Molecular Replacement in CCP4

1. Molecular Replacement in CCP4 Martyn Winn CCP4 group, Daresbury Laboratory

2. Data analysis before MR Matthews coefficient Number copies in a.s.u. Native Patterson (translational NCS) B factor analysis Self RF (rotational NCS)

3. Data analysis before MR Interface to Sfcheck (currently in Validation&Deposition module) completeness, anisotropy, Wilson B, twinning check, pseudo-translation check

4. Finding search models • Need a PDB file for a structurally similar protein. This usually means a homologous protein. • Either you have one already?  • Or you search the Protein Data Bank • Search is based on sequence alignment between target protein and proteins in PDB. • Several bioinformatics tools can help here: • OCA, MSDlite, MSDtarget - all use FASTA • www.ebi.ac.uk/msd • psiBLAST - iterative searching • www.ncbi.nlm.nih.gov/BLAST • FFAS - profile-profile alignment • ffas.ljcrf.edu/ffas-cgi/cgi/ffas.pl

5. Editing search models • Don’t use a raw PDB file for Molecular Replacement unless it is very similar (e.g. same protein, different conditions, ligand, etc.) • Edit it to: • remove residues that don’t occur in the target • remove side chain atoms that don’t occur in the target • (these assume a know alignment from model to target) • remove uncertain regions of model (check B factors, occupancies) • remove flexible loops Note that we don’t add anything!! Homology modelling? Consider use of individual domains and multimers (see MrBUMP below)

6. Chainsaw Norman Stein, Daresbury Lab.

7. MR model preparation: chainsaw • Molecular replacement model preparation utility that edits a PDB search model according to a sequence alignment. • Features: • Removes un-aligned residues from the model • Prunes non-conserved residues back to the gamma atom • Preserves more atoms than in polyalanine model Unmodified template Chainsaw template Polyalanine template Example of 1mr6 used as a template for 1tgx (38% sequence identity)

8. Running Chainsaw: complete PDB file model to target alignment • Alignment from: • original search tool (FASTA, psiBLAST, etc.) • multiple alignment (set of search models, protein family, etc.) • hand-created

9. Molrep Alexei Vagin, York http://www.ysbl.york.ac.uk/~alexei/molrep.html

10. Molrep: overview of functionality Performs complete MR in single step: Expt. data (MTZ) Positioned search model Molrep Search model (PDB) • Individual steps for more difficult cases: CRF, TF, rigid-body • Multi-copy search: locked CRF, dyad search • Self RF • Phased TF, spherically-averaged phased TF • Improve search model • Other search models: electron density map, NMR models • Fit model in electron density map / EM map

11. MR for straightforward case via GUI: title mode MTZ file MTZ labels search model RUN IT!

12. Other parameters DEFAULTS ARE GOOD • Low resolution cut-off • Molrep uses soft cut-off, Boff (BOFF, COMPL, RESMIN) • High resolution cut-off • Molrep uses soft cut-off, Badd (BADD, SIM) • |F|new = |F|input *exp(-Badd*s2)*(1-exp(-Boff*s2) • Defaults estimated • High resolution limit • Absolute cut-off (RESMAX) • Default estimated • Radius of Patterson sphere for CRF • Default is twice radius of gyration of search model, Keyword RAD, Infrequently Used Parameters in GUI

13. Cross Rotation Function Euler angles (CCP4) polar angles R factor List of top RF peaks More details here

14. Translation Function fractional translation R factor Score polar angles List of solutions: top TF for each RF solution contrast of solution

15. Identification of solutions • SCORE = product Correlation Coefficient and maximal value of Packing Function • Packing Function integrated into TF search  removes solutions with overlapping molecules • CONTRAST = ratio of top score to mean score: • >2.5 - definitely solution<2.5 and > 1.8 - solution<1.8 and > 1.5 - maybe solution<1.5 and > 1.3 - maybe not solution, but program accepts it <1.3 - probably not solution

16. Finding more than one copy in the asu By default, Molrep will estimate number of copies to find. Override with NMON keyword Program flow: • CRF • TF for first copy • Fix first copy • TF for second copy • Fix second copy • TF for third copy • . • . • .

