfp label and freer followed by the Rfree label The defaults for the first two are FP and SIGFP respectively Alternatively if the mtz file contains only one column for structure factor amplitudes and only one column for their standard deviations these will be taken The number of cycles default is 20 can be changed with keyword restrcyc The user defined library and the tls tensor for REFMAC can be supplied by using the keywords extralibrary and tlsin CHAPTER 3 USING ARP WARP 49 If auto_solvent sh is called with an option parfile the script will create a pa rameter file and a directory in the workdir whose name will be printed The job can subsequently be launched by warpbin warp_solvent sh NAME_OF_PARFILE If auto_solvent sh is called without an option parfile
29. e still other complaints after the cut you may consider inspecting your data processing The current version of the ARP wARP Wilson plot check might be too stringent Nevertheless the user is advised to visually inspect the Wilson plot and apply his her critical judgement as to whether or not the data should be cut It has sometimes proved beneficial to cut the low resolution data which were flagged as poor 58 5 Citing ARP wARP Please cite the applications of ARP wARP that you have used Please consult the ARP wWARP log file for the most relevant citation The most recent overview of ARP wARP can be found in e Langer G Cohen S X Lamzin V S amp Perrakis A 2008 Automated macro molecular model building for X ray crystallography using ARP wARP version 7 Nature Protocols 3 1171 1179 Applications are presented in e Langer GG Hazledine S Wiegels T Carolan C Lamzin VS 2013 Visual au tomated macromolecular model building Acta Crystallogr D Biol Crystallogr 69 635 641 ARP Navigator e Wiegels T amp Lamzin V S 2012 Use of noncrystallographic symmetry for au tomated model building at medium to low resolution Acta Cryst D 68 446 453 protein model building e Langer G G Evrard G X Carolan C G Lamzin V S 2012 Fragmentation tree density representation for crystallographic modelling of bound ligands J Mol Biol 419 3 4 211 22 ligand building e Hattne J amp Lamzin V S 2008 Pat
30. ed Left mouse button t key pressed and mouse moved an alternative way to trans late the scene in the xy plane CHAPTER 3 USING ARP WARP 55 Left mouse button z key pressed and mouse moved up down the scene is trans lated in z direction perpendicular to screen plane 3 7 2 3 Scaling Middle mouse button pressed and mouse moved left right zooming the scene is scaled and a scale o meter is shown on the right Left mouse button s key pressed and mouse moved analternative way to zoom 3 7 2 4 Clip planes Left mouse button f key pressed and mouse moved left right changes the front clip position Left mouse button b key pressed and mouse moved left right changes the back clip position Left mouse button g key pressed and mouse moved left right changes the front and back clip position together Left mouse button d key pressed and mouse moved left right changes the posi tion of the rotation center similar to translation 3 7 2 5 Map contouring The mouse wheel is used for changing the contour level of a map The map must be activated by pressing the corresponding object button at the bottom of the graphics window Left mouse button c key pressed and mouse moved up down An alternative way to change the contour level 3 7 2 6 Map extent Left mouse button e key pressed and mouse moved map size 3 7 2 7 Mouse Actions Left mouse button pressed in graphics area marks atoms or density switch this in Opt
31. ed X ray data to about 3 5 A resolution or better are required CHAPTER 1 GENERAL INFORMATION 5 5 1 2 Building bound ligands GUI module ARP wARP Ligands command line mod ule auto_ligand sh This constructs a ligand in a difference electron density map after the protein model has been completed and refined It can be given a template search ligand a list of putative ligands cocktail screening or can guess a ligand among the most common ligands in the Protein Data Bank X ray data to 3 0 resolution or better are required Building the solvent structure GUI module ARP wARP Solvent command line module auto_solvent sh This builds a solvent structure after the pro tein model has been refined The procedure is iterative and uses REFMAC for structure refinement X ray data to 2 5 A resolution or better are required A molecular graphics ARP wARP front end which allows the display of mo lecules and electron densities GUI module ARP Navigator executable pro gram arp navigator It is a high quality 3D molecular viewer and a user friendly interface to most of ARP wARP functionalities allowing macromolec ular models secondary structure elements skeletons ligands and solvents to be viewed as they are built Major changes in Version 7 5 Increased performance of protein model building through improved polypeptide recognition NCS restraints atom update SAD refinement option estimation of solv
32. en number only the best are saved to file CHAPTER 3 USING ARP WARP 29 e Launch the ARP wARP Loops window within the CCP4i GUI e Provide required input Building loops Select whether to start from a map or an mtz file Mode loop building Select whether to try to build all loops in the PDB file a sequence file will be needed or to build a specific loop MTZ in X ray data in the MTZ format containing structure factor amplitudes and their standard deviations Fmap PHImap If the MTZ column labels for structure factor amplitudes and their standard deviations have obvious names they will be recognised automatically Otherwise please use the scrolling button navigate to List All Labels and chose appropriate ones Protein model for loop building Provide the PDB file with coordinates of the protein Note that the module will only attempt to build missing loops and will not rebuild any of the existing residues New loops output file Provide the name of the PDB file where the built loops will be written to Protein and new loops combined output Provide the name of the PDB file where the protein model together with the built loops will be written to e Click on Run and choose Run now There are a number of options that can be added A brief description is given below e Definition of loop Build a loop Provide anchor residues of a fragment on the N and the C ter minus side of the protein If you want to rebuild some terminal residue
33. ent content and model accuracy e Improved stability of beta strand DNA RNA and solvent building e Fit Ligand incorporates 84 most common ligands now e and uses cif files defining bond torsion and plane restraints e Incremental improvements in auto depth view and menus of ArpNavigator CHAPTER 1 GENERAL INFORMATION 6 1 3 Latest News Bug Reports and Troubleshooting For the latest news and announcements please visit the ARP wARP page www arp warp org Some problems and tips can be found on the Frequently Asked Ques tions link The developers will greatly appreciate all bug reports or suggested changes 1 4 Distribution The ARP wARP package either for download or for remote execution of protein model building is freely available to academic users provided that they agree to the ARP wARP license conditions and the applications of ARP wARP are properly cited Please consult the ARP wARP log file for most relevant citations Industrial users are requested to obtain a commercial license via the ARP wARP web page 2 Installing ARP wARP The recommended way to obtain and install ARP wARP is through a download and install of a joint CCP4 6 5 ARP wARP 7 5 bundle available from http www ccp4 ac uk The users can also obtain and install a standalone version of ARP wARP either from Se or from the CCP4 download site at http www When using a standalone version of ARP wARP CCP4 6 3 0 is the lowest recommended v
