Rigid body refinement (basics) - EMBL Hamburg

Rigid body refinement (basics)

D.Svergun, EMBL-Hamburg

Shapes from recent projects at EMBL-HHDomain and quaternary structureComplexes and assemblies Domain and quaternary structureComplexes and assemblies

Dcp1/Dcp2 complexS-layer proteins Toxin Bα-synuclein oligomers

She et al, Mol Cell (2008) Fagan et al Mol. Microbiol (2009)

Albesa-Jové et alJMB (2010)

Giehm et alPNAS USA (2011)

In most cases, high resolution models are drawn inside

Structural transitionsFlexible/transient systemsSrc kinaseCytochrome/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 llih b id d lli‘hybrid’ modelling‘hybrid’ modelling

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

The use of high resolution models in SASThe use of high resolution models in SASTh i l d l lTheoretical model or complete

crystal structure availableValidation in solution

Incomplete structure availableAddition of missing

loops/domains

Structure of subunits availableRigid body model of

the complex

Structure of domains and multiple curves available Model of the domain

structurestructure

How to compute SAS from atomic modelHow 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 idistribution Δρ = <ρ(r) - ρs>, where ρs is the scattering density of the solventFurther 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

ΩΩ− 2

bbssa2 )(A+ )(A)(A=)A(=I(s) ssss δρρ

♦ 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 expansionThe use of multipole expansion

ΩΩ− 2

bsa2 )B(+ )E()(A=)A(=I(s) ssss δρρ

If the intensity of each contribution is represented using spherical harmonics

2

0

2 )(2)( sAsI lm

l

lml∑∑−=

∞

=

= π

the average is performed analytically:L l

∑∑= −=

+−=L

l

l

lmlmlmlm sBsEsAsI

0

20

2 )()()(2)( δρρπ

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

CRYSOLCRYSOL andand CRYSONCRYSON:: XX--ray and ray and neutron scattering from macromoleculesneutron scattering from macromolecules

∑∑ +L l

sBsEsAsI 22 )()()(2)( δρρπ

TheThe programsprograms::

∑∑= −=

+−=l lm

lmlmlm sBsEsAsI0

0 )()()(2)( δρρπ

eithereither fitfit thethe experimentalexperimental datadata byby varyingvarying thethe densitydensityofof thethe hydrationhydration layerlayer δρδρ (affects(affects thethe thirdthird term)term) andandyy yy ρρ (( ))thethe totaltotal excludedexcluded volumevolume (affects(affects thethe secondsecond term)term)oror predictpredict thethe scatteringscattering fromfrom thethe atomicatomic structurestructureusingusing defaultdefault parametersparameters (theoretical(theoretical excludedexcluded volumevolumeusingusing defaultdefault parametersparameters (theoretical(theoretical excludedexcluded volumevolumeandand boundbound solventsolvent densitydensity ofof 11..11 g/cmg/cm33 ))provideprovide outputoutput filesfiles (scattering(scattering amplitudes)amplitudes) forfor rigidrigidb db d fi tfi t titibodybody refinementrefinement routinesroutinescomputecompute particleparticle envelopeenvelope functionfunction F(F(ωω))

Scattering components (lysozyme)Scattering components (lysozyme)

1)1) AtomicAtomic1)1) AtomicAtomic2)2) ShapeShape3)3) BorderBorder3)3) BorderBorder4)4) DifferenceDifference

lg I, relative

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

3

Experimental dataFit with shellFit without shell

2

3

Lysozyme

1

2

Hexokinase

0

EPT

-1

PPase

s, nm-10 1 2 3 4

Denser shell or floppy chains:X-rays versus neutronsX 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

oppy 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

0

2♦ Neutrons in D2O (shell):

particle would appear smaller than the atomic model

-2

0

SAXS SANS in H2O SANS in D2O

model

X-rays versus neutrons: experiment

lg I, relativelg I, relative

1

1

Neutrons, D2ONeutrons, H2OX-rays

-1

0X-rays

-1

-3

-2

Neutrons, D2O

Neutrons, H2O-2

0 2 4

-3

s, nm-1

0 1 2 3s, nm-1

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

Other approaches/programs IOther approaches/programs I

TheThe ‘cube‘cube method’method’ ((LuzzatiLuzzati etet al,al, 19721972;; FedorovFedorov andandPavlov,Pavlov, 19831983;; MMüüllerller,, 19831983)) ensuresensures uniformuniform fillingfilling ofof thetheexcludedexcluded volumevolume.. Could/should/mustCould/should/must bebe superiorsuperior overoverthethe effectiveeffective atomicatomic factorsfactors methodmethod atat higherhigher anglesangles..

