Joint Use SAXS MX EM - EMBL Hamburg · X--ray anray andt ttif lld neutron scattering from...

EMBO Global Exchange Lecture Course 2 May 2011 Beijing China

Joint use of SAXS o owith MX and EM

Peter Konarev

European Molecular Biology Laboratory,Hamburg Outstation

BioSAXS group

Information content in SAXSIn SAXS, the molecule’s rotationally averaged scattering pattern is measured as a function of spatial frequency, typicallyt 1 3 l tito 1–3-nm resolution

Because of rotational averaging, the information content of a SAXS spectrum is dramatically reduced compared to aSAXS spectrum is dramatically reduced compared to a diffraction pattern in X-ray crystallography or even a density map from EM.

S lDetector

Monochromatic beamSample

2θ

Log (Intensity)

0

1

2

Scattering vector s=4π sinθ/λ

2θ

s=4π sinθ/λ, nm-10 1 2 3

-1X-ray generator Synchrotron

Nevertheless, SAXS can provide important shape information about proteins and assemblies in the wide size range, which are not amenable to cryo- EM and NMR spectroscopy

Structural methods: resolution, accessible size and speed of experiment/analysissize and speed of experiment/analysis

Time to answer

MonthsNMR (high)

EM Cryo-EM (low)

Weeks

EM, Cryo EM (low)RDC NMR (low)

MX (high)Days

HoursSAXS/WAXS

( g )

Hours

Minutes

SAXS/WAXSSANS (low)

100 101 102 103 104 105 106 MM, kDa(kDa) (MDa) (GDa)

Information content in SAXS

∑∞

⎥⎤

⎢⎡ +

−−

=)(sin)(sin)()( kk

kkssDssDsIssI


A solution scattering curve f ti l ith i

∑=

⎥⎦

⎢⎣ +−1 )()(

)()(k kk

kk ssDssDsIssI

0 2 4 6 8 10 12

Ns0 2 4 6 8 10 12

Ns 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 12s

102

I(s)0 2 4 6 8 10 12s

points (Shannon channels)

sk = kπ/ D

I t i l SAS i t101 101

In a typical SAS experiment, Ns ≈ 5-15

C. E. Shannon & W. Weaver (1949). 100100

( )The mathematical theory of

communication. University of Illinois Press, Urbana.

0.00 0.05 0.10 0.15 0.20s, A-1

0.00 0.05 0.10 0.15 0.20s, A-1

Information content in SAXSSAXS spectrum can be transformed into a radial distributionfunction which is essentially a histogram of all pairwise


function, which is essentially a histogram of all pairwise distances of the atoms in an assembly weighted by their respective atomic numbers.

drsrrpsID

∫=sin)(4)( π

For structure determination, additional information is needed because the radial distribution function alone is relatively

drsr

rpsI ∫0

)(4)( π

yuninformative about the details of molecular structure.

The recent renaissance of SAXS is to a large extent the result ofThe recent renaissance of SAXS is to a large extent the result of efforts on integrating SAXS with other structural information from additional complementary sources (e.g. MX, EM, NMR, bioinformatics etc.).

Integration of SAXS data with other i f iinformation

Similarly to other types of experimental information, SAXS data can be used as a filter for a set of models generated independently by other methodsindependently by other methods.

SAXS data can also be a term in a scoring function that is optimized to obtain a model consistent with the data. (e.g. ab initio modellig, rigid body modelling, addition of missing fragments)

∑+=i

ii PsIsIXE αχ )](),([(})({ exp2

missing fragments)

i

Possible use of solution structure in crystallography

• Determine a solution scattering structure (Dammin/f, Gasbor)

• Place it in unit cell (location & orientation) Place it in unit cell (location & orientation)• Calculate initial phases for phase extension andmolecular replacement

Possible challenges• Resolution and fidelity of initial structure Resolution and fidelity of initial structure• Same solution and crystal structures?• Limitation of using uniform e- densities (flexibleregion hydration layer)region, hydration layer)