34. ersion to use with ARP wARP 7 5 Older versions of CCP4 will probably work but they have not been tested with the latest ARP wARP 7 5 features 2 1 Standalone Intel Mac OSX Installation Unless installing a joint CCP4 ARP wARP bundle CCP4 must be installed before ARP wARP installation is attempted Sudo permissions may be required There could be problems installing ARP wARP when CCP4 is installed using 64 bit Fink 1 Download arpwarp_7 5 dmg from the either ARP wARP or CCP4 website 2 Double click on the downloaded file 3 Double click on the ARPWARP installer 4 Agree to the ARP wARP license 5 Select a destination drive 6 Choose destination directory if the default Applications is not suitable There should not be problems with the installation However if there are any we encourage you to save the installation log that is displayed and send it to the ARP wARP developers using the link on the ARP wARP homepage 2 2 Standalone Command Line Installation on Mac OSX or Linux Unless installing a joint CCP4 ARP wARP bundle CCP4 must be installed before ARP wARP installation is attempted There could be problems installing ARP wARP on Mac OSX when CCP4 is installed using 64 bit Fink 7 CHAPTER 2 INSTALLING ARP WARP 8 1 Download the full ARP wARP package arp_warp_7 5 tar gz from the CCP4 or ARP wARP web site and save it in a location of your choice Next type gunzip arp_warp_7 5 tar gz tar xvf arp_warp_7
35. esidues in the AU not match each other the number of molecules in the AU will be reset accordingly Input MTZ file We have observed that sometimes the MTZ files do not have proper headers e g non standard space group name or zero space group number ARP wARP uses CAD program to always do a header fix thus the MTZ file may have an extension mtz cad Space group number ARP wARP supports all standard non centrosymmetric space groups Plbar and several non standard space groups e g 1017 or 2017 The space group is figured out solely from the symmetry operators stored in the MTZ file header Input files The ASCII files sequence input PDB or input file with heavy atoms are always converted to a Unix line feed thus they have an extension _1f CHAPTER 3 USING ARP WARP 21 Checking whether input PDB contains ligands This check comes up if the initial model is available Should the model contain ligands unknown to the REF MAC library they are renamed to free DUM atoms This should not affect the model building performance but the warning is printed R factor after REFMAC before model building If the initial model is available a number of restrained refinement cycles with REFMAC is carried out until R factor convergence Building cycle one Normally one should expect a considerable part of the struc ture built already at the starting building cycle If this is not the case observe the situation for a few further building cycles If
36. h each subunit having 50 residues then you enter 100 and 2 respectively CHAPTER 3 USING ARP WARP 20 9 Enter MTZ labels FP and SIGFP are compulsory for model building starting from the existing model PHI is additionally needed and FOM is optional for start from experimental phases 10 Click on I agree to cite the required references and would like to proceed with ARP wARP remote services This uploads the files to the cluster in Hamburg launches the job and after a few minutes delay sends you an Email with in structions for viewing 11 Please follow the instructions in the email http link login and password to connect to the Hamburg cluster You can then monitor the log file in your browser window As soon as the job is finished you will be provided with a link to the results that you can then download Please keep in mind that once the job is finished your data will be kept for one week only Make sure that you download your data within that time 3 1 4 Output files short log file The following information could be useful when interpreting the log messages that are produced when running ARP wARP Checking the estimated solvent content Should the solvent content be too high or too low ARP wARP will re set it to approximately 50 The target number of residues will be reset accordingly Checking the provided sequence file Should the sequence length the number of molecules in the AU and the total number of r
37. hat are mis interpreted The expected top performance is the correct location of 90 of the helices and 50 of the strands The procedure is relatively fast and takes only seconds to minutes for proteins of moderate size up to 500 residues The secondary structure recognition module is optimised to address lower reso lution data and hard cases where e g the full model building protocol has not been successful For a resolution higher than 2 6 A the module will automatically trim the resolution and Wilson B factor of the data to approach its design conditions CHAPTER 3 USING ARP WARP 24 e Launch ARP wARP Quick Fold window within the CCP4i GUI e Provide required input MTZ in X ray data in the MTZ format containing structure factor amplitudes and their standard deviations phases and foms Fobs Sigma Phib FOM If the MTZ column labels for structure factor ampli tudes their standard deviations phases and figures of merit have obvious names they will be recognised automatically Otherwise please use the scrolling button navigate to List All Labels and choose appropriate ones Output PDB file Provide the PDB file name where the constructed secondary structure fragments will be output to e Set parameters Number of residues Provide the expected number of residues in the asym metric unit This is optional but if given should be a good guess within 20 of the true number Do NOT build beta strands If you have real doubts abou
38. however there is essentially nothing autotraced for further building cycles please inspect whether the ini tial phases are sufficiently good or the space group is correct Search for helices and strands The module for building helical and beta stranded fragments is invoked if requested or by default with data at 2 7 A resolution or lower The number of built helical stranded residues and chain fragments is printed Rounds within building cycle Each cycle of the main chain tracing is carried out in several rounds Normally each successive round should result in more residues and in fewer fragments The maximum length of the traced fragment and the tracing score of the model built are also printed for information The tracing score is on an arbitrary scale but the higher it is the better Chains residues and estimated correctness of the model The output from the best tracing round is processed further Fragments of 4 residues or shorter as well as the terminal residues of the fragments are converted to free atoms The rest is used to provide restraints for subsequent ARP wARP REFMAC cycles The value of the estimated correctness of the model should steadily approach 100 if the tracing is successful Residues docked into sequence If the sequence is provided the autotraced frag ments are docked into it and the side chains are built and refined in real space The results are printed out If the sequence is not provided side chain guesses only