17. Solving complexes • Choose first component (largest, highest similarity) • Solve for first component (probably need to specify NMON explicitly) • New Molrep job • Model in - second component • Fixed in - positioned first component • Repeat for all other components Possibility to use spherically-averaged phased TF using phases from first component

18. Phaser Randy Read, Airlie McCoy, Cambridge Phaser website: http://www-structmed.cimr.cam.ac.uk/phaser/

19. Performs complete MR in single step: Expt. data (MTZ) Positioned search model Phaser Search model (PDB) Use “MODE MR_AUTO” or “automated search” in the GUI • anisotropy correction • fast rotation function • fast translation function • packing • refinement and phasing loop over models

20. More functionality ... • All steps can be run separately • Search over spacegroups: • MTZ spacegroup and enantiomorph • All spacegroups in MTZ point-group • Selected spacegroups • Ensemble models (see later) • Brute RF and TF - slow and accurate • Normal mode analysis • Generates perturbed models

21. MR for straightforward case via GUI: mode MTZ file target details search model specify search RUN IT!

22. FRF Euler angles (CCP4) Top LLG and Z-scores for FRF

23. FTF fractional translation FRF solution number Top LLG and Z-scores for FRF

24. Packing Phaser does packing check after FTF Clashes = C atoms closer than 2Å Default number of clashes = 0 Think about increasing to 2 or 5

25. Solution files: • .sol file produced at end of job • Contains summary of all solutions • Each solution contains rotations and usually translations - • 3DIM vs 6DIM • One line per model located • .sol file can be read back into Phaser in later jobs Z-scoreHave I solved it? less than 5 no 5 - 6 unlikely 6 - 7 possibly 7 - 8 probably more than 8 definitely RFZ = RF Z-score TFZ = TF Z-score

26. Ensemble models Phaser refers to search models as “ensembles” Often, ensemble contains single model, as in traditional MR But Phaser can use an ensemble of > 1 models, which may work better than any single model Models in an ensemble must be superposed prior to use in Phaser - use e.g. Superpose in CCP4 N.B. Phaser will complain if: • MW of models in ensemble are too different • RMS between models is too large (In Molrep, construct ensemble as pseudo-NMR PDB file)

27. Finding more than one copy in the asu • Specify > 1 in Composition of the asymmetric unit • (keyword COMPOSITION ... NUMBER) • Specify > 1 in Number of copies to search for • (keyword SEARCH ... NUMBER) Phaser will issue warnings if these numbers are wrong. • CRF • TF for first copy • Fix first copy (possibly multiple sets) • CRF for second opy • TF for second copy • Fix second copy (possibly multiple sets) • . • . • .

28. Complexes • As before, but: • Define > 1 type of component • Composition of the asymmetric unit • Define another component • Define > 1 ensemble • Define ensembles • Add ensemble • Specify all searches • Search details • Add another search E.g. beta-blip example in Phaser tutorial: http://www-structmed.cimr.cam.ac.uk/phaser/tutorial/Phaser_MR_tute.html

29. MrBUMP Ronan Keegan, Martyn Winn, Daresbury Lab.

30. The aim of MrBUMP • An automation framework for Molecular Replacement. • Particular emphasis on generating a variety of search models. • Can be used to generate models only. • Wraps Phaser and/or Molrep. • Also uses a variety of helper applications (e.g. Chainsaw) and bioinformatics tools (e.g. Fasta, Mafft) • Uses on-line databases (e.g. PDB, Scop) • In favourable cases, gives “one-button” solution • In unfavourable cases, will suggest likely search models for manual investigation (lead generation)

31. The Pipeline ` Target MTZ & Sequence Target Details ` Template Search ` Model Preparation Check scores and exit or select the next model ` Molecular Replacement & Refinement

32. Search for homologous proteins FASTA search of PDB • Sequence based search using sequence of target structure. • Can be run locally if user has fasta34 program installed or remotely using the OCA web-based service hosted by the EBI. All of the resulting PDB id codes are added to a list These structures are called model templates

33. Search for additional similar structures • Additional structure-based search (optional) • Top hit from the FASTA search is used as the template structure for a secondary structure based search. • Uses the SSM webservice provided by the EBI (a.k.a. MSDfold) • Any new structures found are added to the list. • Provides structural variation, not based on direct sequence similarity to target • Manual addition • Can add additional PDB id codes to the list, e.g. from FFAS or psiBLAST searches • Can add local PDB files

