1. CCP4 helpdesk: ccp4@dl.ac.uk What’s New in CCP4 6.0? Martyn Winn CCP4 CCLRC Daresbury Laboratory Cheshire

2. CCP4 helpdesk: ccp4@dl.ac.uk Background

3. CCP4 helpdesk: ccp4@dl.ac.uk Collaborative Computational Project Number 4 CCP4 software suite: Scope: covers data processing through to refinement and validation Modular: lots of individual programs sharing data via common file formats Keywords control program function and provide additional data Redundancy in choice of programs Also has a graphical interface CCP4i Version 6.0 was released in February 2006 Version 6.0.1 was released in June 2006 CCP4 website: http://www.ccp4.ac.uk/ Old workshops and course: http://www.ccp4.ac.uk/ccp4course.php Bulletin Board http://www.ccp4.ac.uk/ccp4bb.php Newsletters: http://www.ccp4.ac.uk/newsletters.php

4. CCP4 helpdesk: ccp4@dl.ac.uk Scope of the CCP4 Suite ~200 (?) programs covering all aspects of PX structure determination Relatively small subset are significant for most users, e.g.: Data processing and reduction ( mosflm, scala, truncate...) Experimental phasing ( mlphare, acorn, rantan, oasis, bp3...) Molecular replacement ( amore, molrep, phaser ) Density modification ( dm, solomon, pirate ) Refinement ( refmac5, sketcher ) Graphics and building ( ccp4mg, coot ) Validation and analysis ( sfcheck, procheck …) Utility and conversion programs (e.g. fft, cad, revise …)

5. CCP4 helpdesk: ccp4@dl.ac.uk Graphical User Interface CCP4i Sits on top of programs Hides details of individual programs User not locked in Allows mix-and-match approach Philosophy: “Task-driven” rather than “program-driven” Interfaces to tasks use “natural language” approach e.g. Key features: Easy-to-use interfaces to major programs and utilities Integrated tools for file viewing and basic project management Customisable Integrated help system Some non-CCP4 programs also interfaced (e.g. ARP/wARP, SHELX)

6. CCP4 helpdesk: ccp4@dl.ac.uk Navigating the suite Key questions: Which program(s) do I need? How do I run it/them? Documentation (http://www.ccp4.ac.uk/docs.html): Roadmaps Tutorials (html) Individual program documentation (html) Function index General background on twinning, reindexing, … Postscript manual Slightly dated but still useful Content distinct from program documentation Runnable example scripts $CCP4/examples/unix/runnable

7. Automated download and install for 6.0 Downloads divided into a number of packages: Core CCP4 (equivalent to v5.0) Phaser and CCTBX CCP4mg Coot CHOOCH plus dependencies (“tools”): Tcl/Tk/BLT, Python Installation: For Macintosh and Windows: use the self-extracting packages On Windows: remove any previous installation first admin privileges are required to install For Linux, Unix variants use install.sh script in download archive to install automatically

8. CCP4 helpdesk: ccp4@dl.ac.uk CCP4 6.0

9. CCP4 helpdesk: ccp4@dl.ac.uk Updated programs in core CCP4 6.0 New versions of major programs: REFMAC 5.2.0019 MOLREP 9.0.09 SFCHECK 7.0.18 MOSFLM 6.2.5 Updates to minor programs: PDBCUR (PDB editing tool): Summarise model content, choose most likely conformation, delete hydrogens/atoms based on occupancy threshold … MATTHEWS_COEFF : Outputs Kantardjieff & Rupp resolution-based probabilities for number of molecules in ASU

10. CCP4 helpdesk: ccp4@dl.ac.uk New programs in core CCP4 Superpose (Eugene Krissinel, EBI) secondary-structure based alignment program aligns first on secondary structure then on Cα atom positions outputs the transformation matrix plus per-residue listing of the quality of the match & identification of the secondary structure BP3 (Raj Pannu, Leiden) heavy-atom refinement and phasing program uses multivariate likelihood functions to refine and phase heavy and/or anomalously scattering substructure (i.e. S/MIR(AS) and S/MAD) Pdb_merge “Jiffy” program to combine the content from two PDB files

