CCP4 Study Weekend 2013 “Molecular Replacements”

What’s new in CCP4 "What's new in CCP4 6.3.0" "PiMS Update" Ronan Keegan, STFC Rutherford Appleton Laboratory "PiMS Update" Chris Morris, STFC Daresbury Laboratory "New features in Mosflm and iMosflm" Harry Powell, MRC Cambridge "What's new in Crank" Pavol Skubak, Leiden University "Modelling around 10Å resolution using ARP/wARP" Philipp Heuser, EMBL Hamburg

Bytes Lunchtime Bytes Coot - P. Emsley/B. Lohkamp CCP4mg - S. McNicholas MrBUMP / AMPLE - R. Keegan/M. Winn Xia2/Diamond - G. Winter/P. Hathaway Aimless / Pointless - P. Evans Mosflm / iMosflm - H. Powell/O. Johnson Phaser - R. Oeffner Buccaneer / Nautilus (5th of January only)  - K. Cowtan Crank (4th of January only) - P. Skubak PiMS - C. Morris Balbes - F. Long Refmac / ProSMART - G. Murshudov/R. Nicholls Bytes

Research Complex at Harwell STFC Rutherford Appleton Laboratory What’s new in CCP4 6.3.0 Ronan Keegan Research Complex at Harwell STFC Rutherford Appleton Laboratory

About CCP4 CCP4 stands for “Collaborative Computational Project Number 4” One of several CCPs set up in the UK to advance and support scientific software development CCP4 was set up in the late 1970’s to bring together the leading developers of software in the field of protein X-ray crystallography in the UK The aim was to assemble a comprehensive collection of software to satisfy the computational requirements of the relevant UK groups

Hosted by the Science and Technology Facilities Council (STFC) Part of the Research Complex at Harwell (RCaH) next door to Diamond CCP4 funded by: Biotechnology and Biological Sciences Research Council (BBSRC) Medical Research Council (MRC) Industrial funding CCP4

Core Group (Oxford): CCP4 Maintain and support software suite Application and infrastructural software developments Collaborate with Diamond on software deployment on the beamline Educational outreach Maintaining CCP4 resources such as the CCP4 bulletin board CCP4

Core Group (Oxford): Cambridge: Cambridge CCP4 Maintain and support software suite Application and infrastructural software developments Collaborate with Diamond on software deployment on the beamline Educational outreach Maintaining CCP4 resources such as the CCP4 bulletin board Cambridge: Laboratory of Medical Biology Data processing software - Mosflm, Aimless Refinement software – Refmac Model building - Coot University of Cambridge Phaser group Cambridge CCP4

Core Group (Oxford): Cambridge: University of York: York Cambridge Maintain and support software suite Application and infrastructural software developments Collaborate with Diamond on software deployment on the beamline Educational outreach Maintaining CCP4 resources such as the CCP4 bulletin board Cambridge: Laboratory of Medical Biology Data processing software - Mosflm, Aimless Refinement software – Refmac Model building - Coot University of Cambridge Phaser group University of York: Software development – CCP4mg, Buccaneer, Nautilus CCP4 GUI2 development York Cambridge CCP4

Core Group (Oxford): Cambridge: University of York: Maintain and support software suite Application and infrastructural software developments Collaborate with Diamond on software deployment on the beamline Educational outreach Maintaining CCP4 resources such as the CCP4 bulletin board Cambridge: Laboratory of Medical Biology Data processing software - Mosflm, Aimless Refinement software – Refmac Model building - Coot University of Cambridge Phaser group University of York: Software development – CCP4mg, Buccaneer, Nautilus CCP4 GUI2 development Others include the Crank group at Leiden in the Netherlands York Cambridge CCP4

CCP4 Usage

The CCP4 software suite The CCP4 suite is a comprehensive suite of software for protein crystallography New versions of the suite are released about every 18 months New programs Major updates to existing programs Other new features such as changes to CCP4i interface New - Revisions to the current release are made available through the CCP4 updates manager

CCP4 Software Crystallisation Data Collection Data Processing and Reduction Experimental Phasing Molecular Replacement Density Modification Model Building Refinement Structure Analysis Deposition

CCP4 Releases Current Release Version - CCP4 6.3.0 Several new programs: Aimless, Dimple, AMPLE, Zanuda, ProSMART, Nautilus, Ensembler, Sculptor, Jligand In collaboration with the EMBL Hamburg, ARP/wARP is now rolled with the CCP4 suite Updates to many of the major programs Phaser, Refmac, Mosflm/iMosflm, Crank, MrBUMP New infrastructure Downloads manager and Updates Manager

