Solution Scattering from Biological Macromolecules

Hybrid rigid body modelling

Al Kikhney

EMBL Hamburg

Hybrid rigid body modelling in ATSAS 3.0

• CRYSOL – computing SAXS from a model

• SASREF – rigid body modelling

• BUNCH – adding missing fragments

• CORAL – multidomain protein complexes

• SREFLEX – flexible refinement based on normal mode

analysis

• SASBDB – repository for SAS data and models

nm-1

experimental SAXS pattern

experimental SAXS pattern

SAXS data from macromolecules in solution

log I(s)

nm-1

experimental SAXS pattern

experimental SAXS pattern

calculated from model

SAXS data from macromolecules in solution

log I(s)

SAXS data from macromolecules in solution

nm-1

experimental SAXS pattern

experimental SAXS pattern

calculated from model

log I(s)

nm-1

log I(s)

Computing SAS from an atomic model

Aa(s): atomic scattering in vacuum

nm-1

log I(s)

Computing SAS from an atomic model

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

Computing 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

Running CRYSOL

CRYSOL (X-rays): Svergun et al. (1995) J. Appl. Cryst. 28, 768

• Command-line interface

• Web interfacehttps://www.embl-hamburg.de/biosaxs/atsas-online/crysol.php

• PyMOL plugin SASpy

Running CRYSOL

Running CRYSOL

CRYSOL (X-rays): Svergun et al. (1995) J. Appl. Cryst. 28, 768

• Command-line interface

• Web interfacehttps://www.embl-hamburg.de/biosaxs/atsas-online/crysol.php

• PyMOL plugin SASpy

• PRIMUS

reduced

m Significance level α = 1%

100 0.68 < χ2 < 1.41

500 0.85 < χ2 < 1.17

1000 0.89 < χ2 < 1.12

2000 0.92 < χ2 < 1.08

Goodness of fitLog I(s)

χ2 = 2.4

s, nm-1

s s

s

– Ifit(s)

• Iexp(s)

Goodness of fit

Δ/σ

+3

-3

Log I(s)

DATCMP Franke et al. (2015)

Correlation Map…

Nat. Methods 12, 419-422

Error-weighted

residual difference plot

s, nm-1

s, nm-1

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

Rigid body modelling

Log I(s)

s, Å-1

fit

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

Rigid body modelling

χ2 = 54

Log I(s)

s, Å-1

13

fit

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

Rigid body modelling

χ2 =

Log I(s)

s, Å-1

fit

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

Rigid body modelling

χ2 =

Log I(s)

s, Å-1

1.06

Rigid body modellingHuge 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

Rigid body modelling

InterconnectivityAbsence of steric clashesSymmetry

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

SASREF

Rigid body modelling

InterconnectivityAbsence of steric clashesSymmetry

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

SASREF

Rigid body modelling

InterconnectivityAbsence of steric clashesSymmetryIntersubunit contacts

From chemical shifts by NMR or mutagenesis

Distances between residues FRET or mutagenesis

Relative orientation of subunits RDC by NMR

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

SASREF

Rigid body modelling

InterconnectivityAbsence of steric clashesSymmetryIntersubunit 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.

SASREF

Rigid body modelling

InterconnectivityAbsence of steric clashesSymmetryIntersubunit 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.

SASREF

Rigid body modelling

InterconnectivityAbsence of steric clashesSymmetryIntersubunit 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.

SASREF

Rigid body modelling

InterconnectivityAbsence of steric clashesSymmetryIntersubunit 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

Reconstruction of missing fragments

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

SASREF

Immunosuppressive Yersinia Effector YopM and

DEAD Box Helicase DDX3

• YopM: 50 kDa, crystal structure available

• DDX3: 42 kDa, 68% of structure available

• YopM:DDX3 complex

L. Berneking et al. (2016) PLoS Pathog 12(6):e1005660

SASBDB project page

Immunosuppressive Yersinia Effector YopM and

DEAD Box Helicase DDX3

• YopM: 50 kDa, crystal structure available

L. Berneking et al. (2016) PLoS Pathog 12(6):e1005660

Chi2 = 390

Log I(s)