11. CCP4 helpdesk: ccp4@dl.ac.uk Kevin Cowtan, York Clipper a set of crystallographic software libraries developed by Kevin Cowtan see http://www.ysbl.york.ac.uk/~cowtan Pirate statistical phase improvement (replacement for dm ) Nb: executables are actually called cpirate and cmakereference Other Clipper utilities in 6.0 see e.g. the Clipper Utilities module of CCP4i New core programs: pirate

12. CCP4 helpdesk: ccp4@dl.ac.uk General categorisation of protein/solvent (order/disorder vs dense/sparse) Uses phases from known reference structure with features similar to unknown structure (use makereference program for this) Doesn’t require knowledge of solvent content Pirate NCS. Novel feature: each molecule gets its own mask based on agreement with NCS. Pirate in 6.0. Slower than DM. Dependent on quality of HL coeffs, because they describe noise used to generate noisy reference structure. For large reference structures, not so sensitive to choice. Might want something else for protein-DNA. Otherwise 1ajr. New version of pirate in August New core programs: pirate

13. CCP4 helpdesk: ccp4@dl.ac.uk MR model preparation: chainsaw Molecular replacement model preparation utility that mutates a template PDB file according to a sequence alignment. Features: –Prunes non-conserved residues back to the gamma atom –Removes “unlikely” parts of model –Preserves more atoms than in polyalanine model Example of 1mr6 used as a template for 1tgx (38% sequence identity) Unmodified templateChainsaw templatePolyalanine template

14. CCP4 helpdesk: ccp4@dl.ac.uk New core CCP4i tools and features “Greyed out” tasks indicate that you need to install underlying software first e.g. SHELX Database Search/Sort Tool Quick switch between projects Top level help split into topics Custom job view colours

15. CCP4 helpdesk: ccp4@dl.ac.uk New interfaces in CCP4i Crank : Suite of programs for automated structure solution based on the CCP4i infrastructure Current version supports SAD, SIR and SIRAS Covers steps from scaled/merged data to density modification “Translucent box” – also intended as teaching aid Shelx_cde: Interface to the Sheldrick SHELX C/D/E programs for heavy atom location, phasing and refinement Allows programs to be run as a pipeline or individually Performs file format conversions and extracts useful graphs from output Not all options currently interfaced NB#1 scala interface (Scale and merge intensities task) can now outputs data in a form suitable for SHELX and SOLVE NB#2 still need to fetch and install SHELX programs yourself

16. CCP4 helpdesk: ccp4@dl.ac.uk New/updated interfaces in CCP4i Interfaces for new programs: Pirate and Clipper utilities BP3 (in the Experimental Phasing module) Superpose Chainsaw (“Create Search Model” in Molecular Replacement module) Also a launcher task for CCP4mg launcher (see later) Plus updates to existing tasks, for example: areaimol (accessible surface area calculation): generate pairwise area differences for arbitrary groups of chains matthews_coef and pdbset/pdbcur: accommodate new/extended functionality

17. CCP4 helpdesk: ccp4@dl.ac.uk CCP4mg : presentation graphics & analysis CCP4 molecular graphics package compatible with CCP4 environment focused on structure analysis and presentation graphics/movies view coordinates (ball & stick, ribbons, spheres…) show surfaces (electrostatic, solvent accessible …) display maps (electron density…) examine local environment, contacts, packing diagrams structure superposition CCP4 6.0 includes version 0.12 See http://www.ysbl.york.ac.uk/~ccp4mg/

18. CCP4 helpdesk: ccp4@dl.ac.uk Phaser 1.3 Phaser website: http://www-structmed.cimr.cam.ac.uk/phaser/ / Maximum likelihood based phasing Current version has methods for molecular replacement (experimental phasing functions under development) uses ensembles of many models weighted by estimates of similarity Allows searching for multiple molecules in multiple spacegroups Requires CCTBX libraries (included) Full CCP4i interface also provided

