martyn winn, stfc daresbury laboratory. 1. ccp4 as a suite 2. overview of ccp4 functionality 3....
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Martyn Winn, STFC Daresbury Laboratory
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1. CCP4 as a suite2. Overview of CCP4 functionality3. Future directions
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• ~200 independent programs from a wide pool of developers• There may be several ways of doing a similar task
e.g. Molrep/Phaser, DM/Solomon/Parrot• Common routines placed in software libraries• Infrastructure to guide user in applying programs
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• Programs only accessible via command line.• Typically run via shell scripts.• A crystallographic task requires chaining several programs together.• Helped by the early adoption of standard file formats:
LCF → MTZPDBCCP4 map
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• ccp4i released 2000• Program scripts prepared by task interfaces• Convenient access to program output• Management of user data into projects
Big step forward at the time !
Liz Potterton
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• Increasing sophistication of crystallographic programs• Development of software pipelines
some intelligence, but also parameter scanning• Faster computers to support pipelines
CrankXIA2MrBUMPBalbes
Potential to do most of the work for you. But ...Always those difficult casesYou still need to get to know your structure!
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Model buildingRefinement with Refmac / ShelxlValidation with Molprobity tools
(reduce/probe)
Indexing / integrationLaue group determination with
Pointless ("QuickSymm")Scaling with Scala ("QuickScale")
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• Many ways to access CCP4 programs• Programs hidden by graphical interfaces or
become components of pipelines• CCP4 programs used by many 3rd party pipelines
Now look at available programs in more detail ...
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Phase improvement /Model (re)building
Data processing
Experimental phasing
Molecular Replacement
Model completion / refinement
Validation
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Images → unmerged MTZ file• Inspect images • Identify spots and index• Integrate• Mosflm / iMosflm
AutomationXIA2Multiple datasets → merged MTZ file"semi-automatic" (describe experiment and go)
Andrew Leslie
Harry Powell
Graeme Winter
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Identifying Laue group of datapointless
Scaling and merging reflectionsscalapointless/scala can be called from iMosflm
Deriving structure factor amplitudesctruncateI(+) / I(-) → F(+) / F(-) required for map coefficients, but intensities preferred for phasing/refinement
How good is your data ??check for twinning, incorrect spacegroup assignment, anisotropy, etc.scala, ctruncate, sfcheck
NormanStein
PhilEvans
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Substructuredetection
SubstructurePhasing
DensityModification
ModelBuilding
Raj Pannu
HeavyAtoms
Afro/Crunch2ShelxC/D
BP3 SolomonDMPirateShelxE
BuccaneerARP/wARP
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Other CCP4 phasing programs:Mlphare -
Old but fast, still used e.g. in auto-RickshawAcorn -
Ab initio structure determination using small starting structure (even randomly placed atom)
Requires atomic resolution, or artificially extended dataOasis -
Dual-space direct methods for SAD/SIRPhaser -
SAD phasingLog-likelihood-gradient (LLG) maps to find additional anomalous
sitesAlternate cycles of LLG completion and substructure refinementInput anomalous atom sites and/or partial structure
combine experimental phasing with MR
Eleanor Dodson
Randy Read
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MrBUMP
SEQSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCQPGFTGARCTENVPMKVQ
data
target sequence automationpipelines
homologous structures positioned in cell
Ronan Keegan
Balbes
Fei Long
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Automation pipelines link existing programs.
Alexei Vagin
Data analysis:No. copies in cell, rotational/translational NCSmatthews_coef, molrep, polarrfn, sfcheck, fft
Model generation:Find and edit homologous structureschainsaw, molrep
MR programs:molrep - also multi-copy searches, map and NMR search models, phased RF and TF ...phaser - also combined with SAD phasing
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Phase Improvementor Density Modification
DM / Pirate / ParrotSolomon Dmmulti - multicrystal averaging
Model buildingBuccaneerRapperSloop - loop building
CootSequins - validates model side chains against electron density
Kevin Cowtan
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Refinement with Refmac:Optimise model parameters e.g. {x,y,z}You choose form of model (atomic vs group parameters, scaling function, etc.)Refmac included in Arp/Warp, Buccaneer, Coot, etc.Latest developments:
• Twin refinement - automatic detection of significant twin operators and refinement of twin fractions• Refinement against SAD data - anomalous scatterers included in XYZIN• Automatic weighting Xray vs geometry• External restraints, used in latest ARP/wARP
Garib Murshudov
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CootInspection of models and mapsModel buildingValidationReal space refinementRefinement with RefmacNCS toolsSequence assignmentInclusion of Molprobity tools
Paul Emsley
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Model:appropriate level of detail?data/parameter ratiorestraintsgeometry
Fit to data:R / RfreeChoice of free reflectionsElectron density support for ligands
rampage
sfcheck
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CCP4mgNew QT versionPublication quality imagesWide variety of representationsSurfaces and electron density supportedEasy to produce moviesTransfer of structures/views to and from Coot
Liz Potterton
Stuart McNicholas
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A good idea!!• More datasets, collected faster• Can you remember what x2_t7_refmac1.mtz from Dec 08 is?• Automation schemes produce files from many trials• External requirements, e.g. BBSRC Data Sharing policy, journal requirements
Chris Morris
Rob EsnoufTools available:PiMS & xtalPiMSsynchrotron databases, e.g. ISPyBccp4i projectslab databasespublic databases e.g. TARDIS for images
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wiki
You Are Here!
check here for current known issues
ccp4bb
courses
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New programs:PRODRG_ccp4, RAPPERtk, SLOOP, JLigand
New GUI:• Greater support for automation• Better presentation of results
Data management:• Support for back-end databases
Distribution of CCP4:• Monolithic suite & individual program updates• Incremental updates e.g. via rsync• Web services - CPU-intensive applications (e.g. MrBUMP) and/or utilities
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Eugene Krissinel George Pelios Natalie Zhao
Charles Ballard
Aided by the old hands ...
Ronan Keegan
Located in the Research Complex at Harwell
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Small-angle X-ray scattering (SAXS)
Dawson et al., Structure 15, 942–954 (2007)
Cryo-electron microscopy
Ranson et al. (2001) Cell 107, 869-879
Kaestner et al., J. Struct. Biol., 167 117–128 (2009)
MolecularDynamicsSimulations
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Science, 325, 1544 (2009)
Structures in a cellular context - combining structural genomics and systems biology.
System of 478 proteins:120 expt.336 automatic modelling3 manual modelling19 TM proteins
503 metabolites645 reactions
non-random distribution of folds
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DL/RAL/DLS:Charles Ballard, Ronan Keegan, Norman Stein, Peter Briggs, Eugene Krissinel, George Pelios, Natalie Zhao, Graeme Winter, Chris Morris
MRC-LMB:Phil Evans, Andrew Leslie, Harry Powell
Cambridge:Randy Read, Airlie McCoy, Gabor Bunkoczi
York:Eleanor Dodson, Kevin Cowtan, Garib Murshudov, Alexei Vagin, Liz Potterton, Stuart McNicholas, Fei Long
Oxford:Paul Emsley, Martin Noble, Rob Esnouf
Leiden:Raj Pannu, Pavol Skubak