28 th March 2007 MrBUMP – Automated Molecular Replacement Ronan Keegan, Martyn Winn CCP4, Daresbury Laboratory

28 th March 2007 Aim of MrBUMP Automated framework for Molecular Replacement Particular emphasis on generating variety of search models Wraps Phaser, Molrep and Acorn Uses a variety of helper applications (eg Chainsaw) and bioinformatics tools (eg FASTA, Mafft) Uses on-line databases (eg PDB, Scop) In favourable cases, gives “one-button” solution In unfavourable cases, suggests likely search models for manual investigation

28 th March 2007 Pipeline ` ` ` ` Target MTZ & Sequence Target Details Template Search Model Preparation Molecular Replacement & Refinement Check scores and exit or select the next model

28 th March 2007 Template Search Sequence based search (FASTA) Secondary structure based search (SSM) Domain search (SCOP) Identification of possible multimers (PQS & PISA) Users can also enter their own templates by ID or from locally held files.

28 th March 2007 Model Preparation Search models can be prepared for MR in several ways –Chainsaw – non-conserved residues are pruned (sequence provided) –Molrep – pruning of non-conserved side-chains (internal sequence alignment) –Polyalanine – all side chain atoms are pruned beyond the CB atom –PDBclip – models are not modified An ensemble of the best models is also created for Phaser

28 th March 2007 Molecular Replacement & Refinement For each search model, MR is 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 Allow Molrep / Phaser to set resolution limits and weights After MR, models are passed to Refmac for restrained refinement otherwise final Rfree < 0.48 or final Rfree < 0.52 and dropped by 5% final Rfree < 0.35 or final Rfree < 0.5 and dropped by 20%    “ success” “marginal” “failure”

28 th March 2007 MrBUMP on clusters MrBUMP is usually run on a desktop However, 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 other types of queuing system (e.g. LSF, Condor) if there is enough demand. Job control: All nodes terminate when one finds a solution

28 th March 2007 MrBUMP Output Currently produces a long log file listing search results, model preparation steps, summaries from each MR and refinement job and relevant references for programs used. Not ideal, there’s a lot of information to trawl through. Summary of results now provided at the end of log file. Future versions will provide results in marked-up web page format for more clarity.

28 th March 2007 MrBUMP pre-release Beta version first released in Jan’ 06 (current version is 0.3.3) Currently supported on Linux and Mac OSX, Windows version will be available when included in suite. Will be included in next release of CCP4 (version 6.1) MrBUMP paper to be published in Acta Cryst. D in April ‘07 First citations in Obiero et al., Acta Cryst. (2006). F62, ; El Omari et al., Acta Cryst. (2006). F62,

28 th March 2007 New features Run Acorn after refinement for phase improvement (high resolution data) Support for searching in enantiomorphic spacegroups. Users can now specify template models by PDB ID or add local PDB files. “Generate models only” option. XML Output. Additional multiple alignment programs supported – Tcoffee and Probcons.

28 th March 2007 New feartures: Smartie Peter Briggs’ “Smart” CCP4 log file browser. Robust method for reading and extracting information from log files. Parses log files and picks out tables, header information and various other pieces of information. Very useful when generating XML output from a automation wrapper script such as MrBUMP.

28 th March 2007 Future versions Improvements to multimeric search models (using PISA) Supplement multiple alignment with additional sequences and/or structural information Model completion and/or re-building Target complexes.

28 th March 2007 Conclusions Test cases and the examples demonstrated the utility of trying a range of search models, a protocol that can only be attempted adequately by automation. MrBUMP is not meant to compete with careful analysis of the data and model by an experienced crystallographer. However, it may succeed in difficult cases by finding a combination of models and protocols that would not otherwise have been tried. In more straight forward cases the advantage is simply one of convenience. CCP4 web server log files indicate that it’s being used at least 3- 4 times a day.

28 th March 2007 Some thoughts about automation MrBUMP started out as a project to develop a one-button solution for MR (e-HTPX). It has evolved to be more interactive if required whilst retaining the one-button functionality. Users like to have the donkey work done automatically but prefer controlled/transparent automation with the ability to both stop and start the program as they please at given points in the pipeline. Also like to have a large degree of control of the input parameters.