Evaluating the Solution from MrBUMP and Balbes

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Evaluating the Solution from MrBUMP and Balbes

• Ronan Keegan, Fei Long, Martyn Winn, Garib
  Murshudov
• STFC Daresbury Laboratory
• York Structural Biology Laboratory, University of
  York

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PDB Depositions (1999-2009)
1999
2009
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What are MrBUMP and Balbes?

• Automated molecular replacement (MR) and
  beyond....
• MrBUMP
• Brute force approach - try a variety of search
  models prepared in several different ways in MR
• Balbes
• Identifies the best possible search model to be
  used in MR by using a specially grafted version
  of the PDB optimised for use in MR

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MrBUMP

• An automation framework for Molecular
  Replacement.
• Particular emphasis on generating a variety of
  search models.

• Wraps Phaser and/or Molrep.
• Uses a variety of helper applications (e.g.
  Chainsaw) and bioinformatics tools (e.g. Fasta,
  Mafft) to generate search models
• Makes use of up-to-date on-line databases (e.g.
  PDB, Scop)

• In favourable cases, gives one-button solution
• In Complicated Cases, will suggest likely search
  models for manual investigation (lead generation)

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The Pipeline

Target Details
Target MTZ Sequence

Template Search
N templates

Model Preparation
Check scores and exit or select the next model
N x M models

Molecular Replacement Refinement
Phase Improvement
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Search for model templates

• FASTA search of PDB
• Sequence based search using sequence of target
  structure

All of the resulting PDB id codes are added to a
list
These structures are called model templates

• Other templates from
• SSM search using top hit from the FASTA search
• Can add additional PDB id codes to the list
• Can add local PDB files

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Multiple Alignment step
target
model templates
pairwise alignment (used in Chainsaw)
Jalview 2.08.1 Barton group, Dundee
currently support ClustalW, MAFFT, probcons or
T-coffee for multiple alignment
Model template scoring score sequence
identity X alignment quality
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Domain 1
X
Domain 2
SCOP
PQS/PISA
Multimers Better signal-to-noise ratio than
monomer, if assembly is correct for the target.
Domains e.g. if relative domain motion
template chains
superpose
Ensembles Create ensemble of top search models,
for use in additional run of Phaser. Need to be
similar in MW and rmsd
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Search Model Preparation

• Search models prepared in four ways
• PDBclip
• original PDB with waters removed, most probable
  conformations selected and format tidied (e.g.
  chain ID added)
• Molrep
• Molrep model preparation function which aligns
  the template sequence with the target sequence
  and prunes 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

more side chain truncation
deal with deletions
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Molecular replacement step

• Running MR
• For each search model, MR done with Molrep or
  Phaser or both.
• MR programs run mostly with defaults
• MrBUMP provides LABIN columns, MW of target,
  sequence identity of search model, number of
  copies to search for, number of clashes tolerated

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Molecular replacement step

• MR output
• MR scores and un-refined models available for
  later inspection
• ? assess quality of solution, extent of model
  bias
• MrBUMP doesnt use MR scores, but checks for
  output file with positioned model, and passes to
  refinement step

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Testing enantiomorphic spacegroups

• 11 pairs of enantiomorphic spacegroups containing
  screw axes of opposite handedness, e.g. P41 and
  P43)
• usually both need to be tested in MR
• correct spacegroup indicated by TF and packing

• MrBUMP can test both in Molrep and/or Phaser.
• For each search model, best MR results used to
  fix spacegroup for subsequent steps.
• Discrimination good for good search model
  correct MR solution

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Inclusion of fixed models

• MrBUMP will now accept one or more positioned
  models.
• These are included as fixed models in all MR
  jobs.

• Thus, solve complexes through consecutive runs
  of MrBUMP.
• Automation of this in progress ....

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Restrained refinement step

• 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 lt 0.35 or final Rfree lt 0.5 and
dropped by 20
?
success
?
final Rfree lt 0.48 or final Rfree lt 0.52 and
dropped by 5
marginal
?
poor
otherwise
conservative .....
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Refinement results
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Summarised results..
Best search model so far and file location for
this model
List of sorted results so far
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Example (thanks to Elien Vandermarliere)
Target is an arabinofuranosidase Data to 1.55Å in
P212121
Small C domain (144 res) solved with 34 seq
ident model (1w9t_B_MOLREP best out of 4
solutions)
With C domain solution fixed, large N domain (345
res) solved with 28 seq ident model (1gyh_C_CHNSA
W best out of 7 solutions)
Acorn CC increases from 0.04 to 0.18 This step
part of MrBUMP ARP/wARP then builds 457/493
residues to R/Rfree 0.185/0.225
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MrBUMP output

