Rigid body refinement (basics) D.Svergun, EMBL-Hamburg Shapes from - - PowerPoint PPT Presentation

Rigid body refinement (basics)

D.Svergun, EMBL-Hamburg

Shapes from recent projects at EMBL-HH

Domain and quaternary structure Complexes and assemblies Domain and quaternary structure Complexes and assemblies

Dcp1/Dcp2 complex S-layer proteins Toxin B α-synuclein oligomers She et al, Mol Cell (2008) Fagan et al Mol. Microbiol (2009) Albesa-Jové et al JMB (2010) Giehm et al PNAS USA (2011)

In most cases, high resolution models are drawn inside

Structural transitions Flexible/transient systems

Src kinase Cytochrome/adrenodoxin Microbiol (2009) Complement factor H

are drawn inside the shapes

Bernado et al JMB (2008) Xu et al JACS (2008) Morgan et al NSMB (2011)

Using SAXS with MX/NMR: Using SAXS with MX/NMR: h b id d lli h b id d lli ‘hybrid’ modelling ‘hybrid’ modelling

Model building where high resolution portions are Model building where high resolution portions are positioned to fit the low resolution SAXS data

The use of high resolution models in SAS The use of high resolution models in SAS

Th i l d l l Theoretical model or complete crystal structure available Validation in solution Incomplete structure available Addition of missing loops/domains Structure of subunits available Rigid body model of the complex Structure of domains and multiple curves available Model of the domain structure structure

How to compute SAS from atomic model How to compute SAS from atomic model

Isolution(s) Isolvent (s) Iparticle(s)

♦ To obtain scattering from the particles, solvent

scattering must be subtracted to yield effective density di t ib ti Δ ( ) h i th tt i distribution Δρ = <ρ(r) - ρs>, where ρs is the scattering density of the solvent Further the bound solvent density may differ from

♦ Further, the bound solvent density may differ from

that of the bulk

Scattering from a macromolecule in solution Scattering from a macromolecule in solution

Ω Ω

−

2 b b s s a 2

) ( A + ) ( A ) ( A = ) A( = I(s) s s s s δρ ρ

♦ Aa(s): atomic scattering in vacuum

♦ As(s): scattering from the excluded volume ♦ Ab(s): scattering from the hydration ♦ Ab(s): scattering from the hydration shell CRYSOL (X ) 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

The use of multipole expansion The use of multipole expansion

Ω Ω

−

2 b s a 2

) B( + ) E( ) ( A = ) A( = I(s) s s s s δρ ρ If the intensity of each contribution is represented using spherical harmonics

2 2

) ( 2 ) ( s A s I

lm l l m l

∑ ∑

− = ∞ =

= π

the average is performed analytically:

L l

∑ ∑

= − =

+ − =

L l l l m lm lm lm

s B s E s A s I

2 2

) ( ) ( ) ( 2 ) ( δρ ρ π

This approach permits to further use rapid algorithms for rigid body refinement

CRYSOL CRYSOL and and CRYSON CRYSON: : X-

• ray and

ray and neutron scattering from macromolecules neutron scattering from macromolecules

∑ ∑

+

L l

s B s E s A s I

2 2

) ( ) ( ) ( 2 ) ( δρ ρ π

The

The programs programs: :

∑ ∑

= − =

+ − =

l l m lm lm lm

s B s E s A s I ) ( ) ( ) ( 2 ) ( δρ ρ π

either

either fit fit the the experimental experimental data data by by varying varying the the density density

• f
• f the

the hydration hydration layer layer δρ δρ (affects (affects the the third third term) term) and and y y ρ ( ) the the total total excluded excluded volume volume (affects (affects the the second second term) term)

• r
• r predict

predict the the scattering scattering from from the the atomic atomic structure structure using using default default parameters parameters (theoretical (theoretical excluded excluded volume volume using using default default parameters parameters (theoretical (theoretical excluded excluded volume volume and and bound bound solvent solvent density density of

