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 modelMR model

map CC = 0.38

Human angiotensinogen after MR+DM

Final modelMR 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 modelMR model

map CC = 0.44(0.48 with GdCl3)

Rat C2 angiotensinogen after 4-fold multicrystal averaging

Final modelMR 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

Trimminguntrimmed

Trimmingtrimmed

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 1.475ÅNaI 72.5Å 286.4Å 3.2Å(3.

6Å)92Å2 0.9763

Å

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

Los Alamos National Laboratory

Jane & David Richardson, Jane & David Richardson, Vincent Chen, Chris Vincent Chen, Chris

Williams, Bryan Arendall, Williams, Bryan Arendall, Swati Jain, Bradley HintzeSwati Jain, Bradley Hintze

Cambridge University

Duke University