Bucca aneer The buccaneer softwarre for automated model building g of p protein stru uctures across a broad range of re esolutions. Kevin Cowtan C YSBL, Unive ersity of York cowtan@ysb bl.york.ac.uk Kevin Cowtan, [email protected] Buccaneer Statistical model building software based on the use t construct likelihood of a reference structure to targets for protein feature es. N New since i 2007 2007: V Version i 1.0 0 ffor CCP4 v6.1 61 Buccaneer-Refmac pipelline NCS auto-completion Improved p sequencing q g Kevin Cowtan, [email protected] Buccaneer: Method d • Compare simulated map and kno own model to obtain likelihood target, then search for this target in n the unknown map. Reference structure: Work structure: LLK K Kevin Cowtan, [email protected] Buccaneer: Method d Compile statistics for refe erence map in 4A sphere about C => LLK target. g Use mean/variance. mean/variance 4A sphere about Ca also used by 'CAPRA' Ioeger et al. (but different target function). Kevin Cowtan, [email protected] Buccaneer 10 stages: Find candidate C-alpha p p position ns Grow them into chain fragments Join and merge the fragments, resolving branches Link nearby N and C terminii (iff possible) Sequence the chains (i.e. dockk sequence) Correct insertions/deletions Filt based Filter b d on poor d density it NCS Rebuild to complete NCS S copies of chains Prune any remaining clashing chains c Rebuild side chains Kevin Cowtan, [email protected] Buccaneer Use a likelihood function ba ased on conserved density features. The same likelihood function is used several times. This makes the program very simple (<3000 lines), and the whole w calculation works over a range of resolutions. Finding, growing: Look for C C-alpha alpha environment Sequencing: ALA Look for C-beta C environment CYS Kevin Cowtan, [email protected] HIS MET THR ... x20 Buccaneer Case Study: S A difficult loop in a 2.9A map, m calculated using real data from m the JCSG. Kevin Cowtan, [email protected] Find candidate C-a alpha positions Kevin Cowtan, [email protected] Grow into chain fra agments Kevin Cowtan, [email protected] Join and merge ch hain fragments Kevin Cowtan, [email protected] Sequence the cha ains Kevin Cowtan, [email protected] Correct insertions//deletions Kevin Cowtan, [email protected] Prune any remaining clashing chains Kevin Cowtan, [email protected] Rebuild side chain ns Kevin Cowtan, [email protected] Comparison to the e final model Kevin Cowtan, [email protected] Buccaneer Model completion uses “Latteral growing”: Grow sideways y from existing g chain fragments g by y looking g for new C-alphas at an appropria ate distance “sideways” from the existing chain: Kevin Cowtan, [email protected] Unmodeled densitty Kevin Cowtan, [email protected] Lateral growing likkelihood function Kevin Cowtan, [email protected] New C-alpha cand didates Kevin Cowtan, [email protected] Resulting model Kevin Cowtan, [email protected] Buccaneer: Resultss Model completeness not very de ependent on resolution: Kevin Cowtan, [email protected] Buccaneer: Resultss Model completeness dependentt on initial phases: Kevin Cowtan, [email protected] Buccaneer Cycle y BUCCANEER and REFMAC for most complete model Single run of BUCCANEER onlyy ((more options) p ) quick assessment/advanced use Latest versions from York as above Faster and better results Kevin Cowtan, [email protected] Buccaneer Kevin Cowtan, [email protected] Buccaneer What it does: Trace protein chains (trans ns-peptides peptides only) Link across small gaps Sequence q Apply NCS Build side chains (roughly) Refine (if recycled) WORK AT LOW RESOLUTIONS 3 6A (4A?) with 3.6A ith good d pha h ses Kevin Cowtan, [email protected] Buccaneer What it does not do (yet): Cis Cis-peptides peptides Waters Ligands g Loop fitting Move C-alpha to fit rotam mer Kevin Cowtan, [email protected] Buccaneer What you need to do afterw wards: Tidy up with Coot. Or ARP/wARP when reso olution is good. Buccaneer/ARP/wARP be etter+faster than ARP/wARP. T i lC Typical Coot steps: Connect up any broken ch hains. Use density fit and rotame er analysis to check rotamers rotamers. Check Ramachandran, molprobity, m etc. Add waters, ligands, checck un-modeled blobs.. R Re-refine, fi examine i diff differe ence maps. Kevin Cowtan, [email protected] Kevin Cowtan, [email protected] Buccaneer: Latest Buccaneer 1.2 Use of Se atoms, MR mo odel in sequencing. Improved numbering of output o sequences (ins/del) Favour more p probable sidechain rotamers Prune clashing side chains Optionally fix the model in the ASU Performance improveme ents (1.5 x) Including 'Fast mode' (2-3 x for good maps) Multi-threading M lti th di Kevin Cowtan, [email protected] Buccaneer: Summa ary A simple, fast, easy to use (i.e. ( MTZ and sequence) method of model building g which is robust against g resolution. d to 3.7A when User reports for structures down phasing is good. oved by iterating with Results can be further impro refinement in refmac (and d in future future, density modification). Proven on real world proble ems. ems Kevin Cowtan, [email protected] Achnowle edgements g Help: H l JCSG data archive: www.jcsg.org j g g Eleanor Dodson n, Paul Emsley, Randy Read Read, Clemens Vonrhein Vonrhein, Raj Pannu F di Funding: The Royal y Socie etyy Kevin Cowtan, [email protected]
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