39. ile Refinement with REFMAC The R factor and R free if requested are printed after refinement of the protein with REFMAC Check that the value of the R factor is decreasing upon solvent building Job termination The statement Task completed successfully indicates that the job has finished with no error An error statement QUITTING ARP wARP module stopped with an error message name_of_the_program CHAPTER 3 USING ARP WARP 47 indicates that one of the modules of the task has terminated with an error message Please refer to the specified log file CHAPTER 3 USING ARP WARP 48 3 6 2 Running solvent building from command line auto_solvent sh The script auto_solvent sh in the warpbin directory allows you to run the sol vent building as a single line command without the use of the GUI The use of auto_solvent sh is fairly simple The script prints out help information if it is in voked without arguments warpbin auto_solvent sh datafile mtzfile protein starting _PDB_file workdir FULLPATH_WORKING_DIRECTORY solventfileout foutput_PDB_file fp fp_label sigfp sigfp_label freer freer_label restrcyc number_of_cycles default is 20 arpmode noupdate default is update arpmode waters Lextralibrary user_defined_library_for_REFMAC5 tlsin fixed pre refined TLS tensors from REFMAC5 parfile parfilename_if_only_parfile_is_to_be_created FO OE OE FOO OO GO BO Optional command lin
40. imple scaling of the low angle part of the X ray data You can change this to bulk solvent correction if you are sure that your low angle data below about 8 A resolution are complete and correct Scaling B factor The default is to use anisotropic B factor for scaling the X ray data You can choose isotropic scaling B factor if your data are systemati cally incomplete e g a cone is missing in reciprocal space Scaling and cA calculations This parameter also appears if the free R flag is chosen for refinement of the protein part of the model The scaling and calculation of cA coefficients by REFMAC map can be done on the basis of the free reflections this is the default or using all reflections TLS refinement The default is not to do a TLS refinement of the model Input a user defined library file If you already have a REFMAC style cif li brary for e g your already present ligand you can input it here e Crystal parameters Space group Cell ARP wARP asymmetric unit Wilson B factor and Sol vent content are derived automatically from the MTZ and the PDB files displayed for information only and cannot be changed However you may want to check whether their values conform to your expectations Resolution By default all reflections present in the MTZ file will be used You can check the box Use reflections between and then narrow the range if you are aware of certain deficiencies of your data 3 6 1 1 Output files short log f
41. ions menu Double click will also centre on atoms CHAPTER 3 USING ARP WARP 56 Right mouse button pressed on top of an object button opens the Mini menu of the related object Parameters close save etc Right mouse button pressed in graphics area opens the Quick actions menu 3 7 2 8 Keyboard Actions w Hide the menu and all attached information as long as pressed W shift w Lock the function of w and do not show the menu when released To unlock press w or shift w again then the menu will be visible again G shift g Launch a goto atom dialog see goto atom below C shift c Center on the last mark set irrespective of whether this was an atom or a density region D shift d Activate the display of distances between the most recent mark and all other marks set so far m Toggle the control of a detached model move the model only vs move the crystal frame alone with the model fixed k Toggle the control of a detached model move the model and the crystal frame together vs move the crystal frame alone 3 7 3 Object Buttons When a file is loaded and put on display small boxes appear in the bottom left comer representing each of the graphical objects Only one object can be active at a time An object can be made active by clicking on the box with the left mouse button A little eye symbol shows whether this object is currently on display or if it s hidden Clicking with the right mouse
42. lO al gf Optional command line arguments are given in square parentheses Possible combinations of MTZ labels are For start from phases fp sigfp phibest fom or fbest sigfp phibest to build initial free atoms model and fp sigfp to refine the model If fbest is given fom will be ignored For start from a model fp sigfp to refine the model All input files are assumed to be located in working directory unless they are given with full path If workdir is not given the current directory will be assumed All output files will be written into workdir subdirectory Additional useful tips Normally the job runs in a subdirectory called YYYYMMDD_HHMMSS To run the job in the current directory use auto_tracing sh jobId If you invoke auto_tracing sh from another script and the keywords with double word argument are not properly understood e g resol 20 0 2 5 try resol 20 0 2 5 or resol 20 0 2 5 If you have a par file from an earlier version of emph ARP wARP and would like to re run that job now use auto_tracing sh defaults OLD_PAR_FILE This will create a par file compatible with the current emph ARP wARP version and the keywords which are new to OLD_PAR_FILE will take their default values NCS based chain extension and NCS restraints with REFMAC are applied automatically if the resolution of the data is equal to or lower than 2 1 A CHAPTER 3 USING ARP WARP 17 Input ncsextension 1 0
43. molecule and there is one density map on display Furthermore all other models displayed are taken as occupants of space and the binding site can not intersect with them In return a dummy atom model of the located density blob is shown Fit Ligand Here The ligand to be fit is the detached model there is at least one density map on display that has one of its blobs marked The output will replace the detached model Build Helices At least one density map must be on display or activated Helices are built and side chains are modelled up to C gamma atoms 4 Additional Remarks 4 1 Quality of the X ray Data The space group of the X ray data should be correctly determined Incorrect space group is a frequent cause for ARP wARP to build only 50 or less of the model in short fragments The X ray data should be as complete as possible especially in the low resolution range 10 A and worse If the low resolution data are systematically incomplete e g missing or overloaded reflections the density map even in the case of a good model may be discontinuous and inconsistent with the model ARP wARP automatically checks the fit of your data to the expected Wilson plot and report discrepancies if necessary If suggested to cut the data from the high resolution side follow the suggestion If suggested to cut the data from the low resolution side do so but do not cut to a resolution below 8 or 10 A If suggested to ignore all data or there ar
