MrBUMP Molecular Replacement with Bulk Model Preparation

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MrBUMP Molecular Replacement with Bulk Model
Preparation

• Ronan Keegan, Martyn Winn
• CCP4 group, Daresbury Laboratory
• Como May 23rd 2006

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The aim of Mr Bump

• 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)

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Target MTZ Sequence
Target Details

• Currently
• Number of residues and molecular weight
• Matthews Coefficient.
• Estimated number of molecules in the a.s.u.

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Target MTZ Sequence
Target Details

Model Search
Generate a list of structures that are possible
templates for search models
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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.
• Local search is done against the complete list of
  PDB sequences derived from ATOM records in the
  PDB structure files.
• All of the resulting PDB id codes are added to a
  list
• Not interested in the alignment to target at this
  stage.

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Search for similar structures

• Secondary 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.
• Any new structures found that arent included in
  the list of matches from the FASTA search 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

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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

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

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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

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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.

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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
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Target MTZ Sequence
Target Details

Model Search

Model Preparation
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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 IDs 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

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Search Model Preparation

• Ensemble for Phaser
• Top scoring search models are superposed to
  create a ensemble model.
• This may provide a better search model than any
  of the individual models on their own.
• Currently the default is to use the top 5 scoring
  search models but plan to create dynamically
  based on MW and RMSDs of constituent search models

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Target MTZ Sequence
Target Details

Model Search

Model Preparation

Molecular Replacement Refinement
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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 to determine how good the resulting model
  is.
• If the final value for Rfree is less than 0.35 or
  it is less than 0.5 and has fallen by more than
  20 from the initial Rfree, a solution is deemed
  to have been found.
• Models that produce an Rfree below 0.5 and the
  value looks to be falling will be highlighted as
  marginal solutions that are worthy of further
  investigation if no solution is found using the
  other search models.

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Target MTZ Sequence
Target Details

Model Search

Model Preparation
Serial mode Check Scores and exit or select the
next model

Molecular Replacement Refinement
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Target MTZ Sequence
Target Details

Model Search

Model Preparation
Parallel mode Start multiple MR jobs and exit
when one finds a solution





Molecular Replacement Refinement
Molecular Replacement Refinement
Molecular Replacement Refinement
Molecular Replacement Refinement
Molecular Replacement Refinement
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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.

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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
• Good deal of interest and some successes
• Regular updates (currently version 0.3)

http//www.ccp4.ac.uk/MrBUMP
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Example 1
1vlw 3 chains of 205aa. Data in C2221 to 2.3Å.
Using Molrep.
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Example 2
Anon
MrBUMP marginal solution
solution used
yes arp/warp builds and docks entire
molecule no arp/warp fails wrong MR solution
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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!

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Future developments

• Windows support (almost done)
• Complexes (in progress)
• Processing of multiple target sequences
• Improved alignment
• Multiple alignment against larger sequence
  database
• Alignment from profile-based search
• User-supplied alignment
• Incorporate PISA multimer determining service
  (in progress)
• Model generation
• Identification of flexible loops
• Normal mode generated conformations
• Develop web-service version to allow CCP4i users
  to run jobs on CCP4 cluster

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Acknowledgements

• Ronan Keegan, CCP4 _at_ Daresbury
• Thanks to authors of all underlying programs and
  services
• Other suggestions from
• Dave Meredith, Graeme Winter, Daresbury
  Laboratory.
• Eugene Krissinel, EBI, Cambridge.
• Eleanor Dobson, YSBL, York University
• Geoff Barton, Charlie Bond, University of Dundee
• Randy Read, Airlie McCoy, Cambridge
• Funding
• BBSRC (e-HTPX, CCP4)

http//www.ccp4.ac.uk/MrBUMP