• f 1

1. .1 1 g/cm g/cm3 )

provide

provide output

• utput files

files (scattering (scattering amplitudes) amplitudes) for for rigid rigid b d b d fi t fi t ti ti body body refinement refinement routines routines

compute

compute particle particle envelope envelope function function F( F(ω)

Scattering components (lysozyme) Scattering components (lysozyme)

1) 1)

Atomic Atomic

1) 1)

Atomic Atomic

2) 2)

Shape Shape

3) 3)

Border Border

3) 3)

Border Border

4) 4)

Difference Difference

lg I, relative

Effect of the hydration shell, X Effect of the hydration shell, X-

• rays

rays

3

Experimental data Fit with shell Fit without shell

2 3

Lysozyme

1 2

Hexokinase EPT

• 1

PPase

s, nm-1

1 2 3 4

Denser shell or floppy chains: X-rays versus neutrons X rays versus neutrons

12 Scattering length density, 1010cm-2

♦ For X-rays: both lead to

10 12 floppy side chains denser solvent layer solvent density

similar effect (particle appears larger) ♦ Floppy chains would in

6 8 protein density

• ppy s de c a

s

♦ Floppy chains would in all cases increase the apparent particle size ♦ Neutrons in H O (shell):

4 6

♦ Neutrons in H2O (shell): particle would appear nearly unchanged

2

♦ Neutrons in D2O (shell): particle would appear smaller than the atomic model

• 2

SAXS SANS in H2O SANS in D2O

model

X-rays versus neutrons: experiment

lg I, relative

lg I, relative

1

1

Neutrons, D2O Neutrons, H2O X-rays

• 1

X-rays

• 1
• 3
• 2

Neutrons, D2O Neutrons, H2O

• 2

2 4

• 3

s, nm-1

1 2 3

s, nm-1

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

Other approaches/programs I Other approaches/programs I

The

The ‘cube ‘cube method’ method’ (Luzzati Luzzati et et al, al, 1972 1972; ; Fedorov Fedorov and and Pavlov, Pavlov, 1983 1983; ; M Mü üller ller, , 1983 1983) ) ensures ensures uniform uniform filling filling of

• f the

the excluded excluded volume volume. . Could/should/must Could/should/must be be superior superior over

• ver

the the effective effective atomic atomic factors factors method method at at higher higher angles angles. .

CRYDAM

CRYDAM (still unpublished) (still unpublished)

lg I, relative

♦ Represents hydration shell by dummy water atoms

2

dummy water atoms ♦ Handles proteins, carbohydrates, nucleic acids and their complexes ♦ Is applicable for wide angle

1 X-ray data, lysozyme Fit by CRYSOL Fit by CRYDAM

♦ Is applicable for wide angle scattering range Malfois, M. & Svergun, D.I. (2001), to be submitted

CRYSOL 3.0

CRYSOL 3.0 (is coming) (is coming)

s, nm-1 5 10

• be sub

ed

Other approaches/programs II Other approaches/programs II

• J.

. Bardhan Bardhan, , S S. . Park Park and and L L. . Makowski Makowski ( (2009 2009) ) SoftWAXS SoftWAXS: : a a computational computational tool tool for for modeling modeling wide wide-

• angle

angle X X-

• ray

ray solution solution scattering scattering from from biomolecules biomolecules J. . Appl Appl. . Cryst

• Cryst. 42

42, 932 932-

• 943

943 - A program program to to compute compute WAXS WAXS y , p g p g p

• Schneidman

Schneidman-

• Duhovny

Duhovny D, D, Hammel Hammel M, M, Sali Sali A A. . ( (2010 2010) ) FoXS FoXS: : a a web web server server for for rapid rapid computation computation and and fitting fitting of

• f SAXS

SAXS profiles profiles. . Nucleic Nucleic Acids Acids Res Res. . 38 38 Suppl Suppl: :W W540 540-

• 4

4. . - Debye Debye-

• like

like computations, computations, Web Web server server Grishaev Grishaev A Guo Guo L Irving Irving T Bax Bax A (2010 2010) Improved Improved Fitting Fitting of

• f Solution

Solution X

• Grishaev

Grishaev A, A, Guo Guo L, L, Irving Irving T, T, Bax Bax A. (2010 2010) Improved Improved Fitting Fitting of