44. mtz file contains only one column for structure factor amplitudes and only one column for their standard de viations these will be taken The number of ligand building cycles default is 2 can be changed with keyword nligandcycles The approximate location of the binding site can be supplied by the user either by providing the pdb file s of a ligand or a just a list of atoms located at the binding site search model or by specifying the XYZ coordinates of a point defining the binding region using search_position and search_radius default value for the latter is 5 A For test purposes the con structed ligand can be compared to known reference models hand or pre fitted The required keyword is reflist followed by the full path to a text file containing a list of pdb files with the reference ligands and their absolute paths Building of a partially occupied ligand can be requested using the keyword partial followed by the number 4 or higher A user defined ligand library can be input using key word extralibrary Water molecules can be kept prior to placement of the ligand by using the option keepwaters followed by the number 1 To build the ligand from a list of candidates cocktail the coordinates of the ligand candidates should be concatenated into one file specified by the above men tioned keyword ligand The different ligands must be distinguished by their residue name columns 18 20 in the concatenated pdb file different chai
45. n identifiers or residue sequence numbers may also be used to differentiate the ligands but we rec ommend the use of different residue names ARP wARP will automatically choose the best matching ligand candidate and will attempt to build it at the binding site either determined automatically or supplied by the user i e by supplying the bind ing site using search_model or search_position keywords To build the partially occupied ligand enter keyword partial with the appro priate parameter defining the size of the smallest ligand fragment ARP wARP will automatically choose the best matching ligand fragment and will attempt to build it at the binding site either determined automatically or supplied by the user If auto_ligand sh is called with the option parfile the script will create a pa rameter file and a directory in the workdir the names of which will be printed The job can subsequently be launched by typing warpbin warp_ligand sh NAME_OF_PARFILE CHAPTER 3 USING ARP WARP 43 If auto_ligand sh is called without the option parfile it will also launch the job The log files and additional output files as well as the building results can be found in the directory created CHAPTER 3 USING ARP WARP 3 6 Automated Solvent Building 3 6 1 Running solvent building from the GUI ARP wARP Solvent Job title Solvent building wzm oer a Fy ce Fobs Sigma Steing notin ROE J ce tT S Do ARP REF
46. n next to clip to The clip radius can be set Again here the contour level and extension can be set by typ ing them If the contouring is changed in the graphics using the mouse wheel then in the map parameters menu these values will change Model Parameters For the active model object only one can change the pa rameters of display A dialog window appears where various settings can be found to suit a special purpose Show Graphics Status This activates the display of the status information on graphics in a separate little window e g centre and eye position CHAPTER 3 USING ARP WARP 53 Show Scale o meter This is a toggle button to activate deactivate the meter bar on the right to show distance units at the current scale Off by default Show Axes Orientation This is a toggle button to activate deactivate the dis play of xyz axes with letters and in colours in the top right corner of the graphics window On by default Show Contour Levels Switch on the display of all contour levels of maps loaded at the top right On by default Auto activates when a contour level is changed Show Clipping Info Displays graphical information about the clipping planes in relation to centre and eye position Off by default Auto activates when clipping is changed Hide Object Takes the active object off the screen but does not delete it The little eye symbol changes to closed If one changes to a hidden item as active object then the me
47. nu item will read as Show object Hide All But Active Object If for an isolated view you want to just look at the active object then pressing this will take all objects except it off the screen without deleting them All their eye symbols change to the closed state Reset Display This resets the display to a defined hard coded position orien tation and scale factor Observe the status bar on the right e Options Centre On Last Mark This will translate to the position of the last mark set atom or density Centre On Active Model This will translate the centre position of the visible volume to the centre of mass of the model that is currently the active one Centre On New Models This is a toggle button with a little indicator field When clicked it changes the behaviour of the viewer in that it will ac tivate deactivate the automatic centring on every newly loaded model The default of this is activated Fit Active Model To Screen This attempts to set the scale factor and the cen tre position such that the active model is completely visible in x and y direction It also adjusts the orientation to align the model such that its longest principal axes are in the xy plane Mark Atoms This is a toggle button that activates deactivates the single click marking labelling of atoms with the left mouse button CHAPTER 3 USING ARP WARP 54 Clear Atom Marks All atom marks are deleted Clear Atom Distances All distance line
48. o approximately 50 The target number of residues will be reset accordingly Residues and chain fragments The important numbers are highlighted in red and bold in the short log file indicating the number of residues and the number of fragments into which these residues are arranged The higher the values of the Connectivity index and the Tracing score the more complete and reliable the resulting model is The length of the longest chain is also printed Further extension of the model You may try to feed the PDB output of the module into the Classic model building However subject to the resolution of the data this may not provide enough seed for subsequent automatic tracing of the full chain Job termination The statement Task completed successfully indicates that the job has finished with no error An error statement QUITTING ARP wARP module stopped with an error message name_of_the_program indicates that one of the modules of the task has terminated with an error message Please refer to the specified log file CHAPTER 3 USING ARP WARP 26 3 2 2 Building secondary structure from the command line auto_albe sh The script auto_albe sh where albe stands for alpha beta in the warpbin direc tory allows you to run the secondary structure building as a single line command without the use of the GUI The use of auto_albe sh is fairly simple The script prints out help information if it is invoked without arguments Usag