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, lysozymeFit by CRYSOLFit 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-10 5 10

o be sub ed

Other approaches/programs IIOther approaches/programs II

JJ.. BardhanBardhan,, SS.. ParkPark andand LL.. MakowskiMakowski ((20092009)) SoftWAXSSoftWAXS:: aa computationalcomputational tooltoolforfor modelingmodeling widewide--angleangle XX--rayray solutionsolution scatteringscattering fromfrom biomoleculesbiomolecules JJ.. ApplAppl..CrystCryst.. 4242,, 932932--943943 -- AA programprogram toto computecompute WAXSWAXSyy ,, p gp g ppSchneidmanSchneidman--DuhovnyDuhovny D,D, HammelHammel M,M, SaliSali AA.. ((20102010)) FoXSFoXS:: aa webweb serverserver forforrapidrapid computationcomputation andand fittingfitting ofof SAXSSAXS profilesprofiles.. NucleicNucleic AcidsAcids ResRes.. 3838SupplSuppl::WW540540--44.. -- DebyeDebye--likelike computations,computations, WebWeb serverserverGrishaevGrishaev AA GuoGuo LL IrvingIrving TT BaxBax AA ((20102010)) ImprovedImproved FittingFitting ofof SolutionSolution XXGrishaevGrishaev A,A, GuoGuo L,L, IrvingIrving T,T, BaxBax AA.. ((20102010)) ImprovedImproved FittingFitting ofof SolutionSolution XX--rayray ScatteringScattering DataData toto MacromolecularMacromolecular StructuresStructures andand StructuralStructural EnsemblesEnsemblesbyby ExplicitExplicit WaterWater ModelingModeling.. JJ AmAm ChemChem SocSoc.. 132132,, 1548415484--66.. -- GenerateGenerate bulkbulkandand boundbound waterswaters withwith MD,MD, dodo fitfit thethe datadata toto thethe modelmodelPoitevinPoitevin F,F, OrlandOrland H,H, DoniachDoniach S,S, KoehlKoehl P,P, DelarueDelarue MM ((20112011))..AquaSAXSAquaSAXS:: aa webweb serverserver forfor computationcomputation andand fittingfitting ofof SAXSSAXS profilesprofiles withwithnonnon--uniformallyuniformally hydratedhydrated atomicatomic modelsmodels.. NucleicNucleic AcidsAcids.. ResRes.. 3939,, WW184184--WW189189 -- GenerateGenerate waterswaters aroundaround proteinsproteins usingusing MDMD ((AquaSolAquaSol program)program)WW189189 -- GenerateGenerate waterswaters aroundaround proteinsproteins usingusing MDMD ((AquaSolAquaSol program)program)VirtanenVirtanen JJ,JJ, MakowskiMakowski L,L, SosnickSosnick TR,TR, FreedFreed KFKF.. ((20112011)) ModelingModeling thethehydrationhydration layerlayer aroundaround proteinsproteins:: applicationsapplications toto smallsmall-- andand widewide--angleangle xx--rayrayscatteringscattering.. BiophysBiophys JJ.. 101101,, 20612061--99.. -- UseUse aa ““HyPredHyPred solvationsolvation”” modelmodel totogenerategenerate thethe shell,shell, gearedgeared towardstowards WAXSWAXS..

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

http://dara.embl-hamburg.de/

About 15000 atomic models of biologically active molecules are generated from the entriesare generated from the entries in Protein Data Bank and the scattering patterns computed by CRYSOL

Rapidly identifies proteins with similar shape (from lowsimilar 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 modelsValidation of high resolution models

Crystallographic packing forces are Packing forces in the crystal restrict theCrystallographic packing forces arecomparable with the intersubunitinteractions. The solution structuresof multisubunit macromolecules

Packing forces in the crystal restrict theallosteric transition in aspartatetranscarbamylase

of multisubunit macromoleculescould be significantly different fromthose 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 modelsValidation of high resolution modelslg I, relative

2

1

SAXS experimentFit by 1yzbFit by 2aga

0.0 0.2 0.4 0.6 0.8

0

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-1o

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 KinaseClosure of the two domains of PGK upon substrate binding is essential for the enzymeClosure 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 ofA SAXS fingerprint of open/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 9.5603-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 21:09Final 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 licensingcomplex 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

of prereplicative complexes (pre-RCs) onto 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 licensingcomplex regulates DNA replication licensing

In eukaryotes, DNA rereplication is prevented by control of the assembly f li i l ( RC )of prereplicative complexes (pre-RCs)

onto chromatin. Cdt1 is a key component of the pre-RC assembly. Timely inhibition of Cdt1 by Geminin isTimely 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 acan 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 modelingThe idea of rigid body modelingThe idea of rigid body modelingThe idea of rigid body modeling

•The structures of two subunitsin reference positions areknownknown.

