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R J Read, Department of HaematologyCambridge Institute for Medical Research
Experimental phasing in Phaser
Experimental phasing in Phaser
• Current capabilities• SAD phasing, substructure editing and
completion• seeding with MR solution
• Automation features• automation within Phaser• Python scripting
• Status for upcoming release of CCP4• Future plans
Intuitive understanding of SAD phasing
*F ;F , ,O OP H H
Expected difference between F+ and F
FO
*Re F
Expected value of F (H)
*F ,O OP ;H
Intuitive understanding of SAD phasing
Total likelihood is integral of the product of the two distributions under the black circle
FO
Expected value of F (H)
Expected difference between F+ and F
SAD log-likelihood gradient (LLG) map
• Compute derivative of log-likelihood wrt heavy atom structure factor• opposite phase shifts for plus and minus hands• if mixed anomalous substructure, hand of
model matters• Fourier transform gives map of where
likelihood target would like to see changes in anomalous scatterer model
• Very sensitive to minor sites• picks up sites identified as water molecules in
refined structures determined by halide soaks
Automation of SAD phasing
• Log-likelihood-gradient completion• look for one or several types of scatterer
• start from MR model or partial substructure
• analyse map to add sites, make atoms anisotropic
• delete atoms that fade away• repeat to convergence
• Functions are all available from Python• can run from HAPPy• used in new AutoSolve wizard
Iterative model-building with SAD
• Nitrate reductase structure• integral membrane protein, 1976 residues• contains 21 Fe atoms, 1 Mo, 113 S• solved by Natalie Strynadka, using
combination of Fe-MAD, MIRAS• Fe peak SAD data
• find 11 “Fe” sites with phenix.hyss• several are super-sites of Fe4S4 clusters
• phase and complete adding Fe with Phaser• total of 38 sites, some of which are S atoms• still ghosts of super-sites
Round 1 of iterative model-building
• Improve phases by density modification• Build with ARP/wARP (Resolve also
works…)• 798 residues, 18 docked in sequence
• LLG completion in Phaser, using partial polyAla model• Fe sites are now perfectly resolved
Convergence of iterative model-building
• LLG maps are better than random at identifying atom type• resolve any ambiguities by refined occupancy
• Converges after 5 cycles• anomalous scatterer model from Phaser has
21 Fe, 1 Mo, 84 of 113 S
• 1392/1976 residues, 731 docked in sequence
Case from last week
• Alexei Bochkarev (SGC, Toronto)• MR solution: significant conformational
changes, so noisy map• SeMet peak data: anomalous differences too
weak to determine substructure
• Seed SAD phasing with MR solution• identify 8 Se + 1 Fe (defining haem location)• significantly better phases and map
Status for upcoming release of CCP4
• Phaser version 2.1.3• SAD phasing (not in version 1.3.3 in CCP4
6.0.2)• complete partial substructure• seed with MR model as partial structure
• Both GUI and code have been well exercised on test cases and in workshops
• no known bugs
Future plans
• Combine MR and SAD in one mode• Finish implementation of MIRAS
• test stability, make user-friendly, automatic• LLG completion
• Account for translational NCS• use code developed for MR
• Use intensities (preferably unmerged)• replace Truncate (account for anisotropy,
tNCS)• account for and exploit radiation damage
Contributors
• Experimental phasing• Raj Pannu, Airlie McCoy, Laurent Storoni
• ccp4i GUI• Anne Baker, Airlie McCoy, Peter Briggs
• “BEST” data• Sasha Popov
• PHENIX collaboration• Ralf Grosse-Kunstleve, Pavel Afonine, Paul
Adams• Tom Terwilliger
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