• f Solution

Solution X- ray ray Scattering Scattering Data Data to to Macromolecular Macromolecular Structures Structures and and Structural Structural Ensembles Ensembles by by Explicit Explicit Water Water Modeling Modeling. . J Am Am Chem Chem Soc Soc. . 132 132, , 15484 15484-

• 6

6. . - Generate Generate bulk bulk and and bound bound waters waters with with MD, MD, do do fit fit the the data data to to the the model model

• Poitevin

Poitevin F, F, Orland Orland H, H, Doniach Doniach S, S, Koehl Koehl P, P, Delarue Delarue M M ( (2011 2011) ). . AquaSAXS AquaSAXS: : a a web web server server for for computation computation and and fitting fitting of

• f SAXS

SAXS profiles profiles with with non non-

• uniformally

uniformally hydrated hydrated atomic atomic models models. . Nucleic Nucleic Acids Acids. . Res Res. . 39 39, , W W184 184-

• W189

189 - Generate Generate waters waters around around proteins proteins using using MD MD (AquaSol AquaSol program) program) W189 189 - Generate Generate waters waters around around proteins proteins using using MD MD (AquaSol AquaSol program) program)

• Virtanen

Virtanen JJ, JJ, Makowski Makowski L, L, Sosnick Sosnick TR, TR, Freed Freed KF KF. . ( (2011 2011) ) Modeling Modeling the the hydration hydration layer layer around around proteins proteins: : applications applications to to small small-

• and

and wide wide-

• angle

angle x x-

• ray

ray scattering scattering. . Biophys Biophys J J. . 101 101, 2061 2061-

• 9

9. . - Use Use a a “ “HyPred HyPred solvation solvation” ” model model to to generate generate the the shell, shell, geared geared towards towards WAXS WAXS.

DARA, a database for rapid DARA, a database for rapid characterization of proteins characterization of proteins characterization of proteins characterization of proteins

http://dara.embl-hamburg.de/ About 15000 atomic models of biologically active molecules are generated from the entries are generated from the entries in Protein Data Bank and the scattering patterns computed by CRYSOL Rapidly identifies proteins with similar shape (from low similar shape (from low resolution data) and neighbors in structural organization (from higher resolution data) higher resolution data)

Sokolova, A.V., Volkov, V.V. & Svergun, D.I. (2003) J. Appl. Crystallogr. 36, 865-868

Validation of high resolution models Validation of high resolution models

Crystallographic packing forces are Packing forces in the crystal restrict the Crystallographic packing forces are comparable with the intersubunit

• interactions. The solution structures
• f

multisubunit macromolecules Packing forces in the crystal restrict the allosteric transition in aspartate transcarbamylase

• f

multisubunit macromolecules could be significantly different from those in the crystal

Svergun, D.I., Barberato, C., Koch, M.H.J., Fetler, L. & Vachette, P. (1997). Proteins, 27, 110-117

Validation of high resolution models Validation of high resolution models

lg I, relative

2 1 SAXS experiment Fit by 1yzb Fit by 2aga 0.0 0.2 0.4 0.6 0.8

NMR models of the Josephin domain of ataxin-3: red curve and chain: 1yzb, Nicastro et al. (2005) PNAS USA 102, 10493; blue curve and chain: 2aga, Mao et al (2005) PNAS USA 102 12700

s, A-1

• Nicastro, G., Habeck, M., Masino, L., Svergun, D.I. & Pastore, A.

(2006) J. Biomol. NMR, 36, 267.

Mao et al. (2005) PNAS USA 102, 12700.

