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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
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 2 / 27
SAXS and conformational changeCrystalline and solution conformation may differSAXS can provide insight into conformational transition
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 3 / 27
SAS modeling principle
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 4 / 27
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.
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 5 / 27
Time for some movement
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 6 / 27
SREFLEX: SAXS REFinement through FLEXibility
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 7 / 27
SREFLEX: SAXS REFinement through FLEXibility
Input:
SAXS data
PDB coordinates
Program stages:
1 Structure partition
2 Domain level refinement
3 Residue level refinement
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 8 / 27
Automatic domain assignment based on dynamics
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 9 / 27
Assignment comparison, SCOP vs. auto
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 10 / 27
Experimental example: Josephin domain of ataxin-3
Nicastro et al. (2006) J. Biomol. NMR 36, 267-77
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 11 / 27
Experimental example: MurA
Schonbrunn et al. (1998) Eur. J. Biochem. 253, 406-412
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 12 / 27
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
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 13 / 27
SREFLEX benchmark
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scop auto ffs
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χ2 < 2.0
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Δ RMSDtarget > 1.0 Å
closingopening
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A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 14 / 27
Panjkovich A & Svergun DI (2016) Phys. Chem. Chem. Phys. 18, 5707-19
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 15 / 27
SREFLEX, user example
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 16 / 27
SREFLEX, ab-initio model mismatch
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 17 / 27
SREFLEX, conformational change
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 18 / 27
Conformational change fits ligand binding
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 19 / 27
SREFLEX, web interface (ATSAS online)
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 20 / 27
SREFLEX, command line interface
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 21 / 27
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
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 22 / 27
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
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 23 / 27
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)
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 24 / 27
Summary
Motivation
Understanding the approach
Running the programI Standard runI Custom domain definition
Interpreting results
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 25 / 27
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)
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 26 / 27
SAS in structural biology
ab-initio shape determination
missing fragments
rigid-body hybrid modeling
mixtures
ensemble approach
A. Panjkovich (EMBL) SAXS + NMA 20.10.2016 27 / 27