Possible use of solution structure in EM

• Use a solution scattering structure (Dammin/f, Gasbor) as a starting reference for EM reconstruction

Bron, T. et.al. (2008) Biol. Cell 100, 413

Hsp90 heat-shock protein

• Superposition of SAXS models and independent EM reconstructions

Tumour suppressor p53 and its complex with DNA

independent EM reconstructions

Tidow, H et. al. (2007) Proc Natl Acad Sci USA, 104, 12324

Tumour suppressor p53 and its complex with DNA

Possible use of SAXS information in EM

Solution Structure of the E coli 70S RibosomeSolution Structure of the E. coli 70S Ribosomeat 11.5 A° Resolution

In the Cryo-EM density map reconstructionIn the Cryo-EM density map reconstruction,Fourier amplitudes at higher spatial frequencies are always underrepresented due to charging, instrument instabilities, specimen drift.

To compensate for these effects, scattering intensities bt i d i X l ti tt iwere obtained using X-ray solution scattering

measurements for E.coli ribosomes in the range up to 1/8 Å-1, and a correction to the Fourier amplitudes up to the 1/11.5 Å-1 was applied.

I.S. Gabashvili, R.K. Agrawal, C.M.T. Spahn, R.A. Grassucci, D.I. Svergun, J. Frank, P. Penczek (2000) Cell, 100, 537–549

pp

SAS experiment Data analysis Additional information

Complementary techniquesRadiation sources:

Shape determination

EMSearch volume

Detector

X-ray tube (λ = 0.1 - 0.2 nm)Synchrotron (λ = 0.05 - 0.5 nm)Thermal neutrons (λ = 0.1 - 1 nm)

2θ

Rigid body modelling

Crystallography

NMR

Atomic models

Incident beam

Sample

Wave 2θ

Missing fragments

NMR

BiochemistryOrientationsSolvent

Resolution nm:

Wave

vector k, k=2π/λ Scattered

beam, k1

I,

rela

tive

2

3

Oligomeric mixtures

FRET

Interface

Resolution, nm:

3.1 1.6 1.0 0.8

lg

1

2 mixturesmapping

BioinformaticsScattering curve I(s)

s, nm -10 2 4 6 8

Flexible systems Secondary

structure prediction

Scattering vector s=k1-k, s=4π sinθ/λ

Ab initio modelling (DAMMIN/F)g ( )Using simulated annealing, finds a compact dummy atomsconfiguration X that fits the scattering data by minimizing

f (X) = χ2[Iexp(s), I(X,s)] + ΣαiPi(X)

Discrepancy from the experimental data

Set of penalties formulating various restraints

where χ is the discrepancy between the experimental andwhere χ is the discrepancy between the experimental andcalculated curves, P(X) is the penalty to ensure compactness andconnectivity, α>0 its weight.

compactcompactcompactcompact

looseloose

disconnecteddisconnected

Bead (dummy atoms) modelA sphere of radius Dmax is filled by densely packed beads of radiusr0<< Dmax

Vector of model parameters:Solvent Particle

r0 Dmax

Vector of model parameters:

Position ( j ) = x( j ) =

( h i t )⎩⎨⎧

solventif0particleif1

(phase assignments)

Chacón, P. et al. (1998) Biophys. J. 74, 2760-2775.