Domain Closure in 3-Phosphoglycerate Kinase

Closure of the two domains of PGK upon substrate binding is essential for the enzyme Closure of the two domains of PGK upon substrate binding is essential for the enzyme

• function. Numerous crystal structures do not yield conclusive answer, which conditions

are required for the closure A SAXS fingerprint of A SAXS fingerprint of

• pen/closed conformation

(human PGK) SAXS proves that binding of both substrates induces the closure

Pig PGK Bs PGK Pig PGK Tm PGK Tb PGK Ligands/ Parameters Substr. free MgADP binary MgATP binary 3-PG binary

atern1 atern2 atern1 atern2

N 2 746 4 332 3 524 3 158 3 664 4 767 9 135 9 560

the closure

No 2.746 4.332 3.524 3.158 3.664 4.767 9.135 9.560 3-PG 2.678 5.329 3.297 1.958 3.655 4.234 6.052 6.125 MgATP 3.855 2.848 2.409 3.389 7.827 7.766 3.179 3.910 MgADP 1.486 3.235 1.627 1.140 1.780 2.463 5.151 6.193 MgATP*3-PG 6.140 6.044 4.656 5.307 5.146 4.805 2.247 1.611 MgADP*3-PG 2.303 3.522 2.795 2.049 2.712 2.810 2.018 2.922

Varga, A., Flachner, B., Konarev, P., Gráczer, E., Szabó, J., Svergun, D., Závodszky, P. & Vas, M. (2006) FEBS Lett. 580, 2698-2706.

Rg (theor), A 24.25 24.34 24.02 23.97 24.24 24.16 23.26 22.64

Identification of biologically active oligomers Identification of biologically active oligomers

Biologically active dimer of myomesin-1 Experiment started: 24-07-2004 at 21:09 Final result obtained: 24-07-2004 at 21:48

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

Quaternary structure of the human Cdt1- Geminin complex regulates DNA replication licensing complex regulates DNA replication licensing

In eukaryotes, DNA rereplication is prevented by control of the assembly f li i l ( RC )

Lee et al (2004), Nature, 430, 913

• f prereplicative complexes (pre-RCs)
• nto chromatin. Cdt1 is a key

component of the pre-RC assembly. Timely inhibition of Cdt1 by Geminin is

tGeminin dimer + tCdt monomer

Timely inhibition of Cdt1 by Geminin is essential to this DNA replication licensing. SAXS identifies crystallization conditions for the complex

• V. De Marco, P. J. Gillespie, A. Lib, N. Karantzelis, E. Christodouloua, R. Klompmaker, S.

van Gerwen, A. Fish, M. V. Petoukhov, M. S. Iliou, Z. Lygerou, R. H. Medema, J. J. Blow, D. I. Svergun, S. Taraviras & A. Perrakis (2009) PNAS USA, 106, 19807

Quaternary structure of the human Cdt1- Geminin complex regulates DNA replication licensing complex regulates DNA replication licensing

In eukaryotes, DNA rereplication is prevented by control of the assembly f li i l ( RC )

• f prereplicative complexes (pre-RCs)
• nto chromatin. Cdt1 is a key

component of the pre-RC assembly. Timely inhibition of Cdt1 by Geminin is Timely inhibition of Cdt1 by Geminin is essential to this DNA replication licensing. The mechanism of DNA licensing inhibition by Geminin, is analyzed by combining MX, SAXS and functional

• studies. The Cdt1:Geminin complex

can e ist in t o distinct forms a can exist in two distinct forms, a ‘‘permissive’’ heterotrimer and an ‘‘inhibitory’’ heterohexamer.

• V. De Marco, P. J. Gillespie, A. Lib, N. Karantzelis, E. Christodouloua, R. Klompmaker, S.

van Gerwen, A. Fish, M. V. Petoukhov, M. S. Iliou, Z. Lygerou, R. H. Medema, J. J. Blow, D. I. Svergun, S. Taraviras & A. Perrakis (2009) PNAS USA, 106, 19807

The idea of rigid body modeling The idea of rigid body modeling The idea of rigid body modeling The idea of rigid body modeling

• The structures of two subunits

in reference positions are known known.

• Arbitrary

complex can be constr cted b mo ing and constructed by moving and rotating the second subunit.

• This
• peration

depends

• n

three Euler rotation angles and three Cartesian shifts. three Cartesian shifts.

The idea of rigid body modeling The idea of rigid body modeling The idea of rigid body modeling The idea of rigid body modeling

• The structures of two subunits

in reference positions are known known.

