M86-E00078 APEX2 User Manual.book
Contents
1. cients CC are a sensitive indicator of a proba ble solution In general the solution is valid if the correlation coefficient for all data is greater than twenty and the correlation for weak data is greater than ten Correct solutions should corre spond to those with the largest Patterson figure of merit PATFOM Also there should be a good separation of peak heights in the list between the expected peaks and the noise The Patter son self and cross vectors should be large and the inter peak distance should be reasonable Zinc free insulin belongs to space group 2 3 In this space group two non symmetric related but equivalent indexings are possible resulting in forty eight coordinate points for each of the three sulfur Super atom locations XM may also give an inverted xyz solution This may lead to confusion if you want to compare the XM solu tions to the zinc free insulin PDB entry 9INS The disulfide geometry or inter peak distances presented in Figure B 7 correspond to the cor rect insulin disulfide geometry irrespective of indexing Use this to check whether the XM solution from your own data is correct B 10 M86 E000472. Figure 8 2 Lattice statistics 2 XPREP determines the reduced cell based on the lattice entered above Since the lat tice was primitive and the magnitudes of the cell dimensions were proper a lt b lt c the Original and reduced cells are the same see Figure 8 3 Press Enter to search fora higher symmetry cell APEX2 User Manual W XPREP Ver 6 14 W95 98 NT 2000 ME Copyright Bruke Original cell Figure 8 3 Reduced cell For the YLID no higher symmetry cell is found The program has determined that the YLID crys tal has an orthorhombic primitive lattice see Figure 8 4 Press Enter to accept Figure 8 4 Higher symmetry cells M86 E00078 APEX2 User Manual 3 The next logical step is to determine the space group XPREP suggests this see Figure 8 5 Press Enter to determine the space group E XPREP Ver 6 14 W95 98 NT 2000 ME Copyright Bruke urrent dataset Wavelength Original cell Figure 8 5 Determine the space group 4 If the space group is known or if the com pound is known to be chiral enter that infor mation see Figure 8 6 Generally it is sufficient to press Enter to start the space group determination Figure 8 6 Space Group options M86 E00078 Examine Data 5 XPREP has chosen the crystal system O see Figure 8 7 Press Enter to accept Figure 8 7 Choose the crystal system 6 XPREP has chosen the crystal lattice P see Figure 8 8 Press E
3. 397 33 0 62 4 0 0 63 8 Lattice type P re Gy A Olobv Rirev rhomb on hex axes Select option I I Mean E E 1 0 714 expected 968 centrosym and 736 non centrosym Systematic absence exceptions d Identical indices and Friedel opposites combined before calculating R sym Option Space Group No Risym Nf eq Syst dbs CFOM 4 Im 3 204 centro 4 0 000 0 46 6 CB 123 197 chiral i 7 46 6 C I2 1 3 199 chiral 0 7 46 6 select option B c PgUp PgDn scrolls text only graphics window may be resized Figure B 2 XPREP main window Appendix B PROTEUM User Manual B 1 4 Read Modify or Merge Window XPREP Yer 6 12 W95 98 NT 2000 ME Gl Prompts Index Data Filename or Source of Data 1 5249 insulin example O1 s sca lt current dataset 1 Enter A to the Select option S prompt This Sor RGE current data no scaling C Change CURRENT catasert will refresh the window LOr atrec iagd coon ARAE nen Lie 7 yey PEED 1E nner 5 D D DELETE et ACE indexed a rp 15 P PSI s sOrpt corr 2 Enter A to the Select option E prompt Use Copy file TRANSFORM hkl a nes or SIRAS N No X scale sigmas the default parameters by hitting lt CR gt G Generate simulated powder diagrams E EXIT to main menu 0 QUIT program when prompted for the high resolution cut off VY cutoff and target number of reflec MAD Multiple wavelength Anomalous Dispersion tions X 2 Si oe i ect op
4. 8 Examine Data In this step the space group for the sample is determined and optional simulated precession photographs are calculated to further evaluate the overall quality of the data APEX2 provides two tools for this XPREP for space group deter mination and Precession Images for looking at undistorted slices of reciprocal space This is the final step before beginning the structure solution and refinement process 8 1 XPREP 1 Click on XPREP Space Group Determina tion under Examine in the task bar 2 Check that the two files in the pop up win dow are correct see Figure 8 1 and click OK M86 E00078 2 x PAP file quest data_manual ylid_manual work ylid_manual_Om p4p_ g HKL file f guest data_manual ylid_manual work ylid_manual_Om hk g Cancel Figure 8 1 Select files for XPREP input NOTE In this example the scaling process has created two files ylid_manual_Om p4p contain ing the final unit cell parameters from integration and ylid_manual_Om hkl containing the cor rected intensities Typically these are the files to use for determining space groups but you can browse to choose other files Examine Data 8 2 Space Group Determination 1 XPREP evaluates the data and looks at the mean intensities and the mean int sigma Since these are large for all groups except P XPREP suggests that the lattice is P see Figure 8 2 Press Enter to accept E XPREP Ver 6 14 W95 98 NT 2000 ME Copyright Bruk
