Summary, perspectives, Q&A Dmitri Svergun, EMBL-Hamburg - - PowerPoint PPT Presentation

Summary, perspectives, Q&A

Dmitri Svergun, EMBL-Hamburg

Biological SAXS at ICAN in Moscow

• Acad. Boris K. Vainshtein,

ICAN director and Head of the laboratory of protein structure

• Prof. Lev A.Feigin, leader of the

group (then “sector”, then laboratory) of small-angle scattering

ICAN: SAXS diffractometers

AMUR scheme in one channel version (Sosfenov, Feigin, Bondarenko et al (1969)). The detector had to be moved after the measurement of each angular point (1-5 minutes per point) AMUR with a position-sensitive detector (Mogilevslkj, Dembo, Svergun, Feigin, (1984)) Proportional camera with a delay line, size 1* 10 cm2

Computers and people

Area: > 100 m* * 2 Operative memory: 16K 28,000 operations per second (28 kHz) Boris Schedrin, MSU, Faculty of Computational Mathematics and Cybernetics Samsung S10e Dimensions: 142,2 x 69,9 x 7,9 mm Operative memory: 8 GByte Octa-core Exynos 9820, 1800 MHz

SAXS on bacterial virus T7

Rolbin, Y . A., Svergun, D. I., Feigin, L. A., Gashpar, S., and Ronto, G. (1980) Structure of bacterial virus T7 according to small-angle x-ray scattering data, Doklady AN SSSR 255, 1497-1500.

Bacteriophage T7 is a DNA- containing virus that infects most strains of Escherichia coli. Molecular mass 5.6* 107 Da, size nearly 90 nm

• Prof. Györgyi Rontó, Dr. Katalin Tóth

Semmelweis University, Budapest

Spherical harmonics

• Prof. Heinrich Stuhrmann

Mainz University, ILL, EMBL, GKSS Research Center Geesthacht Contrast variation, Stuhrmann & Kirste (1965) Spherical harmonics (1970)

Structure of bacterial virus T7

Svergun, D.I., Feigin, L.A. & Schedrin, B.M. (1982) Acta Cryst. A38, 827 Agirrezabala, J. M. et al. & Carrascosa J.L. (2005) EMBO J. 24, 3820 SAXS, 1982 Cryo-EM, 2005 Pro-head Mature virus

DESY/EMBL, Hamburg

Michel Koch (EMBL) Heinrich Stuhrmann (GKSS)







N n n n

r c r p

1

) ( ) ( 

20 40 1 2 p(r) r, nm

Indirect Fourier transformation

 

)] ( [ ) ( ) ( min

2 exp

r p s I s I

calc

   

0.0 0.5 1.0 1.5 1 2 3 s, nm-1 lg I

Glatter O. (1977). J. Appl. Cryst., 10, 415 Svergun, D. Semenyuk,

• A. Feigin, L. (1988) Acta
• Cryst. A44, 244

Svergun, D. (1992) J.

• Appl. Cryst. 25, 495

Times Cited: 3,363 (from Google Scholar) “Perceptual criteria”

GNOM

Alexander Semenyuk

Position ( j ) = x( j ) = (phase assignments) Bead models were used for trial-and-error modeling in SAS since 1960-ies. Given the large number of model parameters M  (Dmax / r0)3  103 Monte-Carlo like approaches are to be used for automation Genetic algorithm (DALAI_GA)

• P. Chacón, F. Morán, J. F. Díaz, E. Pantos,

and J. M. Andreu (1998). Biophys. J., 74: 2760- 2775 Solvent Particle

2r0

A sphere of radius Dmax is filled by densely packed beads of radius r0<< Dmax

Dmax

   solvent if particle if 1

Bead (dummy atoms) models

Pablo Chacon (Madrid)

Ab initio program DAMMIN

Using simulated annealing, finds a compact dummy atoms configuration X that fits the scattering data by minimizing where  is the discrepancy between the experimental and calculated curves, P(X) is the penalty to ensure compactness and connectivity, > 0 its weight.