• Arbitrary

complex can be constr cted b mo ing and constructed by moving and rotating the second subunit.

• This
• peration

depends

• n

three Euler rotation angles and three Cartesian shifts. three Cartesian shifts.

Equation for rigid body modeling Equation for rigid body modeling

Rotation: A Shift: x, y, z C B

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

Rotation: α, β, γ

Using spherical harmonics, the amplitude(s) of arbitrarily Using spherical harmonics, the amplitude(s) of arbitrarily rotated and displaced subunit(s) are analytically expressed rotated and displaced subunit(s) are analytically expressed via via the initial amplitude and the six positional parameters: the initial amplitude and the six positional parameters: Clm

lm(s) =

(s) = C (B (B β ) Clm

lm(B

(Blm

lm,

, α, , β, , γ, x, y, z). , x, y, z). The scattering from the complex is then rapidly calculated as The scattering from the complex is then rapidly calculated as

( )

[ ]

∑∑

∞

+ + =

* 2

) ( ) ( Re 4 ) ( ) (

l l lm lm B A

s C s A s I s I s I π

− l

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

Constraints for rigid body modelling Constraints for rigid body modelling

Interconnectivity Interconnectivity Absence of steric clashes Absence of steric clashes Symmetry Symmetry Symmetry Symmetry Intersubunit contacts Intersubunit contacts (from chemical shifts by (from chemical shifts by NMR or mutagenesis) NMR or mutagenesis) NMR or mutagenesis) NMR or mutagenesis) Distances between Distances between residues (FRET or residues (FRET or mutagenesis) mutagenesis) g ) g ) Relative orientation of Relative orientation of subunits (RDC by NMR) subunits (RDC by NMR) Scattering data from Scattering data from subcomplexes subcomplexes

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

• Eur. Biophys. J.

. 35 35, , 567. 567.

Interactive and local refinement Interactive and local refinement

♦ ASSA (SUN/SGI/DEC) ♦ MASSHA (Win9x/NT/2000)

Kozin & Svergun (2000). J. Appl.

• Cryst. 33, 775-777

Konarev, Petoukhov & Svergun (2001).

• J. Appl. Cryst. 34, 527-532

EPSPS

Manual refinement: quaternary structure of the Manual refinement: quaternary structure of the dimeric dimeric α crystallin domain crystallin domain dimeric dimeric α-crystallin domain crystallin domain

Feil, I.K., Malfois, M., Hendle, J., van der Zandt, H. & Svergun, D.I. (2001) J. Biol. Chem. 276, 12024-12029

Global rigid body modelling (SASREF) Global rigid body modelling (SASREF)

Fit ( lti l X Fit ( lti l X d t ) tt i ( ) f ti l d t ) tt i ( ) f ti l Fits (multiple X Fits (multiple X-ray and neutron) scattering curve(s) from partial ray and neutron) scattering curve(s) from partial constructs or contrast variation using simulated annealing constructs or contrast variation using simulated annealing Requires models of subunits, builds interconnected models without Requires models of subunits, builds interconnected models without steric clashes steric clashes steric clashes steric clashes Uses constraints: symmetry, distance (FRET or mutagenesis) Uses constraints: symmetry, distance (FRET or mutagenesis) relative orientation (RDC from NMR), if applicable relative orientation (RDC from NMR), if applicable

lg I, relative

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

• Eur. Biophys. J.

. 35 35, 567. , 567.

10 11 9 10

s, nm-1

0.5 1.0 1.5 2.0 8

A global refinement run with distance constraints A global refinement run with distance constraints

A tyrosine kinase MET (118 kDa) consisting of five domains Single curve fitting with Program SASREF distance constraints: C to N C to N termini contacts

Gherardi, E., Sandin, S., Petoukhov, M.V., Finch, J., Youles, M.E., Ofverstedt, L.G., Miguel, R.N., Blundell, T.L., Vande Woude, G.F., Skoglund, U. & Svergun, D.I. (2006) PNAS USA, 103, 4046.