49. o input restraints for the ligand to be modelled rather than using those detected from the input structure such restraints should be included in the cif file input here e Crystal parameters Space group Cell ARP wARP asymmetric unit Wilson B factor and Sol vent content are derived automatically from the MTZ file and the total number of residues in the asymmetric unit They are displayed for infor mation only and cannot be changed However you may want to check whether their values conform to your expectations Resolution By default all reflections present in the MTZ file will be used You can check the box Use reflections between and then narrow the range if you are aware of certain deficiencies of your data CHAPTER 3 USING ARP WARP 40 e Test and comparison parameters Compare with an already fitted ligand If you have the final model of the lig and in the correct orientation and would like to check the installation and the performance of the software you can check this box You will then have to provide a PDB file that will be used for comparison 3 5 1 1 Output files short Log File The following information could be useful when interpreting the log messages that are produced when building ligands Refinement with REFMAC The R factor and R free if requested are printed after refinement of the protein part only with REFMAC A value higher than about 30 may indicate that the computed difference map may be too noisy f
50. or lo cation of the ligand The ligandbuild program The mapping of the difference density synthesis param eterised with grid points onto the ligand atoms is run as many times as defined by the number of ligand building cycles ligandbuild and M_ligandbuild Real space fit Up to 108 top constructed ligand models undergo a real space refine ment with respect to the difference density map The best solution is output If the test and comparison option is selected the r m s d to the reference PDB file XYZREF is also printed There will be a warning given if the stereochem istry of the constructed ligand is poor Also a warning will be given if the constructed ligand molecule has severe steric clashes which may be a sign of an incorrect ligand building You may want to inspect the ligand and the den sity and if there is a clear part of the ligand that is disordered try to either run automatic partial ligand building as described above or manually remove it from the ligand target PDB file and re run the job Job termination The statement Task completed successfully indicates that the job has finished with no error An error statement QUITTING ARP wARP module stopped with an error message name_of_the_program indicated that one of the modules of the task has terminated with an error message Please refer to the specified log file CHAPTER 3 USING ARP WARP 41 3 5 2 Running ligand building from the command line auto_ligand sh
51. ore Close 4 Figure 3 2 Submitting a job to the ARP wARP cluster from the CCP4 GUI Submit a remote job at the Hamburg Cluster T l The options are 1 The data can be archived and made available to any software developer that requests them this is default 2 The data can be made available to ARP wARP AutoRickshaw or REFMAC developers 3 The data must be kept confidential and deleted after the job has finished Option 2 will only allow the data share to the ARP wARP Auto Rickshaw and REFMAC development teams Option 1 will extend the share to anyone who re quests the data In case of option 3 only the short log file Wilson omega log files and the parameter file will be kept by the ARP wARP developers all other data in put PDB PIR and MTZ files as well as log files will be automatically deleted one week after the job has finished In case of any option the ARP wARP developers may inspect the data in case of a job crash and provide a prompt feedback to the user Once the job has been submitted for remote execution the GUI window will indicate that the job has finished Please inspect the log file from the pull down menu option View files from job for further instructions An email will be sent CHAPTER 3 USING ARP WARP 19 to you at the email address that you entered in the GUI window Please follow the instructions in the email http link login and password to connect to the Hamburg cluster You can then
52. ormally need not worry about these except if you want the ligand to be built around a known location or if you would like to screen a list of candidate ligands ie a ligand cocktail A brief description is given below e Optional parameters Build the ligand Binding site location In the most likely place of the complete asymmetric unit default around the same approximate place as a previous ligand The binding site is defined by the position of a compound known to bind at the desired location If you use this option the region is provided by submitting a PDB file specifying the previous ligand coordinates around an approximate XYZ position The binding site is defined by X Y Z Cartesian coordinates and an input search radius option Search for the ligand around It is recommended that the user specify a binding site using this option if partial occupancy of the ligand is to be assessed REFMACS By default the fast protocol is used 1 cycle of refinement If your PDB file needs considerable pre refinement with REFMAC before the dif ference electron density map can be computed you can choose the slow protocol 3 cycles of refinement Free R Flag By default the data flagged as an Rfree set are used in REFMAC refinement You can choose to use R free and this will cause additional options to appear within the section REFMAC parameters CHAPTER 3 USING ARP WARP 39 Ligand building cycles defines the number of grid
53. own in the viewer Open Other File If a file is input containing three columns specifying X Y Z coordinates this option will show these coordinates in the viewer Duplicate Object If an item that is already loaded should be duplicated e g a map that is to be looked at with different contour level and colour Close File Delete an object from memory and remove it from the screen all changes applied will be lost Close All Files The real clean again all changes will be lost Save File As Choose a name for an object and make a copy of what is on the screen for maps and models Open Status File Opens a status file saved previously This will reproduce most of what the screen looked like at the time the status was saved All files must be in their original locations The file name suffix is vst Save Status Saves the current status into a file whose name must be chosen e Actions Fit a Ligand This will run the ARP wARP ligand building as an external soft ware in a separate thread The same files are required as running via the CCP4 GUI When run now is pressed the job starts executing auto_ ligand sh If this is successful i e the parameter file could be made then CHAPTER 3 USING ARP WARP 52 there is a live button in the top right corner where the parameter file can be viewed The short log file of the ligand job appears instantly and the calculated data and structures show up on the screen as soon as they are read
54. phases mzn ocr fa Fobs Sigma PHIB _ FOM qh moor a Sa cal i There are ay total residues in the AU which belong to I molecule s Do 10 cycles of model building 50 cycles in total Use in REFMACS the Maximum Likelihood target function and do notuse the Free R flag Submit the job for remote execution at the Hamburg cluster Figure 3 1 Protein model building using ARP wARP Classic from the CCP4 GUI This module of ARP wARP provides automated protein model building starting from experimental phases or an existing model molecular replacement the so called warpNtrace protocol This module aims to deliver an essentially complete model and an improved density map by utilising the idea of the hybrid model warpNtrace keeps what ever was recognised as protein in a form of polypeptide fragments and the rest as free atoms and refines this hybrid model during a big cycle consisting of sev eral default is 5 ARP wWARP REFMAC update refinement cycles At the end of 10 CHAPTER 3 USING ARP WARP 11 each big cycle the map is interpreted anew using pattern recognition methods new polypeptide model is constructed and if the protocol converges right direction contains more residues in less fragments This whole procedure is iterated default is 10 times The output of warpNtrace is a set of refined polypeptide fragments If the se quence is available the traced
