Using molecular replacement to
exploit multiple crystal forms
Randy Read, Airlie McCoy & Gábor Bunkóczi
Tom Terwilliger
Solving the structure of angiotensinogen
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Human: 1 crystal form
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Rat: 2 crystal forms
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3.3Å, 1 copy, P41212
2.8Å, 2 copies, C2
3.15Å, 2 copies, P3221
Mouse: 2 crystal forms
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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
MR model
Final model
map CC = 0.38
Human angiotensinogen after MR+DM
MR model
Final model
map CC = 0.50
Solving angiotensinogen structures
human
rat C2
rat P3221
+ GdCl3
Rat C2 angiotensinogen after
MR with density + 2-fold averaging
MR model
Final model
map CC = 0.44
(0.48 with GdCl3)
Rat C2 angiotensinogen after
4-fold multicrystal averaging
MR model
Final model
map CC = 0.53
(0.59 with GdCl3)
Renin:angiotensinogen complex
Renin
S-S (18-138)
S-S (18-138)
Solving Drosophila GST2 (1M0U)
Difficult structure from Bogos Agianian (Piet
Gros)
• Find one of two copies with ensemble of 3
structures (28-30% identity)
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search for second copy fails
Find second copy as density from first
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this succeeds: TFZ=10.0
trick to doing this: assign low error to first copy,
higher error to second
How does averaging add information?
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Nyquist-Shannon sampling theorem:
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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
Cell change
Rotated molecule
(Images courtesy of Kevin Cowtan)
Absolute vs. relative cell change
d=6Å
d=3Å
a
h=
d
a + Da
h + Dh =
d
Da
Dh =
d
b=27Å
b=24Å
a=48Å
a=51Å
“Protein X”
525 residues
• Only distant homologues in PDB
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eight models with sequence identity about 20%
Ensemble of 8 possible models
Molecular replacement trials
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MR with individual models failed
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complete or trimmed with FFAS server
MR with ensemble failed initially
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but now works with new “trim” option in
phenix.ensembler
Trimming
untrimmed
Trimming
trimmed
Data sets
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Space group P3112
Crystal
Native
GdCl3
NaI
a= b
70.9Å
68.8Å
72.5Å
c
286.0Å
dmin
2.4Å
289.0Å
286.4Å
3.3Å
3.2Å(3.
6Å)
λ
ΔBaniso
35Å2 0.9728Å
96Å2
92Å2
1.475Å
0.9763Å
Experimental phasing with GdCl3
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GdCl3 derivative diffracts to about 3.3Å
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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
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Use real-space MR to place models in density
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cut out density from SAD map
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back-transform, treat as observed data
rotation search, phased translation
Resolution, phases too poor to rebuild
• Model provides starting mask
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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
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place in large unit cell (4x extent in each direction)
FFT to get molecular transform
Use density for MR solution of native crystal
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RFZ=3.8, TFZ=22.9
or just rigid-body refinement starting from identity
operator
Solve and phase iodide soak
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Diffracts to 3.2Å, but data sparse beyond 3.6Å
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good anomalous signal only to 5.4Å (phenix.xtriage)
Ab initio substructure determination failed
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phenix.hyss
Rigid-body refinement of GdCl3 density model
• Use Fc from MR solution as partial model for
MR-SAD phasing
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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
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Morphing
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Use phenix.morph_model to morph real-space
MR model into native averaged density
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correlation increases from 0.296 to 0.338
Initial model-building
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Build with phenix.autobuild into averaged native
map, start from morphed MR model
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364 residues, 177 assigned to sequence
R=0.42, Rfree=0.48
Iterative averaging and rebuilding
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Carry on with iterative improvement
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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
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GdCl3: 4 sites to 8 sites (2 split)
NaI: 11 sites to 14 sites
Map after iterative process
MR model
Current
model
Tools
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Cutting out density
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Molecular replacement with density
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Molrep
Phaser
SAD phasing starting from density
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phenix.cut_out_density (Tom Terwilliger)
cmapcut (Kevin Cowtan)
Phaser SAD LLG, giving density as partial model
Multi-crystal averaging
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dmmulti
phenix.multi_crystal_average
Acknowledgements
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Phaser:
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Angiotensinogen:
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Airlie McCoy, Gabor Bunkoczi
Penny Stein, Robin Carrell, Aiwu Zhou
Mike Murphy, Fiona Broughton Pipkin
“Protein X”
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Mykhaylo Demydchuk, Aiwu Zhou,
Janet Deane, Penny Stein
The PHENIX Project
Lawrence Berkeley Laboratory
Paul Adams, Ralf Grosse-Kunstleve,
Pavel Afonine, Nat Echols, Nigel
Moriarty, Jeff Headd, Nicholas
Sauter, Peter Zwart
Randy Read, Airlie McCoy,
Gabor Bunkoczi, Rob
Oeffner
Cambridge University
An NIH/NIGMS funded
Program Project
Los Alamos National Laboratory
Tom Terwilliger,
Li-Wei Hung
Duke University
Jane & David Richardson,
Vincent Chen, Chris Williams,
Bryan Arendall, Swati Jain,
Bradley Hintze