19. CCP4 helpdesk: ccp4@dl.ac.uk Model building & validation : Coot 0.0.33 Platform for semi-automated model building and validation tools, including: idealisation real-space refinement manual rotation/translation rigid body fitting ligand and solvation search mutations rotamer analysis & fitting Ramachandran plots and other validation tools e.g. geometry analysis interface to REFMAC See also http://www.ysbl.york.ac.uk/~emsley/coot/

20. CCP4 helpdesk: ccp4@dl.ac.uk Calculate f’/f’’: Chooch Determine values of anomalous scattering factors from raw fluorescence spectra Pinpoint positions of f’ minimum and f’’ maximum Chooch version 5.02 in CCP4 6.0 Not currently interfaced in CCP4i (coming)

21. CCP4 helpdesk: ccp4@dl.ac.uk Beyond CCP4 6.0

22. CCP4 helpdesk: ccp4@dl.ac.uk Ongoing developments within CCP4: programs Available now: MrBUMP : automated Molecular Replacement http://www.ccp4.ac.uk/MrBUMP iMOSFLM : new interface to MOSFLM Other on-going developments within core group: HAPPy (Heavy Atom Phasing in Python): an automated Experimental Phasing pipeline CCP4i/BIOXHIT project database: expanding the data stored by CCP4i and making it available to other programs XIA-DP : an automated data processing system

23. CCP4 helpdesk: ccp4@dl.ac.uk iMOSFLM : new MOSFLM user interface MOSFLM is the CCP4 diffraction image integration program Suffered from a clunky X11 interface – difficult to use (and ugly) Meant that MOSFLM won’t work on MS Windows iMOSFLM : completely redesigned user interface Modern, better controls, easier to learn and use Beta version available at: http://alf1.mrc-lmb.cam.ac.uk/~geoff/mosflm/

24. CCP4 helpdesk: ccp4@dl.ac.uk Some future programs More Clipper-based programs (Kevin Cowtan): Buccaneer : statistical chain-tracing with ML target Privateer : refine crude low-resolution structures See http://www.ysbl.york.ac.uk/~cowtan/ Pointless : Spacegroup determination/(re)indexing for unmerged reflection data (Phil Evans) Othercell: For given cell and lattice type, list alternative indexing and/or cell parameters (Phil Evans) BALBES : Automated MR using REFMAC, MOLREP and SFCHECK (Garib Murshudov, Alexei Vagin and Fei Long)

25. CCP4 helpdesk: ccp4@dl.ac.uk TLS Refinement

26. CCP4 helpdesk: ccp4@dl.ac.uk ● TLS parameterization allows to partly take into account anisotropic motions at medium/low resolution (> 3.5 Å) ● TLS refinement might improve refinement statistics of several percent ● TLS refinement in REFMAC5 is fast and therefore can be used routinely ● TLS parameters can be analyzed to extract physical significance TLS Refinement

27. CCP4 helpdesk: ccp4@dl.ac.uk Displacement of atom (position r w.r.t. origin O) in rigid body: u  t +  r Achieve parameter reduction by constraining atomic displacements to rigid-body model (other models possible):

28. CCP4 helpdesk: ccp4@dl.ac.uk TLS parameters Experiment yields mean square displacements: U TLS  = T + S T  r - r  S - r  L  r T =, L = and S = describe mean square Translation and Libration of rigid body and their correlation (Screw-motion). T  6 parameters, L  6 parameters, S  8 parameters (trace of S is undetermined)

29. CCP4 helpdesk: ccp4@dl.ac.uk Atomic parameters from TLSANL TLS tensors  U TLS for atoms in group (ANISOU records in XYZOUT) U TLS  B TLS and anisotropy A Also have individually refined B res from Refmac B TOT = B TLS + B res (ATOM records in XYZOUT) keyword ISOOUT determines which contribution recorded in ATOM/ANISOU lines