• Log file gives summary of models tried and
  results of MR
• May get several putative solutions
• Ease of subsequent model re-building, model
  completion may depend on choice of solution
• Worth checking poor solutions
• Top solution available from ccp4i

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Using MrBUMP

• Part of CCP4 suite
• CCP4i GUI

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(Balbes)
Fei Long, Alexei Vagin, Paul Young, Garib
Murshudov YSBL, York University
http//www.ysbl.york.ac.uk/fei/balbes/index.html
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Balbes

• Balbes uses a reorganised version of the PDB
  database customised for use in MR.
• Has its own classification of domains most
  suitable for use in molecular replacement
• Database also includes classification of possible
  oligomers based on the basic structures.
• Built upon Molrep for molecular replacement and
  Refmac for doing the refinement

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Balbes Database

• Cut down version of the PDB keeping only single
  copies of unique folds.
• Where 2 entries have sequence ID gt 80 and rmsd
  lt 1 angstrom, the entry with the highest
  resolution is retained
• More than 20000 entries
• More than 23000 domains classified
• For each sequence additional information is
  stored including secondary structure information,
  number of domains and potential to form multimers
• Flexible loops are cut from the stored structures
• Multimers are classified with information from
  the EBI PISA service

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Balbes Workflow
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Assemblies

• Fully automatic support for handling assemblies
• Simply provide additional sequences in input
  sequence file for each additional component in
  the target data
• Balbes will first look for assemblies in its
  database containing all of the sequences but if
  not find it will look for subsets of those
  sequences

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Using Balbes

• Through CCP4i
• included in Linux and Mac OSX releases
• Simple CCP4i interface - provide sequence and
  structure factor file (MTZ)
• Enter multiple sequences in input file for
  complexes

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Using Balbes

• Through the YSBL Web Server
• Balbes is one of several programs available to
  use via the web at the York University YSBL Web
  Server
• Create an account for access

http//www.ysbl.york.ac.uk/YSBLPrograms/index.jsp
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Balbes Web Server

• Enter MTZ and sequence information as a file or
  paste sequence
• Option to check all related space groups
• Option to submit resulting map to ARP/wARP server
  for final model building

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Web Server Results

• Summary of processing for each spacegroup
• Final best result highlighted
• For each spacegroup log file and all output files
  are made available for download (5 days)
• If user opted to use ARP/wARP server a link to
  the ARP/wARP results is provided

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Balbes Output

• Spacegroup specific output
• Download files
• Main summary file showing results of MR and
  refinement for each template model that was used.
• Q value scoring

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Balbes the statistics

• In 2006, 67 of structures deposited in the PDB
  where solved by MR
• Balbes improved this to 80 using
• A better organised database (for MR)
• A better choice of protocols for tackling the MR
  problem
• Improved algorithms in MR and refinement programs

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Balbes Usage

• 90 structures in PDB referencing use of Balbes as
  part of structure solution
• 35 citations
• More than 5000 users of web server version of
  program
• 150 users per month

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Summary

• MrBUMP
• Brute force approach, try everything
• May give lead in borderline cases as well
  automating straight-forward cases
• All results are easily accessible and summarised
• Balbes
• Efficient and quick at solving structures that
  have reasonable homologues
• Deals with assemblies/complexes fully
  automatically
• Combined with ARP/wARP it can take structure
  nearly all the way to completion
• In conclusion
• Try Both!! Compute cycles are very cheap.

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Acknowledgements

• Fei Long, Alexei Vagin, Paul Young, Andrey
  Lebedev, Garib Murshudov Balbes and Refmac
• Martyn Winn, MrBUMP
• Airlie McCoy, Randy Read Phaser
• Alexi Vagin Molrep
• Norman Stein Chainsaw and Ctruncate
• CCP4 Core team Support and Testing

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Downloading Balbes Database

• Balbes database available as a separate download
  (1.6GB)

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PDB Statistics

• The number of structures in the PDB is increasing
  rapidly year on year
• By the end of 2009 there were gt 62000 structures
  deposited
• 7449 structures were deposited in 2009 (12 of
  the total)
• MrBUMP and Balbes seek to exploit this wealth of
  data to improve the success rate of molecular
  replacement