34. Multiple Alignment • After the set of PDB ids are collected in the FASTA and SSM searches, their coordinate-based sequences are collected and put through a multiple alignment with the target sequence • Aims: • Score template structures in a consistent manner, in order to prioritise them for subsequent steps • Extract pairwise alignment between template and target for use in Chainsaw step. Multiple alignment should give a better set of alignments than the original pair-wise FASTA alignments

35. Multiple Alignment target model templates pairwise alignment Jalview 2.08.1 Barton group, Dundee currently support ClustalW or MAFFT for multiple alignment

36. Template Model Scoring • Sequence identity: • Ungapped sequence identity i.e. sequence identity of aligned target residues • Alignment quality: • Dependent on the alignment length, the number of gaps created in the template alignment and the extent of each of these gaps. • The penalties given for gaps and the size of the gaps is biased so that alignments that preserve domains of the structure rather than spreading the aligned residues out score higher. The top scoring models are then used for further processing • Alignment Scoring: • score = sequence identity X alignment quality

37. Domains • Suitable templates for target domains may exist in isolation in PDB, or in combination with dissimilar domains • In case of relative domain motion, may want to solve domains separately

38. Domains • Domains search: • Top scoring templates from multiple alignment are tested to see if they contain any domains. • Uses the SCOP database. This only lists domains that appear more than once in the PDB. • The database is scanned to to see if domains exist for each of the PDBs in the list of templates • Domains are then extracted from the parent PDB structure file and added to the list of template models as additional search models for MR.

39. Multimers • Multimer search: • Search for quaternary structures that may be used as search models. • Better signal-to-noise ratio than monomer, if assembly is correct for the target. • Multimeric structures based on top templates are retrieved using the PQS service at the EBI, and added to the list of search models • PQS will soon be replaced by the use of the PISA service at the EBI (Eugene Krissinel) 1n5a SPLIT-ASU into 4 Oligomeric files of type TRIMERIC 1n5b SPLIT-ASU into 2 Oligomeric files of type DIMERIC 1n5c SYMMETRY-COMPLEX Oligomeric file of type DIMERIC 1n5d SYMMETRY-COMPLEX Oligomeric file of type DIMERIC

40. Search Model Preparation Search models prepared in four ways: • PDBclip • original PDB with waters removed, hydrogens removed, most probable conformations for side chains selected and chain ID’s added if missing. • Molrep • Molrep contains a model preparation function which will align the template sequence with the target sequence and prune the non-conserved side chains accordingly. • Chainsaw • Can be given any alignment between the target and template sequences. • Non-conserved residues are pruned back to the gamma atom. • Polyalanine • Created by excluding all of the side chain atoms beyond the CB atom using the Pdbset program Also create an ensemble model for Phaser based on top 5 models

41. Molecular Replacement and Refinement • The search models can be processed with Molrep or Phaser or both. • The resulting models from molecular replacement are passed to Refmac for restrained refinement. • The change in the Rfree value during refinement is used as rough estimate of how good the resulting model is. final Rfree < 0.35 or final Rfree < 0.5 and dropped by 20%  “success”  final Rfree < 0.48 or final Rfree < 0.52 and dropped by 5% “marginal”  “failure” otherwise • MR scores and un-refined models available for later inspection.

42. MrBUMP on compute clusters • MrBUMP can take advantage of a compute cluster to farm out the Molecular Replacement jobs. • Currently Sun Grid Engine enabled clusters are supported but support will be added for LSF and condor and any other types of queuing system if there is enough demand. • All nodes terminate when one finds a solution

43. Pre-release version of MrBUMP • Pre-release made available in Jan 06 • Simple installation • Currently runs on Linux and OSX. • Windows version almost ready. • Comes with CCP4 GUI . • Can also be run from the command line with keyword input • First citation in Obiero et al., Acta Cryst. (2006). F62, 757-760 • Regular updates (currently version 0.3.2) http://www.ccp4.ac.uk/MrBUMP

44. A few observations ... • In difficult cases, success in MrBUMP may depend on particular template, chain and model preparation method • Nevertheless, may get several putative solutions • Ease of subsequent model re-building, model completion may depend on choice of solution • First solution or check everything? • Expectation that quick solution required - in fact, most users seem happy to let MrBUMP run for long time (hours, days) • Worth checking “failed” solutions!