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

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

SAXS data from macromolecules in solution log I(s) experimental SAXS pattern experimental SAXS pattern nm -1

SAXS data from macromolecules in solution log I(s) experimental SAXS pattern experimental SAXS pattern calculated from model nm -1

SAXS data from macromolecules in solution log I(s) experimental SAXS pattern experimental SAXS pattern calculated from model nm -1

Computing SAS from an atomic model log I(s) A a ( s ): atomic scattering in vacuum nm -1

Computing 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 CRYSOL (X-rays): Svergun et al. (1995) J. Appl. Cryst. 28, 768 nm -1 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 • Command-line interface • Web interface https://www.embl-hamburg.de/biosaxs/atsas-online/crysol.php • PyMOL plugin SASpy CRYSOL (X-rays): Svergun et al. (1995) J. Appl. Cryst. 28, 768

Running CRYSOL

Running CRYSOL • Command-line interface • Web interface https://www.embl-hamburg.de/biosaxs/atsas-online/crysol.php • PyMOL plugin SASpy • PRIMUS CRYSOL (X-rays): Svergun et al. (1995) J. Appl. Cryst. 28, 768

Goodness of fit Log I(s) Significance level α = 1% m 100 0.68 < χ 2 < 1.41 χ 2 = 2.4 500 0.85 < χ 2 < 1.17 1000 0.89 < χ 2 < 1.12 2000 0.92 < χ 2 < 1.08 – I fit ( s ) • I exp ( s ) s, nm -1 s s reduced s

Goodness of fit Log I(s) s, nm -1 +3 Error-weighted DATCMP Franke et al. (2015) Δ/σ residual difference plot Correlation Map… -3 Nat. Methods 12, 419-422 s, nm -1

Rigid body modelling Log I(s) s, Å -1 SASREF: Petoukhov & Svergun (2005) Biophys J. 89 , 1237; (2006) Eur. Biophys. J . 35 , 567.

Rigid body modelling Log I(s) χ 2 = 54 fit s, Å -1 SASREF: Petoukhov & Svergun (2005) Biophys J. 89 , 1237; (2006) Eur. Biophys. J . 35 , 567.

Rigid body modelling Log I(s) χ 2 = 13 fit s, Å -1 SASREF: Petoukhov & Svergun (2005) Biophys J. 89 , 1237; (2006) Eur. Biophys. J . 35 , 567.

Rigid body modelling Log I(s) χ 2 = 1.06 fit s, Å -1 SASREF: Petoukhov & Svergun (2005) Biophys J. 89 , 1237; (2006) Eur. Biophys. J . 35 , 567.

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 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 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 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 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 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.

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 SASBDB project page

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 Log I(s) Chi 2 = 390 s, nm -1

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 Log I(s) Chi 2 = 2.3 s, nm -1 SASBDB: SASDAU8

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 PDBePISA ebi.ac.uk/pdbe/pisa / PDB: 4ow2

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 PDBePISA ebi.ac.uk/pdbe/pisa / PDB: 4ow2

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 Log I(s) s, nm -1

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 Log I(s) s, nm -1 SASBDB: SASDAV8 zhanglab.ccmb.med.umich.edu/I-TASSER/

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 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 Log I(s) SASBDB: SASDAW8 s, nm -1

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

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

CORAL Modelling of multidomain protein complexes against multiple data sets Loops library CORAL: Petoukhov et al. (2012) J. Appl. Cryst. 45, 342-350

CORAL Modelling of multidomain protein complexes against multiple data sets 22 22 34 kDa 13

CORAL

CORAL Log I(s) s, nm -1 +3 Δ/σ -3 SASBDB: SASDDG9 s, nm -1

Flexible refinement 4ake 1ake

SREFLEX SAS RE finement through FLEX ibility based on normal mode analysis Log I(s) s, Å -1 SREFLEX: Panjkovich A. and Svergun D.I. (2016) Phys. Chem. Chem. Phys. 18, 5707-5719