SAXS from biological macromolecules in solution: hybrid ... · macromolecules Large macromolecular...
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Solution Scattering from Biological Macromolecules Hybrid rigid body modelling Al Kikhney EMBL Hamburg
Transcript of SAXS from biological macromolecules in solution: hybrid ... · macromolecules Large macromolecular...
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
