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