Data Processing and Reduction Bytes iMosflm Pointless Determine point-group (& space group) Bytes Aimless (New) Scale-symmetry-related intensities together Produce statistics on data quality Replacement for Scala

Data Processing and Reduction Bytes Automated data-reduction – from diffraction images to merged reflection file Dimple (New) On-beamline rapid calculation of difference maps to ascertain success or failure of ligand binding

Experimental Phasing Bytes Bytes Crank Automated experimental phasing pipeline Heavy atom location through to Model Building Bytes Phaser EP Automated experimental phasing SAD phasing Combined MR and SAD Phasing

Molecular Replacement Bytes Phaser MR Automated Maximum Likelihood method for molecular replacement Twining Translational NCS Molrep Molecular Replacement Phased Rotation/Translation function Multi-copy search

Molecular Replacement - Automation Bytes MrBUMP Automated molecular replacement From model search and preparation through to initial refinement Uses Phaser and Molrep Now includes Model building options Bytes BALBES Automated molecular replacement using Molrep and Refmac Custom DB with search models monomer, domain and multimeric form

BALBES Webservice http://www.ysbl.york.ac.uk/YSBLPrograms/index.jsp or Google for“BALBES”

Molecular Replacement – Search model preparation Sculptor (New) Multi-protocol search model preparation weighted sequence similarity accessible surface area Ensembler (New) Ensemble search model generation tool from the developers of Phaser Molrep & Chainsaw Search model preparation using internal sequence alignment or provided alignment

Molecular Replacement – Ab initio search models Bytes AMPLE (beta version) Generate and prepare ab initio or de novo models using Rosetta and other software for use as search models in molecular replacement Works well for smaller, α-helical proteins (<120 residues) but has been known to work for larger targets Requires installation of several third-party applications such as Rosetta

Density Improvement Parrot Uses maximum likelihood methods and builds on original DM program Fast and fully automated Pirate Statistical-based density modification Better in some cases but slower than Parrot

Model Building Bytes Bytes Buccaneer Chain tracing by identifying connected alpha-carbon positions using a likelihood-based density target Low resolution model building Bytes Nautilus (New) Automatic model building of nucleotide structures in electron density maps Bytes Sloop (New) Loop building by finding gaps in the chain and using fragments from the Richardson's Top500 library of structures to fill the gaps

Model Building Bytes ARP/wARP Automated building of proteins RNA/DNA secondary structure side chains loops solvent ligands Now jointly distributed by CCP4 Bytes

Model Building - Coot Bytes Coot 0.7 RCrane and Cootaneer for RNA building Ligand Builder working in conjunction with ProDRG Jligand interface Wider Linux support

Model Building: ligand building Jligand Generate and regularise ligands Link ligands together and generate link description

Refinement Bytes Refmac 5.7 SIRAS and jelly body refinement refinement of multiple occupancies More reliability at lower resolution Restraints to reference structures Regularised map sharpening Automatic and local NCS restraints New Monomer library Much expanded and corrected library More than 9000 structures in first case Better conformation to PDB 3 format

Refinement Refmac 5.7/5.8 – coming soon Improved jelly body refinement Improved use of restraint information (ProSMART) DNA/RNA base pair restraints Suger/pucker restraints Simultaneous density modification and refinement (initially SAD, more general later) Better anomalous difference maps Beta ensemble refinement Multi-imputation and averaging of structure factors to desired resolution Sampling of conformational space – Gibbs sampling Estimation of individual errors of atoms – less biased electron density calculation

Refinement Bytes ProSMART (New) Structure alignment Generation of external restraints for use in refinement ProDrg Generate restraints for ligands for use in refinement and model building Zanuda (New) Analyse refinement results Check correct space group

Structure Analysis PISA Automatic inference on multimeric states from crystal packing Analysis of macromolecular interfaces and macromolecular interactions QTPisa – new QT based interface

Structure Analysis Bytes CCP4MG 2.7.0 Gesamt (New) Replacement for Superpose Improved secondary structure alignment

Report Viewer - QTRview Graphical log file viewer View graphs, tables and job summary information Launch Coot, CCP4mg and ViewHKL to view data files Can still view original log file

viewHKL Provides detailed graphical interface to reflection data Combines MTZDump with HKLView/HKLPlot Default viewer for MTZ files under ccp4i

Downloading the suite http://www.ccp4.ac.uk Download CCP4 for Windows, Mac or Linux New: Co-distribution of ARP/wARP New: Package Manager – easy installation New: 64-bit Linux version Additional software dependencies also available Download page accessible from http://www.ccp4.ac.uk

