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Using molecular replacement to exploit multiple crystal forms
Randy Read, Airlie McCoy & Gábor Bunkóczi
Tom Terwilliger
Solving the structure of angiotensinogen
• Human: 1 crystal form• 3.3Å, 1 copy, P41212
• Rat: 2 crystal forms• 2.8Å, 2 copies, C2• 3.15Å, 2 copies, P3221
• Mouse: 2 crystal forms• 2.1Å, 1 copy, P6122
• 2.95Å, 4 copies, C2221
Human angiotensinogen: molecular replacement
human
Human angiotensinogen: molecular replacement
human
Human angiotensinogen: molecular replacement
human
Human angiotensinogen after MR
Final model
MR model
map CC = 0.38
Human angiotensinogen after MR+DM
Final model
MR model
map CC = 0.50
Solving angiotensinogen structures
human
rat C2
rat P3221
+ GdCl3
Rat C2 angiotensinogen after MR with density + 2-fold averaging
Final model
MR model
map CC = 0.44(0.48 with GdCl3)
Rat C2 angiotensinogen after 4-fold multicrystal averaging
Final model
MR model
map CC = 0.53(0.59 with GdCl3)
S-S (18-138)
S-S (18-138)
Renin
Renin:angiotensinogen complex
Solving Drosophila GST2 (1M0U)
• Difficult structure from Bogos Agianian (Piet Gros)
• Find one of two copies with ensemble of 3 structures (28-30% identity)• search for second copy fails
• Find second copy as density from first• this succeeds: TFZ=10.0• trick to doing this: assign low error to first
copy, higher error to second
How does averaging add information?
• Nyquist-Shannon sampling theorem:• doubly-sampled Fourier transform can be
reconstructed perfectly
Molecule and molecular transform
(Images courtesy of Kevin Cowtan)
Crystal diffraction samples molecular transform on reciprocal lattice
(Images courtesy of Kevin Cowtan)
Non-isomorphism changes sampling of molecular transform
(Images courtesy of Kevin Cowtan)
Cell change
Rotated molecule
Absolute vs. relative cell change
b=27Åb=24Å
a=48Åa=51Å
d=6Åd=3Å
“Protein X”
• 525 residues• Only distant homologues in PDB
• eight models with sequence identity about 20%
Ensemble of 8 possible models
Molecular replacement trials
• MR with individual models failed• complete or trimmed with FFAS server
• MR with ensemble failed initially• but now works with new “trim” option in
phenix.ensembler
Trimming
untrimmed
Trimming
trimmed
Data sets
• Space group P3112
Crystal
a=b c dmin ΔBaniso λ
Native 70.9Å 286.0Å 2.4Å 35Å2 0.9728Å
GdCl3 68.8Å 289.0Å 3.3Å 96Å2 1.475Å
NaI 72.5Å 286.4Å 3.2Å(3.6Å)
92Å2 0.9763Å
Experimental phasing with GdCl3• GdCl3 derivative diffracts to about 3.3Å
• good anomalous signal (phenix.xtriage) to 4.3Å
• solve substructure with phenix.hyss (4 sites)• phase with Phaser• solvent flattening• see some features of fold, not complete trace
Real-space MR against GdCl3 map
• Use real-space MR to place models in density• cut out density from SAD map
• back-transform, treat as observed data• rotation search, phased translation
• Resolution, phases too poor to rebuild• Model provides starting mask
Real-space MR model in Gd density
Solve native crystal with GdCl3 density
• Use real-space MR model to construct envelope
• Cut out GdCl3 density for one molecule• place in large unit cell (4x extent in each
direction)• FFT to get molecular transform
• Use density for MR solution of native crystal• RFZ=3.8, TFZ=22.9• or just rigid-body refinement starting from
identity operator
Solve and phase iodide soak
• Diffracts to 3.2Å, but data sparse beyond 3.6Å• good anomalous signal only to 5.4Å (phenix.xtriage)
• Ab initio substructure determination failed• phenix.hyss
• Rigid-body refinement of GdCl3 density model
• Use Fc from MR solution as partial model for MR-SAD phasing• SAD log-likelihood-gradient maps yield 11 sites
Multi-crystal averaging
• Get operators from transformations applied to GdCl3 density
• Starting maps from SAD phasing (derivatives) or MR with GdCl3 density (native)
• Carried out with phenix.multi_crystal_average
Morphing
• Use phenix.morph_model to morph real-space MR model into native averaged density• correlation increases from 0.296 to 0.338
Initial model-building
• Build with phenix.autobuild into averaged native map, start from morphed MR model• 364 residues, 177 assigned to sequence• R=0.42, Rfree=0.48
Iterative averaging and rebuilding
• Carry on with iterative improvement• MR-SAD phasing with current model• multi-crystal averaging• AutoBuild, do some manual rebuilding• MR-SAD phasing with updated model• multi-crystal averaging
• Iteration has improved anomalous substructures• GdCl3: 4 sites to 8 sites (2 split)
• NaI: 11 sites to 14 sites
Map after iterative process
Currentmodel
MR model
Tools• Cutting out density
• phenix.cut_out_density (Tom Terwilliger)• cmapcut (Kevin Cowtan)
• Molecular replacement with density• Molrep• Phaser
• SAD phasing starting from density• Phaser SAD LLG, giving density as partial model
• Multi-crystal averaging• dmmulti• phenix.multi_crystal_average
Acknowledgements
• Phaser:• Airlie McCoy, Gabor Bunkoczi
• Angiotensinogen:• Penny Stein, Robin Carrell, Aiwu Zhou• Mike Murphy, Fiona Broughton Pipkin
• “Protein X”• Mykhaylo Demydchuk, Aiwu Zhou,
Janet Deane, Penny Stein
Randy Read, Airlie Randy Read, Airlie McCoy, Gabor Bunkoczi, McCoy, Gabor Bunkoczi,
Rob OeffnerRob Oeffner
Tom Terwilliger, Tom Terwilliger, Li-Wei HungLi-Wei Hung
The PHENIX Project
An NIH/NIGMS funded Program Project
Paul Adams, Ralf Grosse-Paul Adams, Ralf Grosse-Kunstleve, Pavel Afonine, Nat Kunstleve, Pavel Afonine, Nat
Echols, Nigel Moriarty, Jeff Echols, Nigel Moriarty, Jeff Headd, Nicholas Sauter, Peter Headd, Nicholas Sauter, Peter
ZwartZwart
Lawrence Berkeley Laboratory
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Williams, Bryan Arendall, Williams, Bryan Arendall, Swati Jain, Bradley HintzeSwati Jain, Bradley Hintze
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