S D I (1999) Bi h J 762r0 Svergun, D.I. (1999) Biophys. J. 76, 2879-2886

2r0

Dmax

Validation of Electron Microscopy Modelspy

EM2DAMEM2DAM

Contour levellevel

DENSITY MAP (MRC format) from EMDB

BEAD MODEL

13

Validation of Electron Microscopy Modelspy

CRYSOL

BEAD MODEL EM2DAM: surface layer th h ld+

SAXS EXPERIMENTAL DATAthreshold

14

Refinement with DAMMIN

Ab initio modelling (GASBOR)Using simulated annealing, finds a spatialdistribution of K dummy residues within a

g ( )

ysphere with diameter Dmax to fit thescattering data by minimizing

[ ] })({}){,(),(})({ exp2

iiDRi rPrsIsIrf αχ +=

where χ is the discrepancy between theexperimental and calculated curves, P({ri})

Number of neighbours

4

5

6

experimental and calculated curves, P({ri})is the penalty to ensure a chain-likedistribution of neighbors, α>0 its weight.

0.2 0.4 0.6 0.8 1.00

1

2

3

Shell radius, nm

Neighbors distribution

•Has potential for future development (e.g. phase problem in low resolution crystallography)

The use of high resolution models gin SAXS

• Validation of theoretically predicted models

• Analysis of similarities between macromoleculesin solution and in the crystal

• Modelling of the quaternary structure ofmultisubunit particles/complexes by rigid bodyp / p y g yrefinement

Scattering from a Macromolecule in Solutiong

ΩΩ− 2

bbssa2 )(A+ )(A)(A=)A(=I(s) ssss δρρ

♦ Aa(s): atomic scattering in vacuum

ΩΩ

The use of multipole expansionIf the intensity is represented using spherical

♦ As(s): scattering from the excluded l

If the intensity is represented using sphericalharmonics the average is performed analytically:

222 )(2)()( sAAsIl

∑∑∞

πsvolume0

)(2)()( sAAsI lmlml

∑∑−==Ω

== πs

♦Ab(s): scattering from the hydration shell

CRYSOL (X-rays): Svergun et al (1995) J Appl Cryst 28 768

This approach permits to further use rapidalgorithms 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

CRYSOLCRYSOL andand CRYSONCRYSON::XX d t tt i f l ld t tt i f l lXX--ray and neutron scattering from macromoleculesray and neutron scattering from macromolecules

∑∑ +L l

sBsEsAsI 22 )()()(2)( δρρπ

• The programs:

∑∑= −=

+−=l lm

lmlmlm sBsEsAsI0

0 )()()(2)( δρρπ

either fit the experimental data by varying the densityof the hydration layer δρ (affects the third term) andy y ρ ( )the total excluded volume (affects the second term)or predict the scattering from the atomic structure(particle is surrounded by an angular envelope and(particle is surrounded by an angular envelope and0.3 nm thick border layer is built around the envelope)provide output files (scattering amplitudes) for rigidb d fi t tibody refinement routinescompute particle envelope function F(ω)

Scattering components (lysozyme)Scattering components (lysozyme)

1) Atomic1) Atomic2) Shape3) B d3) Border4) Difference

Effect of the hydration shell, X-rayslg I, relative

Effect of the hydration shell, X rays

3

Experimental dataFit with shellFit without shell

2Lysozyme

Hexokinase

0

1

EPT

-1

0

PPase

s, nm-10 1 2 3 4

Other approaches/programs to calculate the scattering Other approaches/programs to calculate the scattering f bi l i l l lf bi l i l l lfrom biological macromolecules:from biological macromolecules:

1) ‘Cube’ method ensures uniform filling of the excluded volume1) Cube method - ensures uniform filling of the excluded volume

2) SoftWaxs (L.Makowski group) – A program to compute WAXS

3) Foxs (A.Sali group) – Debye-like calculations, Web server

4) AXES (A. Bax group) – Explicit water modelling, Web server

Validation of high resolution modelsmodels

lg I, relative

2

SAXS experimentFit by 1yzbFit by 2aga

1

0.0 0.2 0.4 0.6 0.8

0

o

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.

Identification of biologically active oligomersoligomers

Biologically active dimer of myomesin-1Experiment 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

Solution structure of eucaryotic release factor RF1

• For Release factor 1 (RF1), responsible for terminationof translation in E. coli, a more compact form of the protein was

b d b X t ll h d ith th t b d iobserved by X-ray crystallography compared with that observed incomplex with the ribosome by electron microscopy (EM).