55. ps output fle PROJECT e Mew Protein and new loops combined output file PROJECT oo Browse e Definition of the loops required parameters For sequence 1 there are 1 molecules in the AU residues per molecule total in the AU The Methionine residues of this molecule are not Seleno Methionines Use all the chains of the PDB W Build a loop between residues ana the length of the loop is 0 Settings for generating loops Override the default maximum number of CAs per node Force minimum number of CAs 1 Suggest 5 altematives for each loop Selecting best loops 1 Selecting best CAS y Ronorctins fic 4 Run Save or Restore Cose Figure 3 4 Loop building from the CCP4 GUI This module tries to find likely loops to connect fragments of a partial protein structure based on the sequence and the density map It builds the loops in three phases First a tree of possible Ca atoms between the fragments is build next the unlikely ones are removed and the rest of the main chain atoms determined and fi nally the best loops are selected The tree can be build either towards the C terminus of the N terminus of the protein or both The built loops are ordered in descending order according to the density correlation at the main chain atoms including C if present or the correlation of the side chains or a combination of both If the number of loops exceeds the chos
56. r hosting the research group the ARP wARP download servers and remote computational infrastructure funding agencies for research and infrastructure grants and ARP wARP academic and industrial users 61 CHAPTER 6 ACKNOWLEDGEMENTS 62 6 1 Third Party Software The ARP wARP distribution includes an unmodified version of the Open Astex Viewer software http openastexviewer net web license html The ARP wARP distribution also includes a modified version of the smi23d soft ware http www chembiogrid org cheminfo smi23d in the form of executables smi23d and mengine The smi23d software is covered by the Apache License Ver sion 2 0 http www apache org licenses LICENSE 2 0 htm1
57. s you need to remove them from the input PDB file Provide the length of the loop including the two anchor points e Selecting best loops Deviation distance loop connection Set the allowed error in the Ca Ca dis tance Ca density correlation threshold This number sets the number of best loops kept based on the density correlation of the Ca atoms only CHAPTER 3 USING ARP WARP 30 Structural threshold Set the threshold for the minimal value for the log like lihood of this structure Set the minimum value if you want to ensure to keep at least a certain number of loops after pruning Set the maximum value if you want to ensure that the number of loops doesn t exceed a certain amount after structural pruning Main chain density correlation This parameter sets the number of best loops kept e Selecting best Ca atoms Likelihood threshold This is the threshold for a Ca to represent the fifth Ca of a penta peptide based on density correlation Ca Ca distance and struc ture Minimum distance Ca atoms Measures the minimal distance between Ca atoms from the same shell The Ca with the best likelihood is kept e Generating Ca atoms Select generation Ca shell By default a shell with a uniform and regular dis tribution of Ca atoms at exactly Ca Ca distance is generated You can also choose for a uniform and random distribution of the Ca atoms In that case the shell is generated with a given thickness Number of Ca atoms Num
58. s between marks atoms and density are deleted Mark Density Region This is a toggle button that activates deactivates the single click marking of density regions with the left mouse button It can be used to specify density blobs for ligand identification or modelling for example Clear Marked Density Points All marks on density are deleted Create Ligand Topology Provided that a ligand molecule is loaded and se lected this outputs the topological information automatically extracted from the input coordinates to standard output This information will be used for ligand building when such a molecule is provided as input Save Screenshot graphics This will read out the screen pixel buffer and cre ate a bitmap or a png file You can choose or type a file name to use for the new image file Save Screenshot all As above but will also include all elements of the menu status bar and object related buttons e Help Help Screen When clicking on this item a text view window pops up that contains this help text 3 7 2 Mouse and Keyboard functions 3 7 2 1 Rotation Left mouse button pressed and mouse moved the scene rotates about the x and y axes screen plane Left mouse button r key pressed and mouse moved left right the scene rotates about the z axis perpendicular to screen plane 3 7 2 2 Translation Right mouse button pressed and mouse moved the scene is translated in the xy plane screen plane maps are infinitely repeat
59. s for structure factor ampli tudes their standard deviations phases and figures of merit have obvi ous names they will be recognised automatically Otherwise please use the scrolling button navigate to List All Labels and chose the appropriate ones Sequence file in Provide the sequence file in the following format pir The first line should start with gt The second line should be blank CHAPTER 3 USING ARP WARP 12 The sequence 1 letter code starts from the third line The space char acters hereafter are ignored In the case of heteromers separate different sequences with around 10 alanines Dock the autotraced chains Should the sequence be not available please un check this box in ARP wARP flow parameters Total residues in the AU number of molecules Provide the total number of residues in the asymmetric unit ARP wARP may be able to correct ob vious mistakes but it will not replace a human brain The number of molecules is obviously 1 for a monomer In a case of NCS the number molecules should be the number of NCS related molecules e g if you have 2 molecules in the AU with 200 residues each enter 400 for the to tal number of residues and 2 for the number of molecules If you have a hetero multimer e g 3a 3 structure the NCS order is 3 but please make sure that the sequence file contains both a and 3 sequences separated by about 10 alanines SEQUENCE_OF SUBUNIT_AAAAAAAAAA SEQUENCE_OF