Quaternary structure of tetanus toxin

Receptor binding Monomeric fraction Receptor binding H(C) domain reveals concentraton- dependent fraction Dimeric fraction 100 : 0 0 : 100 dependent

• ligomerization

Polydisperse fractions 64 : 36 43 : 57 21 : 79 14 : 86 Mon:Dim Ab initio and rigid body analysis of the dimeric H(C) domain using the structure of the monomer in the crystal (1FV2) and accounting that the mutant Cys869Ala remains Mon:Dim Qazi, O., Bolgiano, B., Crane, D., Svergun, D.I., Konarev, P.V., Yao, Z.P., Robinson, C.V., Brown, K.A. & Fairweather N. (2007) J Mol Biol. 365, 123–134. always monomeric yield a unique model of the dimer

Rigid body modelling of the Xpot ternary Rigid body modelling of the Xpot ternary complex complex

Eleven X-ray and neutron curves Atomic and homology Atomic and homology gy gy models models Distance restrains from tRNA Distance restrains from tRNA footprinting (Arts et al footprinting (Arts et al footprinting (Arts et al. footprinting (Arts et al. (1998) (1998) EMBO J. EMBO J. 17, 17, 7430) 7430)

Fukuhara et al. (in preparation)

Addition of missing fragments Addition of missing fragments Addition of missing fragments Addition of missing fragments

Flexible loops or domains Flexible loops or domains are often not resolved in are often not resolved in high resolution models or high resolution models or genetically removed to genetically removed to facilitate crystallization facilitate crystallization Tentative configuration of Tentative configuration of g such fragments are such fragments are reconstructed by fixing the reconstructed by fixing the known portion and adding known portion and adding th i i t t fit th th i i t t fit th the missing parts to fit the the missing parts to fit the scattering from the full scattering from the full-

• length macromolecule.

length macromolecule.

Building native Building native-

• like folds of missing fragments

like folds of missing fragments

• Using DR

Using DR-type models and protein type models and protein-

• specific penalty functions

specific penalty functions g yp p yp p p p y p p y Primary sequence Secondary structure Excluded volume

Number of neighbours 6 2 3 4 5 6 Shell radius, nm 0.2 0.4 0.6 0.8 1.0 1

Neighbors d b Knowledge-based t ti l Bond angles & dihed als dist ib tion distribution potentials dihedrals distribution

Petoukhov, M.V., Eady, N.A.J., Brown, K.A. & Svergun, D.I. (2002) Biophys. J. 83, 3113

Addition of missing fragments: BUNCH Addition of missing fragments: BUNCH

• BUNCH combines rigid body and

BUNCH combines rigid body and ab initio ab initio modelling to find the positions and orientations modelling to find the positions and orientations modelling to find the positions and orientations modelling to find the positions and orientations

• f rigid domains and probable conformations of
• f rigid domains and probable conformations of

flexible linkers represented as “dummy residues” flexible linkers represented as “dummy residues” chains chains

• Multiple experimental scattering data sets from

Multiple experimental scattering data sets from partial constructs (e.g. deletion mutants) can be partial constructs (e.g. deletion mutants) can be pa t a co st ucts (e g de et o uta ts) ca be pa t a co st ucts (e g de et o uta ts) ca be fitted simultaneously with the data of the full fitted simultaneously with the data of the full-

• length protein.

length protein.

• BUNCH accounts for symmetry, allows one to fix

BUNCH accounts for symmetry, allows one to fix some domains and to restrain the model by some domains and to restrain the model by contacts between specific residues contacts between specific residues

Petoukhov, M. V. & Svergun, D. I. (2005). Biophys. J. 89, 1237-1250

Structure of sensor histidine Structure of sensor histidine-

• kinase PrrB

kinase PrrB

The dimeric sensor histidine-kinase PrrB from Mycobacterium tuberculosis contains ATP y binding and dimerization domains and a 59 aas long (flexible) HAMP linker

Tentative homology model based on Thermotoga maritima CheA Three domain Two domain

PrrB model after rigid body refinement and addition of HAMP linker Nowak, E., Panjikar, S., Morth, J. P., Jordanova R., Svergun, D. I. & Tucker, P. A. (2006) Structure, 14, 275

