SREFLEX: SAXS REFinement through FLEXibility

Alejandro PanjkovichEMBL Hamburg

20.10.2016

A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 1 / 27

Outline

Motivation for flexible refinement

SREFLEX method

Example from user

Running the programI Standard runI Advanced domain definition

Interpreting results

SAXS and conformational changeCrystalline and solution conformation may differSAXS can provide insight into conformational transition

SAS modeling principle

Estimating protein flexibility: normal mode analysis (NMA)

Delarue M, Sanejouand YH (2002) Simplified normal mode analysis of conformational transitions in dna-dependentpolymerases: the elastic network model. J Mol Biol 320: 1011-1024.

Time for some movement

Input:

SAXS data

PDB coordinates

Program stages:

1 Structure partition

2 Domain level refinement

3 Residue level refinement

Automatic domain assignment based on dynamics

Assignment comparison, SCOP vs. auto

Experimental example: Josephin domain of ataxin-3

Nicastro et al. (2006) J. Biomol. NMR 36, 267-77

Experimental example: MurA

Schonbrunn et al. (1998) Eur. J. Biochem. 253, 406-412

SREFLEX: examples from benchmark

This journal is© the Owner Societies 2016 Phys. Chem. Chem. Phys., 2016, 18, 5707--5719 | 5715

backbone chain can be observed at the hinge of the model inFig. 4A. This is a consequence of the automatic partitioning ofthe structure during the restrained refinement stage performedby SREFLEXauto.

3.2.3 Calmodulin, limitations of the approach. The examplein this section is specifically selected to demonstrate the limita-tions of the approach but also of SAXS modeling in general.Calmodulin is a widely studied calcium sensor protein that playsa major role as an intermediate messenger in calcium signallingwithin eukaryotic cells.28 As part of its function, calmodulinundergoes large conformational changes upon calcium binding.Many calmodulin structures are available at the PDB, in differentbinding states and conditions. We have selected two conforma-tions (found in PDB entries 2og5 and 1qx5) to show an examplewhere SREFLEX will not be able to identify the conformationaltransition. Indeed, both conformations differ considerably interms of atomic positions (10.2 Å RMSD after superpositionusing the program MAMMOTH25), but the change provides littlemodification of the overall shape of the protein leading to veryminor alterations in the SAXS profiles (Fig. 5). The particle radiusof gyration (Rg) also changes marginally between the two struc-tures (18.0 Å for 2og5 and 17.7 Å for 1qx5). In such cases,SREFLEX may slightly improve the (already good) consistencywith the SAXS profile, but it is not expected to find a propersolution in terms of RMSD, given that the SAXS profiles aresimilar to each other and would not guide the conformationalsearch. One should however note that this is not a limitation of

SREFLEX but rather an inherent limitation of SAXS-based refine-ment approaches. Furthermore, the complexity of this particularconformational change probably exceeds what can be simulatedwith a limited combination of normal modes.

3.2.4 MurA, fosfomycin antibiotic target. The enzymeUDP-N-acetylglucosamine (UDP-GlcNAc) enolpyruvyltransfer-ase (MurA) catalyzes the committed step in peptidoglycansynthesis and is the target of the broad-spectrum antibioticfosfomycin. MurA undergoes conformational changes uponbinding of UDP-GlcNAc, and these have been investigated byMX and SAXS, with the SAXS data and the crystal structuresavailable for both the liganded and unliganded conformationalstates.29 In this case, we used SREFLEXauto to model theconformational change using the crystallographic structure ofone state and the SAXS profile of the other conformationalstate. SREFLEXscop could not be used, because SCOP assigns theprotein to be a single domain for the entire chain. SREFLEXauto

improved the consistency with the SAXS profiles, as shown inFig. 6. The starting w2

init values were 2.4 for the closing transitionand 3.3 for the opening transition, while the final w2

i values were1.2 and 1.1, respectively. In both directions, the structure was‘opened’ or ‘closed’ as expected from the experimental SAXS profile(as observed from changes in the radius of gyration). The programperformed small rotations of the domains relative to each otherrendering slightly higher RMSD values when comparing theobtained models with the corresponding MX structures. Improve-ments in terms of RMSD were obtained when we applied the iso-lated unrestrained refinement: while starting from RMSDinit = 2.4 Å,

Fig. 4 DNA-binding domain, modeling the unbound-bound transition.SAXS profiles show the better consistency to the target profile (dots) of theSREFLEXscop model over the SREFLEXauto model. Structures shown corre-spond to: (A) the SREFLEXscop model in red superimposed to the targetstructure in green. (B) Pseudo-domains as defined by SREFLEXauto. (C) SCOPdomains used by SREFLEXscop, vectors show the modeled movement.

Fig. 5 Limitations of the approach. Two known structures of calmodulinin different conformation are shown superimposed, even though theydiffer considerably (RMSD is 10.2 Å). The corresponding theoretical SAXSprofiles are very similar, meaning that in this case SREFLEX will not be ableto model the conformational change as explained in the text.

PCCP Paper

SREFLEX benchmark

scop auto ffs

%benchm

arkcases

RMSDtarget < 5.0 Å

closingopening

scop auto ffs

%benchm

arkcases

χ2 < 2.0

closingopening

scop auto fs

%benchm

arkcases

Δ RMSDtarget > 1.0 Å

closingopening

Panjkovich A & Svergun DI (2016) Phys. Chem. Chem. Phys. 18, 5707-19

SREFLEX, user example

SREFLEX, ab-initio model mismatch

SREFLEX, conformational change

Conformational change fits ligand binding

SREFLEX, web interface (ATSAS online)

SREFLEX, command line interface

Divide et impera - split your structure into pieces

The new ATSAS binary partistr may help

Check Pfam or SCOP domains

Use your intuition (particularly for opening structures)

Feed the structure asI d1.pdb,d2.pdb,d3.pdb (command line)I Or as a .zip file to the ATSAS-online version

Interpretation of SREFLEX results

Restrained models (rc), higher χ2 but better stereochemistry

Unrestrained models (uc), lower χ2, more structural distortion

Prioritize lower RMSD against initial structure

Check degree and relevance of clashes and breaks

Make an informed decision on which model is the better choice

Use the proper tool for your question

MW or oligomeric state are way off (better SASREF)

There are long linkers or unstructured regions (better use EOM)

Unrealistic expectations (folding problem not yet solved)

Summary

Motivation

Understanding the approach

Running the programI Standard runI Custom domain definition

Interpreting results

AcknowledgementsDmitri Svergun and the rest of the SAXS team at EMBL Hamburg (https://www.embl-hamburg.de/biosaxs/) Funding:

I EMBL and Marie Curie Actions for postdoc fellowship (EIPOD)

SAS in structural biology

ab-initio shape determination

missing fragments

rigid-body hybrid modeling

mixtures

ensemble approach

SREFLEX: SAXS REFinement through FLEXibility · 2016. 10. 21. · Delarue M, Sanejouand YH (2002)...

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