60. sh NAME_OF_PARFILE If auto_tracing sh is called without an option parfile it will also launch the job The log files and additional output files as well as the building results can be found in the directory created 3 1 3 Remote submission of a model building task This option offers you the following possibilities 1 Your model building will run using external computational facilities where the CPU performance may be superior to your local installation 2 You can be assured that the most recent working executables will be used should you have a problem with your local installation 3 Should the task stop an automatic notification will be forwarded to the ARP WARP developers who can then promptly help you CHAPTER 3 USING ARP WARP 18 4 Upon your wish you can share the results of the completed task with software developers 3 1 3 1 Submitting from the GUI Clicking on the button with Submit the job for remote execution at the Hamburg cluster within the main ARP wARP Classic GUI panel allows one to execute an autotracing task remotely The panel will expand and ask for an email address to be provided Please also choose one of the options from the drop down menu to indicate how you would like your data to be handled W Submit the job for remote execution at the Hamburg cluster Your Email address 0 Job data can be archived and made available to any software developer that requests them Run _ Save or Rest
61. spaced with 5 ARP REF MAC cycles one should expect a job for a structure of 500 residues to be com pleted within about 1 hour subject to the power of the computer you are us ing CHAPTER 3 USING ARP WARP 23 3 2 Automated Construction of Helical and Beta Stranded Fragments 3 2 1 Building secondary structure from the GUI ARP wARP Quick Fold ane IN ARP WARP Version 7 4 0 Secondary Structure Recognition _Help Job title secondary structure recognition MTZin PROJECT mamau Browse LA Fobs Sigma PHIB FOM Output PDB file PROJECT SSS Browse vew T Parameters Number of residues La Do NOT build beta strands Crystal parameters ga Coordinate comparison ays Run Save or Restore Gose Figure 3 3 Running Quick Fold from the CCP4 GUI The procedure for building secondary structural elements is based on the use of discriminant analysis in a successive filtering scheme taking into account the geom etry of alpha helical and beta stranded main chain fragments The built fragments are then regularised and the chain direction is chosen on the basis of their fit to the density Finally the fragments are refined in real space The accuracy of the resulting model depends on many parameters The module should be able to build helices and strands at resolutions as low as 4 5 A However it may not result in complete helical stranded structure and it may also contain parts t
62. ssion This is described below in a separate chapter CHAPTER 3 USING ARP WARP 16 3 1 2 Command line model building auto tracing sh The script auto_tracing sh in the warpbin directory allows running the auto mated model building from the command line without the use of the GUI The use of auto_tracing sh is fairly simple If invoked without arguments the script will print help information Usage auto_tracing sh datafile mtzfile residues number_of_residues_in_AU workdir FULLPATH_WORKING_DIRECTORY fp fp_label sigfp sigfp_label freelabin freer_label fbest weighted_amplitude_label phibest phibest_label fom fom_label modelin input_PDB_file_to_use_as_initial_model seqin sequence_file_for_one_NCS_copy cgr number_of_NCS_copies if seqin is provided default is 1 buildingcycles the_number_of_autobuilding_cycles default is 10 resol rmin rmax default is the full resolution range albe 1 to always invoke albe default is 0 for resol lt 2 7A else 1 restraints 1 to use conditional restraints default is 1 twin 1 to try de twining and twin refinement default is 0 sad 1 to turn on the SAD function refinement needs also wavelength and heavyin on input default is O compareto PDB_file_for_comparison keepjunk 1 to keep intermediate models default is 0 parfile parfilename_if_only_parfile_is_to_be_created OPO LOO E GO POO lO GE OG GOONS
63. t your structure hav ing a fold with a significant content of beta strands you can deactivate their construction by checking the box e Now you are ready to start the job Click on Run and choose Run now There are a number of additional parameters that you normally should not worry about A brief description is given below e Crystal parameters Space group Cell ARP wARP asymmetric unit Wilson B factor and Sol vent content are derived automatically from the MTZ file and the total number of residues in the asymmetric unit They are displayed for infor mation only and cannot be changed However you may want to check whether their values conform to your expectations Resolution By default all data present in the MTZ file will be used You can check the box and then narrow the range if you are aware of certain defi ciencies of your data e Coordinate comparison CHAPTER 3 USING ARP WARP 25 Compare with an already deposited protein for validation or testing If you have the final model and would like to check the installation and the per formance of the software you can check this box You will then have to provide a PDB file that will be used for comparison 3 2 1 1 Output files short log file The following information could be useful when interpreting the log messages that are produced when running Quick Fold Checking the estimated content Should the solvent content be too high or too low ARP wARP will re set it t
64. ta Cryst D49 129 149 model update and solvent For other publications please refer to the references therein or to the ARP wARP web page 6 Acknowledgements The current ARP wARP developers are The Hamburg team European Molecular Biology Laboratory EMBL Hamburg Outstation c o DESY Notkestrasse 85 22603 Hamburg Germany e Victor S Lamzin tel 49 40 89902 121 email victor embl hamburg de e Ciaran Carolan Philipp Heuser e Joana Pereira e loan Vancea e Tim Wiegels Former members e Serge X Cohen Helene Doerksen Guillaume X Evrard Francisco Fernandez Johan Hattne Saul Hazledine Marouane Ben Jelloul Krista Joosten Matheos Kakaris Olga V Kirillova Gerrit G Langer Wijnand Mooij Richard J Morris Parthasarathy Venkataraman Tilo Strutz Petrus H Zwart The authors are especially grateful to e Keith S Wilson York UK one of the originators of the software and Zbyszek Dauter Argonne USA for significant contributions at earlier stages the soft ware development e Anastassis Perrakis Amsterdam NL the former co developer for his great input and encouraging spirit e The REFMAC developers team lead by Garib Murshudov York Cambridge UK e The CCP4 developers currently lead by Eugene Krissinel Didcot UK e Many of our collaborators and active users a comprehensive list is very long We would also like to take this opportunity to thank for the continuing support of ARP wARP the EMBL fo
65. ter recognition based detection of planar objects in 3D electron density maps Acta Cryst D64 834 842 nucleotide building e Joosten K Cohen S X Emsley P Mooij W Lamzin V S amp Perrakis A 2008 A knowledge driven approach for crystallographic protein model com pletion Acta Cryst D64 416 424 loops e Cohen S X Morris R J Fernandez F J Ben Jelloul M Kakaris M Parthasarathy V Lamzin V S Kleywegt G J amp Perrakis A 2004 Towards complete vali dated models in the next generation of ARP wARP Acta Cryst D60 2222 2229 side chains e Morris R J Perrakis A amp Lamzin V S 2003 ARP wARP and automatic interpretation of protein electron density maps In Meth Enz Carter C amp Sweet B eds 374 229 244 protein model building 59 CHAPTER 5 CITING ARP WARP 60 e Morris R J Perrakis A amp Lamzin V S 2002 ARP wARP s model building algorithms I The main chain Acta Crystallogr D58 968 975 protein model building e Perrakis A Morris R and Lamzin V S 1999 Automated protein model building combined with iterative structure refinement Nature Struct Biol 6 458 463 protein model building e Lamzin V S amp Wilson K S 1997 Automated refinement for protein crystal lography In Meth Enz Carter C amp Sweet B eds 277 269 305 protein chain tracing e Lamzin V S and Wilson K S 1993 Automated refinement of protein models Ac
