Hybrid rigid body modelling Al Kikhney EMBL Hamburg Hybrid rigid - - PowerPoint PPT Presentation

▶
hybrid rigid body modelling
SMART_READER_LITE
LIVE PREVIEW

Hybrid rigid body modelling Al Kikhney EMBL Hamburg Hybrid rigid - - PowerPoint PPT Presentation

Dec 07, 2022 162 likes •750 views

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

slide-1
SLIDE 1

Solution Scattering from Biological Macromolecules

Hybrid rigid body modelling

Al Kikhney EMBL Hamburg

slide-2
SLIDE 2

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
slide-3
SLIDE 3

nm-1 experimental SAXS pattern experimental SAXS pattern

SAXS data from macromolecules in solution

log I(s)

slide-4
SLIDE 4

nm-1 experimental SAXS pattern experimental SAXS pattern calculated from model

SAXS data from macromolecules in solution

log I(s)

slide-5
SLIDE 5

SAXS data from macromolecules in solution

nm-1 experimental SAXS pattern experimental SAXS pattern calculated from model log I(s)

slide-6
SLIDE 6

nm-1 log I(s)

Computing SAS from an atomic model

Aa(s): atomic scattering in vacuum

slide-7
SLIDE 7

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

slide-8
SLIDE 8

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

slide-9
SLIDE 9

Running CRYSOL

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

  • Command-line interface
  • Web interface

https://www.embl-hamburg.de/biosaxs/atsas-online/crysol.php

  • PyMOL plugin SASpy
slide-10
SLIDE 10

Running CRYSOL

slide-11
SLIDE 11

Running CRYSOL

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

  • Command-line interface
  • Web interface

https://www.embl-hamburg.de/biosaxs/atsas-online/crysol.php

  • PyMOL plugin SASpy
  • PRIMUS
slide-12
SLIDE 12
slide-13
SLIDE 13
slide-14
SLIDE 14
slide-15
SLIDE 15

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 fit

Log I(s)

χ2 = 2.4

s, nm-1

s s

s

– Ifit(s)

  • Iexp(s)
slide-16
SLIDE 16

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

slide-17
SLIDE 17
slide-18
SLIDE 18
slide-19
SLIDE 19

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

Rigid body modelling

Log I(s) s, Å-1

slide-20
SLIDE 20

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

slide-21
SLIDE 21

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

slide-22
SLIDE 22

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

slide-23
SLIDE 23

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-24
SLIDE 24

Rigid body modelling

Interconnectivity Absence of steric clashes Symmetry

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

SASREF

slide-25
SLIDE 25

Rigid body modelling

Interconnectivity Absence of steric clashes Symmetry

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

SASREF

slide-26
SLIDE 26

Rigid body modelling

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

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

SASREF

slide-27
SLIDE 27

Rigid body modelling

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.

SASREF

slide-28
SLIDE 28

Rigid body modelling

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.

SASREF

slide-29
SLIDE 29

Rigid body modelling

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.

SASREF

slide-30
SLIDE 30

Rigid body modelling

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 Reconstruction of missing fragments

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

SASREF

slide-31
SLIDE 31
slide-32
SLIDE 32
slide-33
SLIDE 33
slide-34
SLIDE 34
slide-35
SLIDE 35

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

slide-36
SLIDE 36

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

slide-37
SLIDE 37

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

slide-38
SLIDE 38

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/

slide-39
SLIDE 39

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/

slide-40
SLIDE 40

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

slide-41
SLIDE 41

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

slide-42
SLIDE 42

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

+

slide-43
SLIDE 43

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

slide-44
SLIDE 44

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

slide-45
SLIDE 45
  • 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 fragments

BUNCH

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

slide-46
SLIDE 46

CORAL

Loops library

Modelling of multidomain protein complexes against multiple data sets

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

slide-47
SLIDE 47

CORAL

Modelling of multidomain protein complexes against multiple data sets

22 22 13 34 kDa

slide-48
SLIDE 48

CORAL

slide-49
SLIDE 49

CORAL

s, nm-1 s, nm-1 Log I(s) Δ/σ +3

  • 3

SASBDB: SASDDG9

slide-50
SLIDE 50

Flexible refinement

4ake 1ake

slide-51
SLIDE 51

SREFLEX

SAS 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

slide-52
SLIDE 52

SREFLEX

Estimating 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

slide-53
SLIDE 53

SREFLEX

SAS 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

slide-54
SLIDE 54

SREFLEX

SAS 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
slide-55
SLIDE 55

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-56
SLIDE 56

1547 2368

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

slide-57
SLIDE 57

Thank you!

www.saxier.org/forum www.sasbdb.org biosaxs.com