• Small-angle X-ray scattering was able to resolve these differencesS a a g e ay sca e g as ab e o eso e ese d e e cesby demonstrating that the more extended (EM) form was present insolution, and that the compaction was an artefact of crystallization.

Vestergaard, B., Sanyal, S., Roessle, M., Mora, L., Buckingham, R. H., Kastrup, J. S., Gajhede, M., Svergun, D. I. & Ehrenberg, M. (2005) Mol. Cell, 20, 929–938.

Solution structure of eucaryotic release factor RF1

• Cryo-EM: extended; spans the distance between the ribosomal decoding and peptidyldecoding and peptidyl transferase centers •Crystal: compact, does not span this distance

lg I, relative2

p

Red: cryo-EMOrange: Xtal

1

(1)(2)(3)(4)(5)(6)(7)

0A

Vestergaard, B., Sanyal, S., Roessle, M., Mora, L., Buckingham, R. H., Kastrup, J. S., Gajhede, M., Svergun, D. I. & Ehrenberg, M. (2005) Mol. Cell, 20, 929–938.

s0.0 0.1 0.2 0.3

Domain closure of 3-phosphoglycerate kinase b d b SAXSobserved by SAXS

3-Phospho-D-glycerate kinase (PGK) is p g y ( )a typical hinge-bending enzyme with two structural domains of about equal

size (MM=43.5 kDa)

OPEN (Pig, Bs)CLOSED (Tb, Tm)

PGK catalyses the phospho-transfer from 1,3-bisphosphoglycerate (1,3-BPG) to MgADP and produces 3 phospho-glycerate (3-PG) and MgATP during the carbohydrate metabolism.

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

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


Numerous crystal structures do not yield

OPEN (Pig, Bs)CLOSED (Tb, Tm)

conclusive answer, which conditions are required for the closure.

The known X ray structures of open and

CRYSOL fits

The known X-ray structures of open and closed conformations were compared to SAXS data.

CRYSOL fits



A SAXS fingerprint of open/closed conformation for humang PGK

(1) – no ligand (2-4) binary PGK complexes (5-6) ternary PGK complexesSAXS data supports that the simultaneous binding of both


pp gsubstrates to PGK are required for complete domain closure.

Interaction of human 3-phosphoglycerate kinase with L M ADP th i i f D M ADPL-MgADP, the mirror image of D-MgADP.

hPGK can accommodate the mirror image L-enantiomer of MgADP into itsof MgADP into its nucleotide-binding siteand can phosphorylate it, almost as effectively as the natural D-enantiomer.

L-MgADP D-MgADP

A. Varga, J. Szabó, B. Flachner, B. Roy, P.V. Konarev, D.I. Svergun, P. Závodszky, C. Périgaud, T. Barman, C. Lionne, & M. Vas, M. (2008) Biochem. Biophys. Res. Comm. 366, 994-1000.

Idea of Rigid Body Modelling• Large macromolecular complexes are

more 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

• Assuming the tertiary structure is notAssuming the tertiary structure is notchanged by complex formation,arbitrary complex can be constructedby moving and rotating the subunits.

• For each subunit this operationdepends on three orientational anddepends on three orientational andthree translational parameters.

