AMPLE
 Ab Initio Modelling of Proteins for
 Molecular Replacement
 
 Jens Thomas Institute of Integrative Biology December 2014 Outline
 • What is AMPLE? • How it works • When it works • Why it works • How to use it AMPLE Ab initio Modelling of Proteins for moLEcular replacement ! • Joint development by CCP4 and the University of Liverpool ! • AMPLE is a comprehensive project to assess the suitability of using cheaply obtained ab initio models in molecular replacement ! • An additional goal of the project is to make AMPLE into an automated software tool that can be made generally available to potential users through CCP4 Molecular Replacement Sequence search for a suitable homolog target electron density map • Phases from homolog • Intensities/positions from target homolog A replacement for Molecular Replacement • No suitably similar homolog ! • Small changes in sequence or crystallisation conditions can lead to different polymorphs crystallising (coiled-coils) ! • Not sure exactly what is in the crystal Ab Initio Modelling • Create database of 3- and 9- residue fragments. • Start from linear chain and splice in fragment geometry. • Randomly move the fragments under simplified forcefield • Score the resulting structures. • 1000’s of “decoys” created. • Decoys are clustered and centroid representatives of largest cluster are considered candidate fold predictions • Refinement under a more realistic physics-based force field Decoys vs. final models • for normal usage > 20,000 decoys are recommended • refinement stage can take weeks on a supercomputer ! This is beyond the capabilities of most crystallography groups. ! But: ! • creating 1000 decoys ~ 1/2 day on a single CPU with ROSETTA • QUARK modelling is available online and takes ~ 1 day Ab Initio Models and Molecular Replacement Simple ab initio modelling Molecular Replacement Clusters of similar structures Works well with superposed ensembles approximating the target Within/between clusters, similarity May only require a partial model indicates accuracy ∴ trim inaccurate regions leaving more reliable core trim to accurate core The AMPLE pipeline Bibby, J., Keegan, R.M., Mayans, O., Winn, M.D., Rigden, D.J., 2012. Acta Crystallographica Section D Biological Crystallography 68, 1622–1631. Pathways to ensembles decoys
 cluster
 truncate
 cluster
 side chains ROSETTA/
 (SPICKER) (THESEUS) (MAXCLUSTER) QUARK m o t a - l l a A 1 reliable … 2A p E % o 1 0 … ly N a 1 S 30% 3 E … A … M … 5 2 0 B % L E … … S A 1 … 2A … m o t a 3 - l A al reliable p o l y a up to 120 ensembles from … 1000 decoys