•Arbitrary complex can beconstr cted b mo ing andconstructed by moving androtating the second subunit.

•This operation depends onthree Euler rotation angles andthree Cartesian shifts.three Cartesian shifts.

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

•The structures of two subunitsin reference positions areknownknown.

•Arbitrary complex can beconstr cted b mo ing andconstructed by moving androtating the second subunit.

•This operation depends onthree Euler rotation angles andthree Cartesian shifts.three Cartesian shifts.

Equation for rigid body modelingEquation for rigid body modeling

Rotation:A

Shift: x, y, z

CB

Using spherical harmonics the amplitude(s) of arbitrarilyUsing 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 viaviathe initial amplitude and the six positional parameters: the initial amplitude and the six positional parameters: CClmlm(s) = (s) = CC (B(B ββ ))CClmlm(B(Blmlm, , αα, , ββ, , γγ, x, y, z)., x, y, z).The scattering from the complex is then rapidly calculated asThe scattering from the complex is then rapidly calculated as

( ) [ ]∑∑∞

++=0

*2 )()(Re4)()(l

llmlmBA sCsAsIsIsI π

−0 l

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

Constraints for rigid body modellingConstraints for rigid body modelling

InterconnectivityInterconnectivityAbsence of steric clashesAbsence of steric clashesSymmetrySymmetrySymmetrySymmetryIntersubunit 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 subcomplexessubcomplexes

Petoukhov & Svergun Petoukhov & Svergun (2005)(2005) Biophys JBiophys J 8989 1237;1237;(2005) (2005) Biophys J.Biophys J. 8989, 1237; , 1237; (2006) (2006) Eur. Biophys. JEur. Biophys. J. . 3535, , 567.567.

Interactive and local refinementInteractive 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 dimericdimeric αα crystallin domaincrystallin domaindimeric dimeric αα--crystallin domaincrystallin 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 XFit ( lti l X d t ) tt i ( ) f ti ld t ) tt i ( ) f ti lFits (multiple XFits (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 clashessteric clashessteric clashes steric clashes Uses constraints: symmetry, distance (FRET or mutagenesis) Uses constraints: symmetry, distance (FRET or mutagenesis) relative orientation (RDC from NMR), if applicablerelative orientation (RDC from NMR), if applicable

lg I, relative

Petoukhov & Svergun (2005) Petoukhov & Svergun (2005) Biophys J.Biophys J. 8989, 1237;, 1237;(2006) (2006) Eur. Biophys. JEur. Biophys. J. . 3535, 567., 567.

10

11

9

10

s, nm-10.5 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

ProgramSASREF

distance constraints:

C to NC 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) domainreveals concentraton-dependentfraction

Dimeric fraction

100 : 0

0 : 100

dependent oligomerization

Polydispersefractions

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 complexcomplex

Eleven X-ray and neutron curves

Atomic and homology Atomic and homology gygymodelsmodels

Distance restrains from tRNA Distance restrains from tRNA footprinting (Arts et alfootprinting (Arts et alfootprinting (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 fragmentsAddition of missing fragmentsAddition of missing fragmentsAddition 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 crystallizationfacilitate crystallizationTentative configuration of Tentative configuration of ggsuch 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 thth i i t t fit ththe missing parts to fit the the missing parts to fit the scattering from the fullscattering from the full--length macromolecule.length macromolecule.

Building nativeBuilding native--like folds of missing fragmentslike folds of missing fragmentsUsing DRUsing DR--type models and proteintype models and protein--specific penalty functions specific penalty functions gg yp pyp p p p yp p y

Primary sequence

Secondary structure

Excluded volume

Number of neighbours6

2

3

4

5

6

Shell radius, nm

0.2 0.4 0.6 0.8 1.00

1

Neighbors d b

Knowledge-based t ti l

Bond angles & dihed als dist ib tiondistribution 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: BUNCHAddition of missing fragments: BUNCH

BUNCH combines rigid body and BUNCH combines rigid body and ab initioab initiomodelling to find the positions and orientationsmodelling to find the positions and orientationsmodelling to find the positions and orientations modelling to find the positions and orientations of rigid domains and probable conformations of of rigid domains and probable conformations of flexible linkers represented as “dummy residues” flexible linkers represented as “dummy residues” chainschains

