small angle scattering data Al Kikhney EMBL Hamburg, Germany - - PowerPoint PPT Presentation

▶
small angle scattering data
SMART_READER_LITE
LIVE PREVIEW

small angle scattering data Al Kikhney EMBL Hamburg, Germany - - PowerPoint PPT Presentation

Aug 19, 2023 352 likes •716 views

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

slide-1
SLIDE 1

Modelling against small angle scattering data

Al Kikhney

EMBL Hamburg, Germany

slide-2
SLIDE 2

Outline

  • Validation of atomic models
  • CRYSOL
  • Rigid body modelling
  • SASREF
  • BUNCH
  • CORAL
  • Oligomeric mixtures
  • OLIGOMER
  • Flexible systems
  • EOM
slide-3
SLIDE 3

SAXS studies of biological macromolecules

Rg MM Volume

Shape

Validation in solution

slide-4
SLIDE 4

Validation in solution

Compute SAS from an atomic model

log I(s) s, Å-1

slide-5
SLIDE 5

Compute SAS from an atomic model

log I(s) s, Å-1

A(s): atomic scattering

slide-6
SLIDE 6

Compute SAS from an atomic model in solution

slide-7
SLIDE 7

Compute SAS from an atomic model

log I(s) s, Å-1

Aa(s): atomic scattering in vacuum E(s): scattering from the excluded volume B(s): scattering from the hydration shell

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

slide-8
SLIDE 8

Compute SAS from an atomic model

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

Using spherical harmonics to perform the average analytically: ...permits to further use rapid algorithms for rigid body modelling.

slide-9
SLIDE 9

SAXS studies of biological macromolecules

Rg MM Volume Rigid body modelling

Shape

slide-10
SLIDE 10

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.

Why

slide-11
SLIDE 11

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

slide-12
SLIDE 12

Rigid body modelling

SASREF

Interconnectivity Absence of steric clashes Symmetry

SASREF: Petoukhov & Svergun (2005) Biophys J. 89, 1237; (2006) Eur. Biophys. J. 35, 567.

slide-13
SLIDE 13

Rigid body modelling

SASREF

Interconnectivity Absence of steric clashes Symmetry

SASREF: Petoukhov & Svergun (2005) Biophys J. 89, 1237; (2006) Eur. Biophys. J. 35, 567.

slide-14
SLIDE 14

Rigid body modelling

SASREF

Interconnectivity Absence of steric clashes Symmetry

SASREF: Petoukhov & Svergun (2005) Biophys J. 89, 1237; (2006) Eur. Biophys. J. 35, 567.

slide-15
SLIDE 15

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.

slide-16
SLIDE 16 s , n m
  • 1
0 .5 1 .0 1 .5 2 .0 lg I, r e la tiv e 8 9 1 0 1 1

Can fit multiple X-ray and neutron scattering curves from partial constructs or contrast variation

Rigid body modelling

SASREF

slide-17
SLIDE 17

Rigid body modelling

http://www.embl-hamburg.de/biosaxs/atsas-online/sasref.php

slide-18
SLIDE 18

SAXS studies of biological macromolecules

Rg MM Volume

Shape Rigid body modelling

Add missing fragments

slide-19
SLIDE 19

Add missing fragments

Flexible loops/domains

  • Not resolved in high resolution models
  • Genetically removed to facilitate crystallization

Reconstruct the missing part to fit the experimental data BUNCH

slide-20
SLIDE 20
  • 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)

Add missing fragments

BUNCH

slide-21
SLIDE 21

CORAL

Loops library

Modelling of multidomain protein complexes against multiple data sets Combines the algorithms of SASREF and BUNCH

slide-22
SLIDE 22

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

  • ne must cross-validate SAS models against all

available biochemical/biophysical information

  • The sample is never perfect
slide-23
SLIDE 23

SAXS studies of biological macromolecules

Rg MM Volume

Shape Rigid body modelling Missing fragments Oligomeric mixtures

slide-24
SLIDE 24

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.

Monomer-dimer equilibrium of tetanus toxin

Receptor binding H(C) domain reveals concentration-dependent oligomerization The model of the dimeric H(C) domain was reconstructed by rigid body modelling using the atomic structure of the monomer (1FV2). 100 : 0 0 : 100 60 : 40 40 : 60 20 : 80 10 : 90 Mon : Dim

slide-25
SLIDE 25

SAXS studies of biological macromolecules

Rg MM Volume

Shape Rigid body modelling Missing fragments Flexible systems Oligomeric mixtures

slide-26
SLIDE 26

Flexible systems

slide-27
SLIDE 27

Flexible systems

slide-28
SLIDE 28

Multiple conformations in solution

Flexible systems

Ensemble Optimization Method

slide-29
SLIDE 29

Multiple conformations in solution

Flexible systems

Ensemble Optimization Method

slide-30
SLIDE 30

Multiple conformations in solution

Flexible systems

Ensemble Optimization Method

slide-31
SLIDE 31

Flexible systems

Ensemble Optimization Method

Rg Rg Rg

slide-32
SLIDE 32

Rg

Flexible systems

Ensemble Optimization Method

Rg distribution for the selected models compared to the Rg distribution for the whole pool

slide-33
SLIDE 33

http://www.embl-hamburg.de/biosaxs/atsas-online/eom.php

slide-34
SLIDE 34

SAXS studies of biological macromolecules

Rg MM Volume

Shape Rigid body modelling Missing fragments Flexible systems Oligomeric mixtures

ATSAS

software package

www.embl-hamburg.de/biosaxs/software.html

slide-35
SLIDE 35
  • 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

Summary

slide-36
SLIDE 36

Thank you!

www.saxier.org/forum biosaxs.com wenmr.eu www.embl-hamburg.de/biosaxs