Structure of sensor histidine Structure of sensor histidine-

• kinase PrrB

kinase PrrB

The dimeric sensor histidine-kinase PrrB from Mycobacterium tuberculosis contains ATP

Tentative homology model

y binding and dimerization domains and a 59 aas long (flexible) HAMP linker

based on Thermotoga maritima CheA Three domain Two domain

Superposition with the independently determined sensor histidine-kinase from PrrB model after rigid body refinement and addition of HAMP linker Superposition with the independently determined sensor histidine kinase from Thermotoga maritima (Marina A. et al. (2005) Embo J. 24, 4247) Nowak, E., Panjikar, S., Morth, J. P., Jordanova R., Svergun, D. I. & Tucker, P. A. (2006) Structure, 14, 275

Tumour suppressor p53 and its complex with DNA

The homotetrameric p53 plays a central role in the cell cycle and maintaining genomic The homotetrameric p53 plays a central role in the cell cycle and maintaining genomic

• integrity. It consists of folded core and tetramerization domains, linked and flanked by

intrinsically disordered segments. Cross-shaped extended p53 from SAXS and NMR Compact and NMR p53/DNA from SAXS and an independent EM reconstruction Tidow, H., Melero, R., Mylonas, E., Freund, S.M., Grossmann, J.G., Carazo, J.M., Svergun, D.I., Valle, M. & Fersht, A.R. (2007) Proc Natl Acad Sci USA, 104, 12324

Addition of missing fragments: CORAL Addition of missing fragments: CORAL

• A merger of SASREF and BUNCH: advanced methods to account for

A merger of SASREF and BUNCH: advanced methods to account for missing loops in multi missing loops in multi-subunit protein structures (RANLOGS CORAL) subunit protein structures (RANLOGS CORAL) missing loops in multi missing loops in multi subunit protein structures (RANLOGS, CORAL) subunit protein structures (RANLOGS, CORAL)

M.V. Petoukhov, D. Franke, A. Shkumatov, G. Tria, A.G. Kikhney, M. Gajda, C. Gorba, H.D.T. Mertens, P.V. Konarev, D.I. Svergun (2012). J. Appl. Cryst. 45, 342-350.

Some words of caution

Or Always remember about ambiguity!

Information content in SAS: simple explanation Information content in SAS: simple explanation

∑

∞

⎥ ⎦ ⎤ ⎢ ⎣ ⎡ + + − − − = ) ( ) ( sin ) ( ) ( sin ) ( ) (

k k k k

s s D s s D s s D s s D s I s s I

A solution scattering curve f ti l ith i

=

⎦ ⎣ + −

1

) ( ) (

k k k

s s D s s D

from a particle with maximum size D can be represented by its values taken at discrete points (Shannon channels)

10

2

I(s)

2 4 6 8 10 12

Ns

points (Shannon channels) sk = kπ/ D I t i l SAS i t

10

1

In a typical SAS experiment, Ns ≈ 5-15

10

• C. E. Shannon & W. Weaver (1949).

0.00 0.05 0.10 0.15 0.20

s, A

• 1

( ) The mathematical theory of

• communication. University of Illinois

Press, Urbana.

Simple explanations do not work in SAS Simple explanations do not work in SAS

Shape determination: M≈ 103 variables (e.g. 0 or 1 bead assignments in DAMMIN Rigid bod methods M 101 a iables (positional and otational Rigid body methods: M≈ 101 variables (positional and rotational parameters of the subunits) From the informational point of view, rigid body modeling should provide unique or at least much less ambiguous models than shape determination NO WAY NO WAY As all the problems are non-linear, the number of Shannon channels does t i t b f t hi h i ibl t t t not give you exact number of parameters, which is possible to extract from the scattering data (depending on accuracy, this number varies between zero and infinity). Further, uniqueness of reconstruction depends largely on the complexity

• f the function f(x) to be minimized

Ambiguity of rigid body analysis Ambiguity of rigid body analysis g y g y y g y g y y