Scattering from a Complex ParticleScattering from a Complex ParticleScattering amplitudes from individual subunits in referencepositions/orientations are evaluated using CRYSOL/CRYSON

Shift: x, y, z

positions/orientations are evaluated using CRYSOL/CRYSON

Rotation:α, β, γ

AA0

The partial amplitudes of arbitrarily rotated and displacedsubunit are analytically expressed via the initial amplitudes

, β, γ

and the six positional parameters (three Euler rotation anglesand three Cartesian shifts):

(i) ( ) (i) ( ) { (i) ( ) (i) (i) (i) (i) (i) (i) }A(i)lm(s) = A(i)

lm(s) { A0(i)

lm(s), α (i), β (i), γ (i), x (i), y (i), z (i) }.Svergun, D.I. (1991). J. Appl. Cryst. 24, 485-492

Interactive and local refinementScattering amplitudes of the subunits are pre-computed and positionalparameters are refined to fit the scattering from the complex

Kozin & Svergun (2000). J.Appl. Cryst. 33, 775-777

Konarev, Petoukhov & Svergun(2001) J Appl Cryst 34(2001). J. Appl. Cryst. 34,527-532

♦ MASSHA (Windows PC)

♦ ASSA (SUN/SGI/DEC)

Global rigid body modelling (SASREF)Fit ( lti l X d t ) tt i ( ) f ti lFits (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 clashessteric clashes Uses constraints: symmetry, distance (FRET or mutagenesis) relative orientation (RDC from NMR), if applicable

lg I, relative

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

10

11

9

10

s, nm-10.5 1.0 1.5 2.0

8

SASREF Restraints2

Set of penalties formulating various restraints


• To ensure the interconnectivity of the entire complexeach subunit should have a contact with at least oneother subunit.The contact distance between C atoms of distinct

Interconnectivity and steric clashes

• The contact distance between Cα atoms of distinctsubunits: 4-7 A.

• Overlap: distance < 4 A.• Not interconnected arrangements of subunits and• Not interconnected arrangements of subunits and

those with steric clashes are penalized.

• From binding affinity studies or from mutagenesisInformation on contactsdata the information on contacting subunits andeven individual residues can be available.

• SASREF allows one to account for thisinformation by specifying the ranges of residuesinformation by specifying the ranges of residuesor nucleotides which can be involved ininteractions between the partners.

Building native-like folds of missing fragments

2Set of penalties formulating various restraints


• Using DR-type models and protein-specific penalty functions

Number of neighbours

5

6

0

1

2

3

4

Excluded volume

Shell radius, nm

0.2 0.4 0.6 0.8 1.00

Neighbors distribution

Knowledge-based potentials

Bond angles & dihedrals distributionp

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

Modelling of multidomain proteins (BUNCH)

• BUNCH combines rigid body and ab initiomodelling to find the optimal positions and orientations of rigid domains and probable conformations of flexible linkers represented as “dummy residues” chains attached to the appropriate termini of domains.pp p

• Multiple experimental scattering data sets from partial constructs (e.g. deletion mutants) can be f f ffitted simultaneously with the data of the full-length protein.

• BUNCH permits to account for the symmetry (the• BUNCH permits to account for the symmetry (the same for all constructs) and offers the possibility to fix some domains.

• Contacts between specific residues can be used as restrains

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

Contacts between specific residues can be used as restrains.

CORAL – analog of BUNCH for complexes

Structure and RNA interactions of polypyrimidine tract binding protein

PTB is an important regulator of alternative splicing, which allows the production of multiple mRNA transcripts from a single pre-mRNA species. PTB contains four domains (RNA recognition motifs, RRMs), whose structure is solved by NMR.

H62

F98

L136

K92

Q96

K137K134H133

4

13

2N

N

D

R185 K266

K94

R122K65

K64

C

L255R185

K238

K271

K266

K259

25

31

4

C

N

B

I187

R254F216

K218

Q223

NMR: high resolution structures

A

Multiple scattering curves fromNMR: high resolution structures of RRM1 and RRM2

Multiple scattering curves from deletion mutants fitted simultaneously

Petoukhov, M. V., Monie, T. P., Allain, F. H., Matthews, S., Curry, S., and Svergun, D. I. (2006). Structure 14, 1021-1027.

Structure and RNA interactions of polypyrimidine tract binding proteinp ypy g p

Overlap of the ab initio and rigid body models

Multiple scattering curves from deletion mutants fitted simultaneouslyand rigid body models

Petoukhov, M. V., Monie, T. P., Allain, F. H., Matthews, S., Curry, S., and Svergun, D. I. (2006). Structure 14, 1021-1027.