5. Setup the Program on a Windows 2000 XP Computer 1 Copy the xprep exe executable to the work ing directory 2 To execute the program double click on the xprep exe icon or use the Command Prompt to cd change directory to the working directory and then type xprep The XPREP window will appear see Figure B 1 B 1 2 XPREP Window Prompts 1 Enter insulin_example_01_s sca for the file name prompt 2 Enter D for the Option prompt 3 Enter Y for the Use from Denzo Scalepack prompt 4 Enter Ifor the Select option prompt This will bring up the XPREP main window see Fig ure B 2 PROTEUM User Manual XPREP Yer 6 12 W95 98 NT 2000 ME ta FREER EE EE EEEEE EEE EEE EEE EEE EEE EEE EEE EEE EEE EEE EEEEEEEEEEEEEEEE EEE EEE EEE EEE EET XPREP Reciprocal space exploration Version 6 12 W95 98 NT 2000 ME COPYRIGHTi c 2001 Bruker AXs ll Rights Reserved FAFA EAAAAEFAFAEEEAEFAFEEFEEEFEFEEFEFEEFEFEEFEEEFEAEEEEEEAEFEEEFAFEFE AEE EAE EAE H HE Screen size Window size Font siz Number of colo Filename lt CR gt if none hkl assumed insulin example O01 s sca SHELZ HKLF 4 format or SAINT rf file SHELX HELF 3 format ondensed data HKLF 1 LIST mu file F ACK or HKL2000 sca file Generate idea data from res or ins EXIT to main menu Q QUIT program ect option D D 77 793 77 793 77 793 Data multiplied by 0 1000 to bring onto reasonable scale 2818 Reflections read from file insulin example _01_
6. The program asks for an output file name Since this data has been cut off at a resolu tion of 0 75A it makes sense to change the output file name to ylid_res75 to reflect the cutoff see Figure 8 19 Press Enter to accept F O00 Figure 8 19 Changing the file name 5 After entering the file name or hitting Enter to accept the default file name an input file for the structure solution module is created and displayed on the screen and the pro gram asks Do you wish to over write the intensity data file ylid_res75 hkl Since the file name has been changed this question must be answered with a y see Figure 8 20 APEX2 User Manual R Figure 8 20 The input file for structure solution and a final question 6 Exit XPREP see Figure 8 21 Press Enter to exit the program R Figure 8 21 Exit XPREP from the general menu NOTE There are many other features in XPREP that can be accessed from the general menu reciprocal space plots simulated powder patterns and a test for merohedral twinning are very useful tools M86 E00078 Appendix B Finding Heavy Atoms or Anomalous Scatterers This section demonstrates the use of the pro grams XPREP and XM SHELXD to determine the locations of the three insulin disulfide groups from the set of anomalous scattering data The sample data accompanying this manual is from a crystal of zinc free bovine insulin Zinc free bovine insulin was chosen beca
7. n in the command prompt window to execute the M86 E00047 Appendix B program The program will display output in the Command Prompt window as well as write a copy of the output to the disk as file name I st See Figure B 7 PSUM 57 86 PSMF Peaks 63 61 43 17 13 10 4 3 Iry 9 20 Peaks 99 96 94 35 34 R 0 334 Min fun 0 472 lt cos gt 0 341 Ra 0 339 Try 9 CC All Weak 36 91 18 36 best 36 91 18 36 best PATFOM 40 51 PATFOM 40 51 X y Z sof U height 0 48441 0 33655 0 23803 20 0 1 0 99 90 0 64152 0 63899 0 07690 20 0 1 0 98 43 0 46549 0 91776 0 16196 20 0 1 0 97 60 0 78958 0 18511 0 04419 20 0 1 0 59 45 0 22447 0 02254 0 12924 20 0 1 0 58 95 Minimum distances top row if special position and PSMF bottom row Peak x y Z self cross vectors 99 9 0 4844 0 3365 0 2380 13 7 inter peak distance 18 4 98 4 0 6415 0 6390 0 0769 20 7 Did 710 2 47 4 To Oy 4655 09178 05 1620 Agape duet To Gao 290 T23 59 4 0 7896 0 1851 0 0442 E oo be Ot 1 0 0 0 0 0 0 0 DO Orgad O OO Qed Lel desc leis Bie adh Fa Figure B 7 Section of the XM output file Appendix B PROTEUM User Manual Since XM does not try to identify whether a solu tion has been found there can be several cor rect solutions among the twenty requested The best solution is the last solution where the Patterson peak list self and cross vector peak heights and inter peak distances are output The all data and weak data correlation coeffi
