Molecular replacement in Phaser
R J Read, Department of Haematology
Cambridge Institute for Medical Research
Molecular replacement in Phaser
Current capabilities
• Automation features
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automation within Phaser
Python scripting
Status for next release of CCP4
• Future plans
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Current capabilities
Anisotropy correction, absolute scaling
• Cell content analysis
• Normal modes perturbation
• Ensemble averaging of multiple models
• Brute force rotation and translation searches with
likelihood targets
• Fast rotation and translation targets with rescoring
• Packing, rigid body refinement
• Automated structure solution
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Automated molecular replacement
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Automated tree search with pruning
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multiple choices of model or ensemble for each
component
multiple components, in specified or permuted order
multiple space groups
One job carries out all steps
• Python scripting
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all functions available from Python
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used for AutoMR wizard in Phenix
could be used for MrBUMP
Status for next release of CCP4
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Phaser version 2.1
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Molecular replacement
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more forgiving clash test
exercised combinations of options
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one known bug using density for model
debugged ccp4i interface
Future plans
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Finish implementation of translational NCS
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speed, stability, generality
Combine MR and SAD in one mode
• Account for internal symmetry of models
• “Greedy” algorithm for placing multiple copies
• Use intensities
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replace Truncate (account for anisotropy, tNCS)
Translational NCS
Non-crystallographic symmetry is found in about
1/3 to 1/2 of crystal structures
• Often parallel to crystallographic symmetry axis
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combination gives translational NCS (tNCS)
Largest class of problems where Phaser fails
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changes expected intensities, but not modelled
Translational NCS: generalise e
In general case, contributions of atoms from
symmetry-related molecules are independent
• For certain hkl values, contributions have the
same or opposite phase
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number of these is called e
Centering applies to all hkl
Df=2ph·t
h odd:
even:Df=p
Df=0
Interference effects from tNCS
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Phase difference of two copies depends on hkl
and translation vector
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Df=2ph·t
Pseudo-translational NCS
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tNCS is not perfect
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Perfect tNCS
There is usually a rotational
component (ncsR)
There is non-isomorphism
between structures
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Differences in coordinates and
scattering
Gives rise to D values (ncsD)
Pseudo-tNCS
Merging Patterson peaks
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Vector (ncsT) is often pseudo-crystallographic
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differing slightly from cell or centering transation
Peaks in Patterson map merge
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have to refine exact translation, perhaps test
alternatives
Modelling pseudo-translational NCS
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Generalised e-factor
e hkl  f  ncsDs , Gs ,ncsR , ncsT ,symmetry 
The e-factors are no longer integers
• The e-factors are found by maximizing the
probability of the data
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Probability described by the Wilson distribution
Similar to anisotropy correction
2
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2 Fhkl
Fhkl
PtNCS  Fhkl  
exp  
hkl
hkl 
e hkl  N
 e hkl  N 
Sensitivity of likelihood to tNCS model
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Likelihood is very sensitive to details of tNCS
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simple model predicts exact cancellations
likelihood can be excessively small for simple model
p(FO)
FO
MR Likelihood Functions
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Use refined values of ncsT, ncsR and ncsD to
calculate the structure factor of the “dimer”
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“implicit” copy
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FC   F1 1  ncsDs Gs ,ncsR exp 2p i hkl  Risym ncsT
isym
Account for tNCS in the variances
• Make second copy explicit for final packing
check and refinement
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Contributors
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Molecular replacement
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ccp4i GUI
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Airlie McCoy, Laurent Storoni
feedback from Kay Diederichs, David Schuller,
Eleanor Dodson, Phil Evans…
Anne Baker, Airlie McCoy, Peter Briggs
PHENIX collaboration
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Ralf Grosse-Kunstleve, Paul Adams
Tom Terwilliger
Sponsors
Wellcome Trust
• NIH
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PHENIX package for automated crystallography
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Paul Adams, Tom Terwilliger, Tom Ioerger, Jim Sacchetini
CCP4
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GUI development for Beast and Phaser