) ( )] , ( ), ( [ ) (

exp 2

X P X s I s I X f    

compact loose disconnected

Svergun, D.I. (1999) Biophys. J. 76, 2879-2886

Ribosome

Joachim Frank (Albany) Heinrich Stuhrmann (Geesthacht) Michel Koch (Hamburg) Igor Serdyuk (Pouschino) Knud Nierhaus (Berlin) Jan Skov Pedersen (Aarhus) Vladimir Volkov (Moscow)

Shape analysis for multi-component systems: MONSA principle

If multple scattering patterns are available for multi-component particle: not only shape but also internal structure can be reconstructed

Svergun, D.I. & Nierhaus, K.H. (2000) J. Biol. Chem. 275, 14432-14439

A+ B A B

Ribosome as seen by scattering

X-rays (EMBL) X-rays (EMBL)

Neutrons (Risoe, DK) Neutrons (Risoe, DK)

Svergun, D.I. & Nierhaus, K.H. (2000) J.Biol. Chem. 275, 14432

42 curves fitted simultaneously

Search volume: cryo-EM model (Frank, 1995)

Contrast variation in heavy water: 0% D2O 40% D2O 70% D2O

Native ribosome Hybrid ribosome

Solution crystal

X-ray and neutron scattering map of protein-RNA distribution in the 70S ribosome E. coli (left, resolution 3 nm) compared with later crystallographic models (right). Top, 30S subunit from Th. thermophilus, resolution 0.33 nm (Schluenzen, F , et al, & Yonath, A. (2000) Cell, 10, 615). Bottom, 50S subunit from H. marismortui, resolution 0.24 nm (Ban, N., Nissen, P ., Hansen, J., Moore, P .B. & Steitz, T.A. (2000) Science, 289, 905). 5 nm

Scattering from a macromolecule in solution

 Aa(s): atomic scattering in vacuum

 As(s): scattering from the excluded volume  Ab(s): scattering from the hydration shell

 



2 b b s s a 2

) ( A + ) ( A ) ( A = ) A( = I(s) s s s s  

CRYSOL (X-rays): Svergun, D., Barberato, C., Koch, M. H. (1995).

• J. Appl. Cryst. 28, 768 (3,043 citations in Google Scholar)

22 Sept 2018

Denser shell or floppy chains?

Giuseppe Zaccai Zehra Sayers

CRYSON (neutrons): Svergun DI, Richard S, Koch MHJ, Sayers Z,

Kuprin S, Zaccai G. (1998) P.N.A.S. USA, 95, 2267

Lysozyme: appears larger for X-rays Thioredoxine reductase : CRYSOL and smaller for neutrons in D2O and CRYSON fits with denser shell

Utilizing high resolution information

Pinotsis, N., Lange, S., Perriard, J.-C., Svergun, D.I. & Wilmanns, M. (2008) EMBO J . 27, 253-264

Biologically active dimer of myomesin-1

SAXS Experiment started: Sat 24-07-2004 at 21:09 Result obtained: Sat 24-07-2004 at 21:48

Nikos Pinotsis Matthias Wilmanns Crystallographic dimers

• f myomesin-1

Rigid body modelling

 Using spherical harmonics, the amplitude(s) of arbitrarily

rotated and displaced subunit(s) are analytically expressed via the initial amplitude and the six positional parameters: Clm(s) = Clm(Blm, , , , x, y, z).

 The scattering from the complex is then rapidly calculated as

Rotation: , ,  A Shift: x, y, z C B

 

 



 

  

* 2

) ( ) ( Re 4 ) ( ) (

l l lm lm B A

s C s A s I s I s I 

Svergun, D.I. (1991). J. Appl. Cryst. 24, 485-492

Interactive and local refinement

ASSA (SUN/SGI/DEC; 2000) MASSHA (Windiows, 2001)

Peter Konarev Mikhail Kozin Sasha Pajnkovich

SASpy (Pymol plugin, 2016)

s, nm-1

0.5 1.0 1.5 2.0

lg I, relative

8 9 10 11

Automated rigid body modelling

 SASREF fits (multiple X-ray and neutron) scattering curve(s) from partial constructs or contrast variation using simulated annealing  Requires models of subunits, builds interconnected models without steric clashes  Uses symmetry, distances (FRET or mutagenesis) relative orientation (RDC from NMR), if available

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

Maxim Petoukhov

 More recent additions are CREDO (adds missing portions), SASREFMX (accounts for partial dissociation).

