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
psiBLAST - iterative searching
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

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. |F|new = |F|input *exp(-Badd*s2)*(1-exp(-Boff*s2)
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 > solution <1.8 and > maybe solution <1.5 and > 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:

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-score Have I solved it?
less than 5 no
unlikely
possibly
probably
more than 8 definitely
RFZ = RF Z-score
TFZ = TF Z-score

26. Phaser refers to search models as “ensembles”
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:

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. Molecular Replacement
The Pipeline
Target MTZ
&
Sequence `
Target
Details `
Template
Search `
Check scores and exit or select the next model
Model
Preparation `
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 Barton group, Dundee
currently support ClustalW or MAFFT for multiple alignment

36. Template Model Scoring
Alignment Scoring:
score = sequence identity X alignment quality
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

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 search: Domains
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. Search for quaternary structures that may be used as search models.
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 final Rfree < 0.35 or
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,
Regular updates (currently version 0.3.2)

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!