Modelling against small angle scattering data Al Kikhney EMBL Hamburg, Germany
Outline • Validation of atomic models CRYSOL • • Rigid body modelling SASREF • BUNCH • CORAL • • Oligomeric mixtures OLIGOMER • • Flexible systems EOM •
SAXS studies of biological macromolecules R g MM Volume Shape Validation in solution
Compute SAS from an atomic model log I(s) s, Å -1 Validation in solution
Compute SAS from an atomic model A( s ): atomic scattering log I(s) s, Å -1
Compute SAS from an atomic model in solution
Compute SAS from an atomic model log I(s) A a ( s ): atomic scattering in vacuum E( s ): scattering from the excluded volume B( s ): scattering from the hydration shell s, Å -1 CRYSOL (X-rays): Svergun et al. (1995) J. Appl. Cryst. 28, 768 CRYSON (neutrons): Svergun et al. (1998) P.N.A.S. USA 95, 2267
Compute SAS from an atomic model Using spherical harmonics to perform the average analytically: ...permits to further use rapid algorithms for rigid body modelling. CRYSOL (X-rays): Svergun et al. (1995) J. Appl. Cryst. 28, 768 CRYSON (neutrons): Svergun et al. (1998) P.N.A.S. USA 95, 2267
SAXS studies of biological macromolecules R g MM Volume Shape Rigid body modelling
Why Rigid body modelling Huge amount of structural information about individual macromolecules Large macromolecular complexes are difficult to study by high resolution methods High resolution models of subunits can be used to model the quaternary structure of complexes based on low resolution methods SASREF: Petoukhov & Svergun (2005) Biophys J. 89 , 1237; (2006) Eur. Biophys. J . 35 , 567.
Rigid body modelling Global refinement with distance constraints A tyrosine kinase MET (118 kDa) consisting of five domains Gherardi, Sandin, Petoukhov, Finch, Youles, Ofverstedt, Miguel, Blundell, Vande Woude, Skoglund, & Svergun (2006) PNAS USA, 103 , 4046.
Rigid body modelling SASREF Interconnectivity Absence of steric clashes Symmetry SASREF: Petoukhov & Svergun (2005) Biophys J. 89 , 1237; (2006) Eur. Biophys. J . 35 , 567.
Rigid body modelling SASREF Interconnectivity Absence of steric clashes Symmetry SASREF: Petoukhov & Svergun (2005) Biophys J. 89 , 1237; (2006) Eur. Biophys. J . 35 , 567.
Rigid body modelling SASREF Interconnectivity Absence of steric clashes Symmetry SASREF: Petoukhov & Svergun (2005) Biophys J. 89 , 1237; (2006) Eur. Biophys. J . 35 , 567.
Rigid body modelling SASREF Interconnectivity Absence of steric clashes Symmetry Intersubunit contacts (from chemical shifts by NMR or mutagenesis) Distances between residues (FRET or mutagenesis) Relative orientation of subunits (RDC by NMR) Scattering data from subcomplexes SASREF: Petoukhov & Svergun (2005) Biophys J. 89 , 1237; (2006) Eur. Biophys. J . 35 , 567.
Rigid body modelling SASREF Can fit multiple X-ray and neutron scattering curves from partial constructs or contrast variation lg I, r e la tiv e 1 1 1 0 9 8 0 .5 1 .0 1 .5 2 .0 - 1 s , n m
Rigid body modelling http://www.embl-hamburg.de/biosaxs/atsas-online/sasref.php
SAXS studies of biological macromolecules R g MM Volume Shape Rigid body modelling Add missing fragments
Add missing fragments BUNCH Flexible loops/domains • Not resolved in high resolution models • Genetically removed to facilitate crystallization Reconstruct the missing part to fit the experimental data
Add missing fragments BUNCH • Positions/orientations of rigid domains • Probable conformations of flexible linkers represented as “dummy residue” chains • Fits multiple scattering curves from partial constructs (e.g. deletion mutants) • Symmetry • Allows to fix domains • Restrain the model by contacts between specific residues • Only single chain proteins (no complexes)
CORAL Modelling of multidomain protein complexes against multiple data sets Loops library Combines the algorithms of SASREF and BUNCH
Words of caution • SAS is a low resolution method • Several shapes may yield an identical scattering pattern • Even with – information about contacting residues from other methods (spin labelling, site-directed mutagenesis, FRET, chemical shifts etc.) – symmetry – no steric clashes one must cross-validate SAS models against all available biochemical/biophysical information • The sample is never perfect
SAXS studies of biological macromolecules R g MM Volume Shape Rigid body modelling Missing fragments Oligomeric mixtures
Monomer-dimer equilibrium of tetanus toxin Receptor binding H(C) domain reveals concentration-dependent oligomerization Mon : Dim 100 : 0 0 : 100 60 : 40 40 : 60 20 : 80 10 : 90 The model of the dimeric H(C) domain was reconstructed by rigid body modelling using the atomic structure of the monomer (1FV2). Qazi, O., Bolgiano, B., Crane, D., Svergun, D.I., Konarev, P.V., Yao, Z.P., Robinson, C.V., Brown, K.A. & Fairweather N. (2007) J Mol Biol. 365, 123 – 134.
SAXS studies of biological macromolecules R g MM Volume Shape Rigid body modelling Missing fragments Oligomeric Flexible mixtures systems
Flexible systems
Flexible systems
Flexible systems Ensemble Optimization Method Multiple conformations in solution
Flexible systems Ensemble Optimization Method Multiple conformations in solution
Flexible systems Ensemble Optimization Method Multiple conformations in solution
Flexible systems Ensemble Optimization Method R g R g R g
Flexible systems Ensemble Optimization Method R g R g distribution for the selected models compared to the R g distribution for the whole pool
http://www.embl-hamburg.de/biosaxs/atsas-online/eom.php
SAXS studies of biological macromolecules R g MM Volume ATSAS Shape Rigid body modelling software package Missing www.embl-hamburg.de/biosaxs/software.html fragments Oligomeric Flexible mixtures systems
Summary • Nothing known: ab initio low resolution structure • Complete high resolution structure known: validation in solution and biologically active oligomers • High resolution structure of domains/subunits known: quaternary structure by rigid body refinement • Incomplete high resolution structure known: probable configuration of missing portions • Mixtures/assemblies: volume fractions of components • Flexible systems: quantitative analysis of configurational ensembles
Thank you! www.embl-hamburg.de/biosaxs www.saxier.org/forum biosaxs.com wenmr.eu
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