Petoukhov, M.V. et al (2012)

• J. Appl. Cryst. 45, 342-350

Normal modes refinement

C.Gorba, O.Miyashita, F.Tama (2008) Biophys J. 94: 1589-99 A.Panjkovich, D.I.Svergun (2016) Phys Chem Chem Phys. 18, 5707-19

Sasha Pajnkovich Christian Gorba Karen Manalastas

Not only biological systems: magnetic iron oxide nanoparticles

Highly monodisperse NPs are coated by phospholipids with PEG tails to become soluble. Shtykova, E.V , Huang, X., Remmes, N., Baxter, D., Dixit, S., Stein, B., Dragnea, B., Svergun, D. I. & Bronstein, L. M. (2007) J. Phys. Chem. C, 111, 18078-18086

s, nm-1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 lg I, relative

• 1

1 2 3 4 5 1 2 s, nm-1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 lg I, relative

• 1

1 2 3 4 5 1 2 s, nm-1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 lg I, relative

• 2
• 1

1 2 3 4 5 1 2 3

shoulder 1st minimum

R, nm 5 10 15 20 p(R), relative 2 4 6 8 10 12

s, nm-1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 lg I, relative

• 2
• 1

1 2 3 4 5 1 2 3

shoulder 1st minimum

R, nm 2 4 6 8 10 p(R), relative 0.0 0.5 1.0 1.5

Ab initio analysis: peculiarities of

• rganization of different NPs

Rigid body analysis: equilibrium clusters of the NPs stabilized by magnetic interactions

Eleonora Shtykova ICRAS, Moscow Lyudmila Bronstein Indiana University

Assessment of flexibility: EOM



One generates a large pool covering the conformational space and selects sub-ensemble(s) such that their mixture fits the available experimental data The structural properties of the selected ensemble(s) are compared to those of the pool

Bernadó P , Mylonas E, Petoukhov MV , Blackledge M, Svergun DI. (2007) J Am Chem Soc. 129: 5656-64. Tria G, Mertens HD, Kachala M, Svergun DI. (2015) IUCrJ. 2: 207-17. Pau Bernadó Stratos Mylonas Giancarlo Tria Mikhail Kachala

All That SAS

Data processing and manipulations Ab initio modeling suite Rigid body refinement Analysis of mixtures

Franke et al (2017)

• J. Appl. Cryst. 50,

1212–1225

Distributed since 1999, on-line access since

• 2006. More than

15,000 users from

• ver 50 countries.

About 60% of all biological SAS publications cite ATSAS programs

First BioSAXS sample changer at X33

A liquid handling robot manufactured by Fraunhofer Institute, Stuttgart. Capacity: 2x96 samples (PCR stripes or well plates) plus 2x24 buffers. Sequential loading of a sample/buffer and cleaning/drying of the cell yields ca 6 min measuring circle.

Round, A. R., Franke, D., Moritz, S., Huchler, R., Fritsche, M., Malthan, D., Klaering, R., Svergun, D. I. & Roessle, M. (2008). J. Appl. Crystallogr. 41, 913. Adam Round Manfred Roessle

First Remote SAXS Experiment at X33

May, 26th, 2009, 12:30 CET: A remote SAXS experiment in the Nanyang Technological University's lobby (Singapore), during a biological SAXS Course. Left: remote access interface with cameras displaying the sample cell and the SAXS robot; right: a Skype window showing the members of the BioSAXS EMBL group monitoring the experiment in Hamburg.