8. nce this formula is incorrect it must be modified now The cor rect chemical formula for the YLID crystal is 2 1 At Select Option do not accept the default answer of E Type in F to enter a new formula 2 2 In response to the question Enter For mula type the correct formula C11 H10 O2 S1 and press Enter 2 3 Check that the information is correct Check that Z seems reasonable for the space group that the density is as expected 1 1 to 1 4 for organic mole cules higher for inorganic compounds and that the atomic volume is around 17 or 18 Significant variation from the expected values may indicate an incor rect molecular formula or missing counter ions or solvates The values at the bottom of the window look fine for the YLID Press Enter to accept M86 E00078 Examine Data E XPREP Ver 6 14 W95 98 NT 2000 ME Copyrig Current dataset Wavelength Original cell Esds Current cell Matrix Crystal system E Formula Formula wt cs Density At vol i Mu rim 1 Figure 8 17 Defining and checking the unit cell contents 3 The next default action for XPREP is to write out the files necessary for the structure solution process see Figure 8 18 Press Enter to accept Current dataset Wavelength Original cell Esds Current cell Matrix Crystal system Space group Formula Formula wt Density Atwood J Mu ren 1 Figure 8 18 Requesting output files Examine Data 4
9. nique heavy atoms 12 3 Se prompt and 2 3 at the Number of Wavelength 1 54178 I unique heavy atoms 12 prompt PoUn PODn scroks text only graphics window may be resized Figure B 3 XPREP read modify or merge window B 4 M86 E00047 PROTEUM User Manual Appendix B 8 Accept the wavelength by clicking lt CR gt at the Wavelength 1 54178 prompt The con tents of the insulin _example ins file will dis play 9 Enter lt CR gt to exit the window which will refresh the XPREP Read Options window 10 Enter E at the Option S prompt to return to the main window and to view the Patterson sections Otherwise enter Q at the Option S prompt to exit the program 11 To display the anomalous difference Patter son maps enter P at the Select option H prompt to access the Patterson window see Figure B 4 M86 E00047 B 5 Appendix B PROTEUM User Manual B 1 i 5 Patterson Window O pt ions i XPREP Ver 6 12 W95 98 NT 2000 M Current dataset SAS deltalF Wavelength 1 54178 Chiral Enter R at the Select option E prompt to Original cell 77 793 77 793 77 793 90 00 9 90 00 Vol 470763 set the resolution limits Esds 0 00 Oo 0 00 Lattice Current cell 7 793 77 793 77 793 90 00 Vol 470783 Enter 99 and 3 5 at the prompt to set the low and high resolution limits Matrix 1 0000 0 0000 0 1 0000 0 0000 0 0000 1 000 Space group I2 1 3 199 chi Laue 1 Enter O at the Select option E prompt to x
10. nter to accept Figure 8 8 Choose the lattice 7 XPREP evaluates the data and looks at the systematic absences for all possible glide planes and screw axes see Figure 8 9 These are displayed across the middle of the figure By examining the number of reflections with I gt 3 sigma l the mean intensities and the mean int sigma which should all be very small for a systematic absence XPREP derives a suggested space group P2 1 2 1 2 1 Press Enter to accept Examine Data 8 3 Reflection Statistics Figure 8 9 Systematic absences and a suggested space group 1 XPREP returns to the general menu seen previously See Figure 8 5 This time D is chosen to evaluate the data set Press Enter to accept 2 There are multiple choices for data manipu lation See Figure 8 10 S chooses a dis play of statistics E XPREP Ver 6 14 W95 98 NT 2000 ME Copyright Bruke ata Filename or Source of Data current dataset Figure 8 10 The data manipulation menu APEX2 User Manual 3 The data can be merged in several ways Choose the Merge ALL equivalents includ ing Friedel opposites option A see Figure 8 11 Press Enter to accept NOTE This merge will not average the reflec tions in the final data file It is only for the calcu lation of statistics Figure 8 11 The merge data menu 4 A table of statistics appears see Figure 8 12 Examine the data presented Is the completi