66. the model from helix strands tracing be more complete than the model from warpNtrace the appro priate message will be printed at the end of the short log file Pre weighted Fobs for initial map calculation Checking this box will result in a pool down menu asking for the FBEST label CHAPTER 3 USING ARP WARP 14 Number of ARP REFMAC refinement cycles between autobuilding The de fault is 5 In cases of poor convergence you can try to increase this number to 10 Skip the autobuilding for the first cycles Checking this box will disable the autotracing for the provided number of cycles This was sometimes ad vantageous with earlier ARP wARP versions when the initial phases were poor Randomisation of atomic positions This also was sometimes advantageous with earlier ARP wARP versions when the initial model bias was high The default is not to randomise Iterate the tracing Each protein chain tracing is carried out in several rounds against the same density map The default number of rounds is 5 and it is not recommended to change this value Density thresholds for atom removal and addition These parameters are fixed to 3 2 and 1 0 respectively In cases of poor convergence partic ularly when the number of both added and removed atoms is consider ably less than the number requested as can be seen from the log file the threshold for atom removal can be slightly increased Also at resolution of 2 5 A and lower it may be advantageous
67. tructure Nucleotides and chain fragments The important numbers are highlighted in red and bold in the short log file indicating the number of nucleotides and the CHAPTER 3 USING ARP WARP 34 number of fragments into which these residues are arranged The length of the longest chain is also printed Job termination The statement Task completed successfully indicates that the job has finished with no error An error statement QUITTING ARP wARP module stopped with an error message name_of_the_program indicates that one of the modules of the task has terminated with an error message Please refer to the specified log file 3 4 2 Running nucleotide building from the command line auto_nuce sh The script auto_nuce sh in the warpbin directory allows you to run the secondary structure building as a single line command without the use of the GUI The use of auto_nuce sh is fairly simple The script prints out help information if it is invoked without arguments Usage warpbin auto_nuce sh datafile mtzfile residues number_of_protein_residues_in_AU nucleotides number_of_nucleotides_in_AU workdir FULLPATH_WORKING_DIRECTORY fp fp_label sigfp sigfp_label fbest weighted_amplitude_label phib phib_label fom fom_label resol rmin rmax default is the full resolution range compareto PDB_file_for_comparison parfile parfilename_if_only_parfile_is_to_be_created 2A AAA AAA AZ Option
68. uld be omitted if Fobs are already FOM weighted Output PDB file Provide the PDB file name where the constructed polynu cleotide fragments will be output to e Click on Run and choose Run now There are a number of options that can be added A brief description is given below e Space group Cell ARP wARP asymmetric unit Wilson B factor and Solvent content are derived automatically from the MTZ file and the total number of residues in the asymmetric unit They are displayed for information only and cannot be changed However you may want to check whether their values conform to your expectations Obviously if you entered ze ros as the expected number of residues and nucleotides the solvent con tent will be displayed as 1 0 but you should not worry about this Resolution By default all reflections present in the MTZ file will be used You can check the box Use reflections between and then narrow the range if you are aware of certain deficiencies of your data 3 4 1 1 Output files short Log File The following information could be useful when interpreting the log messages that are produced when building DNA RNA Checking the estimated content Should the solvent content be too high or too low ARP WARP will re set it to approximately 50 The target number of residues will be reset accordingly Phosphate candidates The identified number of phosphate candidates is typically 100 times higher than the number of nucleotides in the s
69. with good quality phase error estimates in the form of HL coefficients The default is not to use Rfree since the number of traced residues serves as excellent indicator of the success of the job You can certainly turn the use of Rfree on e Click on Run and choose Run now There are a number of additional parameters that you normally should not worry about A brief description is given below e ARP wARP flow parameters Use conditional restraints for free atoms This allows restraints to be used to keep free atoms in reasonable places The default is on Use Non Crystallographic Symmetry Restraints Indicate to REFMAC that it should use NCS restraints At resolution 1 5 A or better this is on by default Use Non Crystallographic Symmetry information to extend chains Extend chains using information provided by related parts of the structure At resolution 1 5 A or better this is on by default Use Loopy to build loops This option allows the loop filling mode to be in voked throughout the iterations The default is on Dock the autotraced chains to sequence The default is to dock the fragments starting from building cycle 0 Se Methionine If you have Se methionine substituted protein regardless of the use of the refinement function you can check the box thus asking ARP wARP to build and refine Se Met residues Search for helices and strands before each building cycle This is the default for resolution of 2 7 A or worse Should
70. y A job may be killed as long as it is running The window cannot be closed when a job is running Build Helices and Strands This will run the ARP wARP secondary structure modeller The same files are required as running via the CCP4 GUI When run now is pressed the job starts executing auto_albe sh Model Solvent This runs the solvent building module of ARP wARP The same files are required as running via the CCP4 GUI When run now is pressed the job starts executing auto_solvent sh Classic Model Building Run the Classic protein model building protocol The same files are required as running via the CCP4 GUI When run now is pressed the job starts executing auto_tracing sh Start CCP4 Start the CCP4 GUI Note Unlike the CCP4i GUI the Fit a ligand and Build Helices and Strands dialogs will also accept density maps as input e Display Global Parameters This allows you to change the background colour and the depth fog You can also switch on and off the perspective distortions These become active only when the Apply button is pressed Map Parameters For the active map object only one can change the param eters of display The window can stay alive even when the map is no longer the active object and it will vanish when the map is deleted Here you can change the map colour whether it is displayed as a mesh or a solid body You can clip the density to a model of a ligand too The struc tures will show up in a browse butto

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