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 bepa t a co st ucts (e g de et o uta ts) ca befitted simultaneously with the data of the fullfitted 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 residuescontacts between specific residues

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

Structure of sensor histidineStructure of sensor histidine--kinase PrrBkinase PrrBThe dimeric sensor histidine-kinase PrrB from Mycobacterium tuberculosis contains ATP ybinding and dimerization domains and a 59 aas long (flexible) HAMP linker

Tentative homology model based on Thermotoga maritima CheA

Threedomain

Twodomain

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 histidineStructure of sensor histidine--kinase PrrBkinase PrrBThe dimeric sensor histidine-kinase PrrB from Mycobacterium tuberculosis contains ATP

Tentative homology model

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

based on Thermotoga maritima CheA

Threedomain

Twodomain

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 DNAThe homotetrameric p53 plays a central role in the cell cycle and maintaining genomicThe 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-shapedextended 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: CORALAddition 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 multimissing loops in multi--subunit protein structures (RANLOGS CORAL)subunit protein structures (RANLOGS CORAL)missing loops in multimissing 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 explanationInformation content in SAS: simple explanation

∑∞

⎥⎦

⎤⎢⎣

⎡++

−−−

=)(

)(sin)(

)(sin)()( kkkk ssD

ssDssDssDsIssI

A solution scattering curve f ti l ith i

= ⎦⎣ +−1 )()(k kk ssDssD

from a particle with maximum size D can be represented by

its values taken at discrete points (Shannon channels)102

I(s)0 2 4 6 8 10 12

Ns

points (Shannon channels)

sk = kπ/ D

I t i l SAS i t101

In a typical SAS experiment, Ns ≈ 5-15

100

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 SASSimple explanations do not work in SASShape determination: M≈ 103 variables (e.g. 0 or 1 bead assignments in

DAMMINRigid bod methods M 101 a iables (positional and otationalRigid 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 WAYNO 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 tnot 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 of the function f(x) to be minimized

Ambiguity of rigid body analysisAmbiguity of rigid body analysisg y g y yg y g y y

A synthetic example: two different orientations ofA synthetic example: two different orientations ofA synthetic example: two different orientations of A synthetic example: two different orientations of tRNA in a dimeric complex with aspartyltRNA in a dimeric complex with aspartyl--tRNA tRNA synthetase obtained by rigid body modelling and synthetase obtained by rigid body modelling and compatible with Xcompatible 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 proceduresin global modelling proceduresin global modelling proceduresin global modelling procedures

InformationInformation aboutabout contactingcontacting residuesresidues fromfrom otherotherexperimentsexperiments (spin(spin labellinglabelling sitesite directeddirectedexperimentsexperiments (spin(spin labelling,labelling, sitesite--directeddirectedmutagenesis,mutagenesis, FRET,FRET, chemicalchemical shiftsshifts etc)etc)InformationInformation aboutabout symmetrysymmetryInformationInformation aboutabout symmetrysymmetryAvoidingAvoiding stericsteric clashesclashesForFor missingmissing loopsloops andand linkerslinkers:: contiguouscontiguous chain,chain,ForFor missingmissing loopsloops andand linkerslinkers:: contiguouscontiguous chain,chain,excludedexcluded volume,volume, RamachandranRamachandran plotplot forfor CCαα,,knowledgeknowledge--basedbased potentialspotentials etcetc

ANDAND STILL,STILL, oneone mustmust alwaysalways crosscross--validatevalidate SASSASd ld l i ti t llll il blil bl bi h i l/bi h i lbi h i l/bi h i lmodelsmodels againstagainst allall availableavailable biochemical/biophysicalbiochemical/biophysical

informationinformation

Structural bases for the function of frataxinR d d l l f f t i ti l t i f t k f tiReduced 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

pscaffold protein Isu/IscU.

IscS IscSdimeric, 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)

IscUIscS/CyaYIscS/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

CyaYIscS/CyaY/IscU

Recent hybrid collaborative projects at EMBL Recent hybrid collaborative projects at EMBL Nuclear receptors Complement factor HFlt3 signaling complexPDH 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 GbpAMyomesin NanocompositesFEBS 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 XBy 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 nearLysozyme and its near--native scattering matesnative scattering matesLysozyme and its nearLysozyme and its near native scattering matesnative 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

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6

6.5

7

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003004005

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0 0.2 0.4 0.6 0.8 1 1.2 1.4XScales : 1.00 1.00 1.00 1.00 1.00

And now, let us awake for the And now, let us awake for the handshands--on practical on practical