11. olution limit of 0 75 see Fig ure 8 14 and press Enter to accept the low resolution cutoff of infinity Figure 8 14 The resolution cutoff menu Examine Data APEX2 User Manual 3 Applying the cutoff removes approximately 5 After the cutoff all of the statistics look bet 3000 reflections from the data set see Fig ter see Figure 8 16 Press Enter to con ure 8 15 The next step is to see if the cut tinue off has improved the statistics Type S and fesolution Data Theory Complete Redundancy Mean I Mean I s Rint Rsigma press Enter E XPREP Ver 6 14 W95 98 NT 2000 ME Copyrig Index Data Filename or Source of Data lt current dataset Figure 8 16 Statistics after high resolution cutoff Figure 8 15 Requesting statistics after applying a high 6 This response will return to the data manip Roo EUa ulation menu see Figure 8 10 Figure 8 13 4 As in step 3 of Section 8 3 enter A to or Figure 8 15 The default answer should merge all equivalents be E for Exit Press Enter to accept this default and return to the general menu 8 6 M86 E00078 APEX2 User Manual 8 5 Preparing an Output File 1 Inthe general menu chose C to define the unit cell contents 2 A window will open displaying the current formula Z the density and the atomic vol ume see Figure 8 17 In this example the formula is incorrect and Z has been set to six to try to achieve a reasonable density and atomic volume Si
12. on near 100 Is the redundancy good Are Rint and Rsigma small and increasing smoothly from top to bottom In particular look at the last two lines which compare all of the data with the high resolu tion data The completion should be near 100 for both the high resolution shell and the complete data set The redundancy and Rint should be similar for the two The Mean Intensity and the Mean I sigma l will usually be quite different The Mean l sigma l for the high resolution data should be greater than 3 0 M86 E00078 APEX2 User Manual For this data set when integrated to 0 67A reso lution the difference between the Rint for the high resolution data and the complete data set is slightly larger than expected Press Enter to continue i XPREP Ver 6 14 W95 98 NT 2000 ME Copyrig Figure 8 12 Intensity statistics 8 4 Applying High Resolution Cutoff NOTE For the YLID crystal if the resolution limit for integration was changed to 0 75 as sug gested on Page 7 3 then there will be no need to apply a High Resolution Cutoff at this point M86 E00078 Examine Data 1 Since the difference between the Rint for the high resolution data and the complete data set is slightly larger than expected it is reasonable to apply a high resolution cutoff using the H option in the data manipulation menu see Figure 8 13 Type H and press Enter Figure 8 13 Requesting a high resolution cutoff 2 Enter a high res
13. program for locating heavy atoms or anomalous scatterers using the anomalous difference hkl and input ins files generated by XPREP For the insulin_example you must modify the XM input file by adding three additional parame ters see Figure B 6 TL INSULIN_EXAMPLE in I2 1 3 iL 1 54178 77 7930 77 7930 77 7930 90 000 90 000 90 000 48 00 0 0090 0 0090 0 0090 0 000 0 000 0 000 T 2 0 5 X 7 2542 5 2 Z 7 Xx oan t mx 4 gt x lt ae 4 OAR p r 6 w U ol N i OunN N S oo NITA P S OFX NDnooi inKtloonNnNo Pages 3 5 99 3 5 NTRY 20 WEED 0 HKLF 3 END Figure B 6 XM input file 1 Insert the SHEL 99 3 5 command line to modify the resolution range of the analysis to use data in the range of 99 to 3 5 A reso lution PROTEUM User Manual 2 Insert the NTRY 20 command line to limit the total number of solutions to twenty Omitting this line will allow the program to run indefinitely 3 Insert the WEED 0 command line to speed up the calculations since we are looking for only three sulfur super atoms NOTE Since the expected XM input file has the ins extension Windows interprets this as a spe cial file Use the Open with the option to edit the file M86 E00047 PROTEUM User Manual B 2 1 Start XM 1 Copy xm exe to the working directory and run the program from there 2 Type the XM filename no extensio
14. s sca Mean I sigma 63 01 Use cell from Denzo Scalepack Y Y Lattice exceptions P N total N f int gt 3si option I PgUp PaDn scrolls text only graphics window may be resized Figure B 1 Utilities gt XPREP M86 E00047 PROTEUM User Manual B 1 3 XPREP Main Window Prompts 1 Enter S for the Select option S prompt to determine the space group This will refresh the XPREP main window 2 Enter C for the Select option C prompt 3 Enter I for the Select option I prompt 4 Enter C for the Select option B prompt to define the space group as 21 3 and to refresh the XPREP main window 5 Inthe refreshed XPREP main window enter D to the Select option H prompt This will pop up the Read Modify or Merge window see Figure B 3 M86 E00047 Appendix B 1 XPREP Yer 6 12 W95 98 NT 2000 ME Gf Current dataset insulin example 01_s sca Wavelength 0 71073 Chiral ar Aaa E a an a a a E 90 00 90 00 Original cell Esds 0 000 0 00 0 00 Current cell 7 793 77 793 7 793 90 00 90 00 Matrix 1 0000 0 0000 1 0000 5 Determine SPACE GROUP C Mu oe CHIRAL sample is optically active SARILY chiral eg may be racemate I JPU nown Space group E EXIT to main menu or Q QUIT program Select option 5 S 4 Triclinic M Monoclinic 0 Orthorhombic T Tetragonal H Trigonal Hexagonal C Cubic or E EXIT select option C C Lattice exceptions P 4 B 366 1447 et 1