Package Manager Easy installation mechanism for CCP4 and associated packages (Coot, ARP/wARP, CCP4mg) Available on Linux, Mac and Windows Sets up system variables automatically if you wish

Package Manager Easy installation mechanism for CCP4 and associated packages (Coot, ARP/wARP, CCP4mg) Available on Linux, Mac and Windows Sets up system variables automatically if you wish

Package Manager Easy installation mechanism for CCP4 and associated packages (Coot, ARP/wARP, CCP4mg) Available on Linux, Mac and Windows Sets up system variables automatically if you wish

Update Manager Automatically install latest fixes and new features Allows for rolling back to previous versions Updates made available once every two weeks or as and when they are needed Available on Linux, Mac and Windows Recent updates: ARP/wARP Phaser 2.5.2 AMPLE Ensembler interface QTPisa Refmac 5.7.0032

Online Documentation & Resources CCP4Wiki: http://ccp4wiki.org Documentation of programs and detailed guides on how to use the software to solve structures General CCP4 Crystallography Wiki Knowledge base for protein crystallographers Linked to from the CCP4Wiki page CCP4 Bulletin Board and ccp4 helpdesk (ccp4@ccp4.ac.uk)

CCP4 Newsletter Back after a 4 year hiatus Detailed articles about many of the new software features and upcoming developments Bi-annual Archive of all previous newsletters now available online (since 1979)

CCP4 Schools/Workshops Main aim is to give education and expert help to researchers working with collected crystal data Some schools also provide expert help with collecting crystal data (APS & Hamburg) Applicants with challenging crystals and/or data are given strong consideration but this is not mandatory Software developers from CCP4, Phenix (APS), Shelx, ARP/wARP, XDS and HKL as well as others give lectures, tutorials and are also available to help with data processing and structure solution throughout the meetings http://www.ccp4.ac.uk – Courses & Events

2013 CCP4 Schools/Workshops CeBEM/CCP4 South American Workshop: Montevideo, Uruguay April 9th – 17th 2013 http://www.pasteur.edu.uy/mx2013 APS/CCP4 Summer School: APS Synchrotron, Chicago, USA June 17th – 26th 2013 http://www.ccp4.ac.uk/schools/APS-2013 BCA Summer School OIST/CCP4 School: Okinawa, Japan – November 2013 ECM 28 - Introduction to Software Development for Crystallographers Warwick University, UK August 23rd-24th Watch out for other CCP4 supported schools in the UK, Germany (Hamburg), India and China

Bytes Lunchtime Bytes Coot - P. Emsley/B. Lohkamp CCP4mg - S. McNicholas MrBUMP / AMPLE - R. Keegan/M. Winn Xia2/Diamond - G. Winter/P. Hathaway Aimless / Pointless - P. Evans Mosflm / iMosflm - H. Powell/O. Johnson Phaser - R. Oeffner Buccaneer / Nautilus (5th of January only)  - K. Cowtan Crank (4th of January only) - P. Skubak PiMS - C. Morris Balbes - F. Long Refmac / ProSMART - G. Murshudov/R. Nicholls Bytes

Acknowledgements CCP4 Core group: Andrey Lebedev, Eugene Krissinel, Charles Ballard, David Waterman, Marcin Wojdyr, Ville Uski, Karen McIntyre, Martyn Winn LMB/MRC: Andrew Leslie, Phil Evans, Garib Murshudov, Rob Nicholls, Harry Powell, Owen Johnson, Fei Long, Paul Emsley Phaser Group: Airlie McCoy, Randy Read, Rob Oeffner, Gabor Bunkoczi YSBL York: Keith Wilson, Kevin Cowtan, Liz Potterton, Stuart McNicholas, Eleanor Dodson Leiden: Raj Pannu, Pavol Skubak Others: Bernhard Lohkamp, Clemens Vonrhein, Ruslan Sanishvili, Frank Von Delft, Martin Noble, Jaclyn Bibby, Daniel Rigden, Graeme Winter, Alun Ashton, David Brown, Gwyndaf Evans, Arwen Pearson, Andrea Thorn, Tim Gruene, George Sheldrick and many more..

AMPLE Automated pipeline Generates 100’s of possible “decoys” based on a target sequence Decoys are clustered to determine most likely fold Top cluster gives rise to a set of ensemble search models for use in molecular replacement MrBUMP is used underneath to feed search models to Phaser and Molrep and on to refinement and model building