October 21, 2012, 19:35

Last bunch of X33 users (EMBO SAXS Course) on the last day of DORIS-3 operation

High brilliance beamline P12 (Petra-3)

30

P12, a X33 reincarnation

• About 1013 photons/seconds in 200* 120 µm2
• Energy between 4 and 20 keV (0.6 to 3 Å)
• Divergence below 0.05* 0.05 mrad2
• Sample-detector distance between 1.5 and 6 m

Automation at P12 beamline

All the developments in high throughput and automation are first of all aimed at facilitating the operation and improving the life of the users

Daniel Franke Clement Blanchet Martin Schroer Nelly Hajizadeh Andrey Gruzinov

SEC-SAXS development

Coupling size-exclusion chromatography (SEC) with synchrotron SAXS, originally proposed by Mathew et al. (2004) J. Synch. Rad. 11, 314, and David & Pérez (2009) J. Appl. Cryst. 42, 892, helps to significantly improve the data quality. SEC-SAXS timeline at P12

Cy Jeffries Daniel Franke Sasha Panjkovich Rob Meijers Melissa Graewert

Dissemination and training

 Biannual EMBO practical

courses on Solution scattering from biological macromolecules (Hamburg, from 2001)

 Biannual EMBO Global Lecture Courses

• n Structural and biophysical methods

for biological macromolecules in solution (from 2010; in Brazil, China, India, Korea, Singapore)

Plus regular lectures and tutorials by BioSAXS group members at numerous courses and conferences worldwide

SAXIER forum (2005-now)

Moderated by Al Kikhney

• Over 1300 registered users,
• ver 900 active users
• Over 6300 posts in 1800 topics

DARA: a database with over 150,000 SAXS patterns for rapid screening

• A. Sokolova et al. (2003). J Appl.
• Cryst. 36, 865-868

A.G. Kikhney, A. Panjkovich, A.V. Sokolova, D.I. Svergun (2016) Bioinformatics, 32, 616-618.

Al Kikhney Anna Sokolova

Valentini E, Kikhney AG, Previtali G, Jeffries CM & Svergun DI. (2015) Nucleic Acids Res. 43, D357-63.

Erica Valentini Cy Jeffries Al Kikhney

Future of biological SAS

 Structural methods, especially MX,

now face the ‘resolution revolution’ challenge from cryo-EM

 For SAS, studies of the overall structure

and transitions will stay important thanks to non-restrictive sample requirements, speed, simplicity of use and automation of modern instruments

 Expected priority directions:

 High throughput screening with synchrotron SAXS/WAXS  Equilibrium mixtures and membrane proteins, with online SEC-

SAXS/SANS and contrast variation SANS

 Flexible fragments and intrinsically disordered proteins  Time-resolved studies of processes, from sub-ms ((un)folding) to

hours (oligomerization, fibrillation)

Acknowledgments



All BioSAXS Group and especially C. Blanchet, C. Jeffries, D.

Franke, A. Gruzinov, H.Mertens, A.Kikhney, M. Graewert



Former BioSAXS Group members and especially

• M. Roessle, M. Petoukhov, P

. Konarev, E.Valentini, M.Kachala, G.Tria, A.Tuukkanen, A. Panjkovich, P . Bernadó, A. Round



EMBL TL/ GLs and especially S.Fiedler, M. Garcia-Alai, T.Schneider, R. Meijers, C. Loew, M. Wilmanns, E. Lemke,

G.Kleywegt, F .Cipriani



External collaborators and especially P

. Vachette (Paris), K.Tóth (Budapest), J. Pedersen (Aarhus), J.Zaccai (Grenoble),

• K. Nierhaus (Berlin), J. Trewhella (Sydney), P

. Tompa (Brussels), J. Westbrook (RCSB Rutgers), V . Volkov and E. Shtykova (Moscow), L. Bronstein (Indiana), C.Betzel (Hamburg), S. Savvides (Gent), K. Djinovic-Carugo (Vienna),

• M. Vanoni (Milan), Z. Sayers (Istanbul)

Courtesy of F.Gabel, IBS, Grenoble

Future of biological SAS

Courtesy of M.Graewert, EMBL-HH

SAXS/SANS. The Choice of the New Generation

Thank you!