15. tion 5 a attering nt with Anomalous Scattering XPREP will then display a table of the anoma OOIT frasan lous signal to noise ratios as a function of reso Select option E A lution This table is useful in determining the High resolution limit in Angstroms for this calculation 0 0 0 0 resolution cutoff for the anomalous scattering I sigma threshold for rejecting after merging 0 5 0 5 data i e the point at which the signal to noise Target number of reflections in local scaling sphere o 0 if no local scaling 100 100 ratio becomes random in this case 3 8 A 3 Continue by using the defaults for the high resolution limit for output data and effective B val u e Rint anom 0 0303 before and 0 0296 after local scaling 66 4 Neighbors used on average for F t F local scaling High resolution limit in Angstroms for saved data 4 At the Filename prompt enter Enter effective B value e g 20 to normalize delta F or Fa values lt CR gt for no renormalization insulin _example Filename to write FA and phi T phi A lt CR gt for none INSULIN EXAMPLE Enter Y to the Write ns fi le prom pt Current dataset contains 2417 545 delta F Write ins file for SHELXD XD Y or N Y Y 6 Accept the filename by clicking lt CR gt at the Filename insulin _example ins prompt Filename INSULIN_EXAMPLE ins Element type for heavy atoms Se S 7 Enter S atthe Element type for heavy atoms Number of u
16. ular to X P Write Postscript file remove the origin n ceton perpendicular to Y R Set reso ion limits ion perpendicular to Zz G Draw GRID lines now on rpening now off 0 Origin removed now yes Enter Z at the Select option E prompt to c Set gaal E EXIT to main menu display the Patterson Z section Select option z play the Z 0 Patterson section igin removed Patterson layer zZ 0 380 eaks in origin removed Patterso Z Enter lt CR gt to use the default for the Z Bee ee coordinate for sigma estimation A list of Patterson peaks on the Z 0 section dis play Use Page Up and Page Down to scroll eee through the peak list a aie T Enter lt CR gt to display the contoured Patter o son section see Figure B 5 0 0000 0 0000 6 4 6 4 6 4 6 6 0 6 0 6 0 4 8 4 4 4 4 4 4 4 3 f 4 4 4 0 0000 0 0000 PgUp PgDn scrolls text only graphics window may be resized Figure B 4 XPREP Patterson window M86 E00047 PROTEUM User Manual PREP Patterson section Figure B 5 Contoured Patterson section The Patterson section for the insulin sulfur SAS example data is noisy due to the weak sulfur signal and the limited redundancy of the 90 data set However the data is sufficient to locate the three disulfide positions using XM SHELXD M86 E00047 Appendix B Appendix B B 2 XM SHELXD XM SHELXD written by George Sheldrick is an extremely efficient
17. use e itis inexpensive and readily available e crystal growth is easy and straightforward e the crystal system is cubic which provides high redundancy in data measurements e there are three disulfide S S bonds in the structure The goal of this exercise is to determine the positions of the three disulfide bonds from the single wavelength anomalous scattering SAS M86 E00047 data produced by PROTEUM ProScale Sulfur has a weak Af 0 56 anomalous scattering sig nal for SAS data recorded using Cu Ka radia tion However by collecting highly redundant data the anomalous signal to noise level in the resulting data set is increased to the point where it can be recorded accurately enough to yield both the positions of the anomalous scatterers and by solvent flattening noise filtering Wang 1985 the protein phases The 24 fold symmetry of the cubic zinc free insulin crystals provides the needed data redun dancy for the SAS analysis without needing to collect a large amount of data Also by using low 3 5 A data the two sulfur positions in the disulfide bond appear as a single sulfur super atom which also aids in the analysis This sec tion briefly describes the procedures used for the analysis Appendix B B 1 XPREP Setup XPREP written by George Sheldrick is a versa tile program for analyzing the scaled structure factors merging data producing Patterson maps and preparing files for SHELXTL and XM B 1 1
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