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 Prof. Lev A.Feigin, leader of the Acad. Boris K. Vainshtein, group (then “sector”, then ICAN director and Head of the laboratory) of small-angle scattering laboratory of protein structure

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 cm 2

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

SAXS on bacterial virus T7 Bacteriophage T7 is a DNA- containing virus that infects most strains of Escherichia coli . Molecular mass 5.6* 10 7 Da, size nearly 90 nm Prof. Györgyi Rontó, Dr. Katalin Tóth Semmelweis University, Budapest 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.

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

DESY/EMBL, Hamburg Michel Koch Heinrich Stuhrmann (EMBL) (GKSS)

Indirect Fourier transformation Svergun, D. Semenyuk, Glatter O. (1977). J. Appl. Cryst., 10, 415 A. Feigin, L. (1988) Acta Cryst. A44, 244 N    ( ) ( ) lg I p r c r n n  1 3 n p(r) 2 2 1 0.0 0.5 1.0 1.5 s, nm-1 1   0 2     0 20 40 min ( ) ( ) [ ( )] I s I s p r r, nm Alexander Semenyuk exp calc “Perceptual criteria” GNOM Svergun, D. (1992) J. Appl. Cryst. 25, 495 Times Cited: 3,363 (from Google Scholar)

Bead (dummy atoms) models A sphere of radius D max is filled by densely packed beads of radius r 0 << D max  1 if particle Position ( j ) = x ( j ) =   0 if solvent Solvent (phase assignments) Pablo Chacon (Madrid) Bead models were used for trial-and-error modeling in SAS since 1960-ies. Given the large number of model parameters M  (D max / r 0 ) 3  10 3 Particle Monte-Carlo like approaches are to be used for automation Genetic algorithm (DALAI_GA) 2r 0 P. Chacón, F. Morán, J. F. Díaz, E. Pantos, D max and J. M. Andreu (1998). Biophys. J. , 74: 2760- 2775

Ab initio program DAMMIN Using simulated annealing, finds a compact dummy atoms configuration X that fits the scattering data by minimizing     2 ( ) [ ( ), ( , )] ( ) f X I s I s X P X exp where  is the discrepancy between the experimental and calculated curves, P(X) is the penalty to ensure compactness and connectivity,  > 0 its weight. compact loose disconnected Svergun, D.I. (1999) Biophys. J. 76 , 2879-2886

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

Shape analysis for multi-component systems: MONSA principle A+ B A B 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

Ribosome as seen by scattering Neutrons Neutrons (Risoe, DK) (Risoe, DK) Contrast variation in heavy water: 42 curves fitted 0% D 2 O 40% D 2 O 70% D 2 O simultaneously Native ribosome Hybrid ribosome X-rays X-rays (EMBL) (EMBL) Search volume: cryo-EM model (Frank, 1995) Svergun, D.I. & Nierhaus, K.H. (2000) J.Biol. Chem. 275 , 14432

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 5 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).

Scattering from a macromolecule in solution 2 2    I(s) = A( s ) = A ( s ) A ( s ) + A ( s ) a s s b b    A a ( s ) : atomic scattering in vacuum  A s ( s ) : scattering from the excluded volume  A b ( s ) : scattering from the hydration shell 22 Sept 2018 CRYSOL (X-rays): Svergun, D., Barberato, C., Koch, M. H. (1995). J. Appl. Cryst. 28 , 768 ( 3,043 citations in Google Scholar)

Denser shell or floppy chains? Zehra Sayers Giuseppe Zaccai Lysozyme: appears larger for X-rays Thioredoxine reductase : CRYSOL and smaller for neutrons in D 2 O and CRYSON fits with denser shell CRYSON ( neutrons): Svergun DI, Richard S, Koch MHJ, Sayers Z, Kuprin S, Zaccai G. (1998) P.N.A.S. USA , 95 , 2267

Utilizing high resolution information 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 Crystallographic dimers of myomesin-1 Nikos Pinotsis Matthias Wilmanns Pinotsis, N., Lange, S., Perriard, J.-C., Svergun, D.I. & Wilmanns, M. (2008) EMBO J . 27, 253-264

Rigid body modelling Shift: x, y, z A B C Rotation:  ,  ,   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: C lm (s) = C lm (B lm ,  ,  ,  , x, y, z).  The scattering from the complex is then rapidly calculated as    l        2 * ( ) ( ) 4 Re ( ) ( ) I s I s I s A s C s A B lm lm  0 l Svergun, D.I. (1991). J. Appl. Cryst. 24 , 485-492

Interactive and local refinement ASSA (SUN/SGI/DEC; 2000) Peter Konarev Mikhail Kozin Sasha Pajnkovich SASpy (Pymol plugin, 2016) MASSHA (Windiows, 2001)

Automated rigid body modelling  SASREF fits (multiple X-ray and neutron) scattering curve(s) from partial constructs or contrast variation Maxim using simulated annealing Petoukhov  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.  More recent additions are lg I, relative CREDO (adds missing 11 portions), SASREFMX (accounts for partial 10 dissociation). 9 Petoukhov, M.V. et al (2012) 8 J. Appl. Cryst. 45, 342-350 0.5 1.0 1.5 2.0 s, nm -1

Normal modes refinement Sasha Pajnkovich Christian Gorba Karen Manalastas 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

Not only biological systems: magnetic iron oxide nanoparticles lg I, relative lg I, relative Lyudmila Bronstein Eleonora Shtykova shoulder 5 5 1 Indiana University ICRAS, Moscow 1 shoulder 2 4 4 1st minimum 2 3 3 Highly monodisperse NPs are coated 3 3 1st minimum 2 2 by phospholipids with PEG tails to p(R), relative p(R), relative 1 1 become soluble. 12 1.5 10 8 0 0 1.0 6 4 Rigid body analysis: equilibrium 0.5 2 -1 -1 0 0.0 0 5 10 15 20 R, nm 0 2 4 6 8 10 R, nm clusters of the NPs stabilized by -2 -2 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 magnetic interactions s, nm -1 s, nm -1 Ab initio analysis: peculiarities of lg I, relative lg I, relative organization of different NPs 5 5 1 1 2 2 4 4 3 3 2 2 1 1 0 0 -1 -1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 s, nm -1 s, nm -1 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

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 Giancarlo Tria Pau Bernadó Stratos Mylonas 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. Mikhail Kachala

All That SAS Franke et al (2017) J. Appl. Cryst. 50, 1212–1225 Data processing and manipulations Ab initio modeling suite Rigid body refinement Analysis of mixtures Distributed since 1999, on-line access since 2006. More than 15,000 users from over 50 countries. About 60% of all biological SAS publications cite ATSAS programs