A synthetic example: two different orientations of

A synthetic example: two different orientations of

A synthetic example: two different orientations of

A synthetic example: two different orientations of tRNA in a dimeric complex with aspartyl tRNA in a dimeric complex with aspartyl-

• tRNA

tRNA synthetase obtained by rigid body modelling and synthetase obtained by rigid body modelling and compatible with X compatible with X-

• ray and contrast variation neutron

ray and contrast variation neutron

Petoukhov, M.V. & Svergun, D. I. (2006) Eur. Biophys. J. 35, 567-576

scattering data scattering data

Constraints and restrains used Constraints and restrains used in global modelling procedures in global modelling procedures in global modelling procedures in global modelling procedures

Information

Information about about contacting contacting residues residues from from other

• ther

experiments experiments (spin (spin labelling labelling site site directed directed experiments experiments (spin (spin labelling, labelling, site site-directed directed mutagenesis, mutagenesis, FRET, FRET, chemical chemical shifts shifts etc) etc)

Information

Information about about symmetry symmetry

Information

Information about about symmetry symmetry

Avoiding

Avoiding steric steric clashes clashes

For

For missing missing loops loops and and linkers linkers: contiguous contiguous chain, chain,

For

For missing missing loops loops and and linkers linkers: contiguous contiguous chain, chain, excluded excluded volume, volume, Ramachandran Ramachandran plot plot for for C Cα, knowledge knowledge-

• based

based potentials potentials etc etc AND AND STILL, STILL, one

• ne must

must always always cross cross-

• validate

validate SAS SAS d l d l i t i t ll ll il bl il bl bi h i l/bi h i l bi h i l/bi h i l models models against against all all available available biochemical/biophysical biochemical/biophysical information information

Structural bases for the function of frataxin

R d d l l f f t i ti l t i f t k f ti Reduced levels of frataxin, an essential protein of yet unknown function, cause neurodegenerative pathology. Its bacterial orthologue (CyaY) forms functional complexes with the two central components to iron–sulphur cluster assembly: desulphurase Nfs1/IscS SAXS: free IscS is p scaffold protein Isu/IscU. IscS IscS dimeric, free IscU and CyaY are monomeric IscS IscU CyaY IscS IscU IscS/IscU Ab initio and rigid body models of complexes: I U bi d th (sol) IscU IscS/CyaY IscS/CyaY IscS/IscU IscU binds on the periphery of IscS dimer, CyaY binds close to the dimerization interface IscU (MX) C Y IscS/CyaY/IscU Prischi F, Konarev PV, Iannuzzi C, Pastore C, Adinolfi S, Martin SR, Svergun DI & Pastore A. (2010) Nat Commun. 1, 95-104 CyaY IscS/CyaY/IscU

Recent hybrid collaborative projects at EMBL Recent hybrid collaborative projects at EMBL

Nuclear receptors Complement factor H Flt3 signaling complex PDH complex E2 core Rochel et al NSMB (2011) Morgan et al NSMB (2011) Verstraete et al Blood (2011) Marrott et al FEBS J (2011) ( ) ( ) HCV NS3/4A inhibitor Colonization factor GbpA Myomesin Nanocomposites FEBS J (2011) Schiering et al PNAS USA (2011) Wong et al Plos Pathog (2012) Pinotsis et al Plos Biol (2012) Shtykova et al JPC (2012)

By the way, can X By the way, can X-

• ray scattering

ray scattering i ld h f ld? i ld h f ld? yield the fold? yield the fold?

Lysozyme and its near

Lysozyme and its near-native scattering mates native scattering mates

Lysozyme and its near

Lysozyme and its near native scattering mates native scattering mates

7.5 No scale LYZ23.FIT LYZ23.FIT LYZ58.FIT FOOL01.FIT FOOL03.FIT 17-Oct-2001 04:24:12 Close window to continue 001 002 003 6 6.5 7 Y 003 004 005 5 5.5 6 0.2 0.4 0.6 0.8 1 1.2 1.4 X Scales : 1.00 1.00 1.00 1.00 1.00

And now, let us awake for the And now, let us awake for the hands hands-

• on practical
• n practical