s, nm-1

Immunosuppressive Yersinia Effector YopM and

DEAD Box Helicase DDX3

• YopM: 50 kDa, crystal structure available

L. Berneking et al. (2016) PLoS Pathog 12(6):e1005660

SASBDB: SASDAU8

Chi2 = 2.3

Log I(s)

s, nm-1

Immunosuppressive Yersinia Effector YopM and

DEAD Box Helicase DDX3

• YopM: 50 kDa, crystal structure available

L. Berneking et al. (2016) PLoS Pathog 12(6):e1005660

PDB: 4ow2

PDBePISA

ebi.ac.uk/pdbe/pisa/

Immunosuppressive Yersinia Effector YopM and

DEAD Box Helicase DDX3

• YopM: 50 kDa, crystal structure available

L. Berneking et al. (2016) PLoS Pathog 12(6):e1005660

PDB: 4ow2

PDBePISA

ebi.ac.uk/pdbe/pisa/

Immunosuppressive Yersinia Effector YopM and

DEAD Box Helicase DDX3

• YopM: 50 kDa, crystal structure available

• DDX3: 42 kDa, 68% of structure available

L. Berneking et al. (2016) PLoS Pathog 12(6):e1005660

Log I(s)

s, nm-1

Immunosuppressive Yersinia Effector YopM and

DEAD Box Helicase DDX3

• YopM: 50 kDa, crystal structure available

• DDX3: 42 kDa, 68% of structure available

L. Berneking et al. (2016) PLoS Pathog 12(6):e1005660

SASBDB: SASDAV8 zhanglab.ccmb.med.umich.edu/I-TASSER/

Log I(s)

s, nm-1

Immunosuppressive Yersinia Effector YopM and

DEAD Box Helicase DDX3

• YopM: 50 kDa, crystal structure available

• DDX3: 42 kDa, 68% of structure available

• YopM:DDX3 complex

L. Berneking et al. (2016) PLoS Pathog 12(6):e1005660

+

Immunosuppressive Yersinia Effector YopM and

DEAD Box Helicase DDX3

• YopM: 50 kDa, crystal structure available

• DDX3: 42 kDa, 68% of structure available

• YopM:DDX3 complex

L. Berneking et al. (2016) PLoS Pathog 12(6):e1005660

SASBDB: SASDAW8

Log I(s)

s, nm-1

Adding 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

BUNCH: Petoukhov & Svergun (2005) Biophys J. 89, 1237-1250

• 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)

Adding missing fragmentsBUNCH

BUNCH: Petoukhov & Svergun (2005) Biophys J. 89, 1237-1250

CORAL

Loops library

Modelling of multidomain protein complexes against multiple data sets

CORAL: Petoukhov et al. (2012) J. Appl. Cryst. 45, 342-350

CORALModelling of multidomain protein complexes against multiple data sets

22

22

13

34 kDa

CORAL

CORAL

s, nm-1

s, nm-1

Log I(s)

Δ/σ

+3

-3

SASBDB: SASDDG9

Flexible refinement

4ake 1ake

SREFLEXSAS REfinement through FLEXibility based on normal mode analysis

s, Å-1

Log I(s)

SREFLEX: Panjkovich A. and Svergun D.I. (2016) Phys. Chem. Chem. Phys. 18, 5707-5719

SREFLEXEstimating protein flexibility: normal mode analysis (NMA)

Delarue & Sanejouand (2002) Simplified NMA of conformational transitions in DNA-dependent polymerases: the elastic network model. J Mol Biol 320:1011-1024

SREFLEXSAS REfinement through FLEXibility based on normal mode analysis

SASDC36 – Structural and functional dissection of the DH and PH domains of oncogenic Bcr-Abl tyrosine kinase

s, nm-1

s, nm-1

Log I(s)

Δ/σ

+3

-3

χ2 = 1.5

SREFLEXSAS REfinement through FLEXibility based on normal mode analysis

SASDC36 – Structural and functional dissection of the DH and PH domains of oncogenic Bcr-Abl tyrosine kinase

χ2 = 1.0

s, nm-1

s, nm-1

Log I(s)

Δ/σ

+3

-3

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

15472368

Kikhney AG, Borges CR, Molodenskiy DS, Jeffries CM, Svergun DI (2020) Protein Science 29(1); 66-75

Thank you!

www.saxier.org/forumwww.sasbdb.orgbiosaxs.com