Dimer model for Filamin C (domains 23-24) obtained by SAXS

Filamins are dimeric actin-binding proteins that contribute to organization of the actin based cytoskeleton and to its remodelling by integrating differentthe actin-based cytoskeleton and to its remodelling by integrating different signalling pathways.

The crystal structure of domain 23 of filamin C e c ys a s uc u e o do a 3 o a Cshowed that the protein adopts the expected immunoglobulin (Ig)-like fold.

Filamin C domain 24 forms an antiparallel dimer exploiting strands C and D, and it was proposed that these two strands create a dimerization interface in all vertebrate filamins.

In order to investigate if the domain 23 influences dimerization of filamins the tandem domains 23 and 24 of filamin C were used for structural studies.

L.Sjekloca, R. Pudas, B. Sjoblom, P. Konarev, O. Carugo, V. Rybin, T.R. Kiema,D. Svergun, J. Ylanne, & K.D. Carugo, (2007) J Mol Biol. 368, 1011-1023.

Dimer model for Filamin C (domains 23-24) bt i d b SAXSobtained by SAXS

DAMMIN and BUNCH models

No symmetry5 nm

o sy e y

P2 symmetry

The results of the SAXS study on construct 23–24 clearly indicate that domain 23 is not involved in dimerization but protrudes away from the dimer core

L.Sjekloca, R. Pudas, B. Sjoblom, P. Konarev, O. Carugo, V. Rybin, T.R. Kiema,D. Svergun, J. Ylanne, & K.D. Carugo, (2007) J Mol Biol. 368, 1011-1023.

Quaternary structure of complexes of tyrosine kinase Met with ligands

Extracellular domain of receptor tyrosine kinase Met60 kDa

Sema + PSI Ig1 Ig2 Ig3 Ig4

Extracellular domain of receptor tyrosine kinase Met 60 kDa

g g g g

Hepatocyte growth factor / scatter factor

Met is a receptor tyrosine kinase with hepatocyte growth factor / scatter factor (HGF/SF) as its natural

4*10 kDa N K1 K2 K3 K4 SP

growth factor / scatter factor (HGF/SF) as its natural ligand. HGF/SF induced Met signaling promotes a complex cellular response including the stimulation

of cell division and cell migrationof cell division and cell migration.

HGF/SF (6 structural domains) controls the growth of epithelial cells through the receptor tyrosine kinase MET (5 structural domains).

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.

Ab initio and Rigid Body Models of Met928Ab initio and Rigid Body Models of Met928

DAMMINSASREF


3D Modelling of Sc and Tc HGF/SF3D Modelling of Sc and Tc HGF/SF

• TC HGF/SF - MetTC HGF/SF Met

X

N K1 K2 K3 K4 SP

Conversion of pro(single-chain)-HGF/SF into the active two-chain form is associated with a major structural transition from a compact, closed

X

j p ,conformation to an elongated, open one.


Combining SAXS, MX and CryoEM datag yThe active two-chain

form of HGF/SF forms a 1:1complex with Met anddisplays HGF/SF wrappedaround the β-propellerβ p p(Sema) domain of MET928.


Hepatocyte growth factor/scatter factor and MET signalling

A truncated Metectodomain ( Met5 = Sema+PSI ) builds a 2:2 complexwith two-chain HGF/SFwith two chain HGF/SFassembled around thedimerization interface seenin the crystal structure ofin the crystal structure ofthe NK1 fragment ofHGF/SF, which displays thefeatures of a functionalfeatures of a functional,signaling unit.


SAXS and EM study of Lymazine synthaseThis enzyme catalyzes the formation of 6,7-dimethyl-8-ribityllumazine in the penultimate step of riboflavinbiosynthesisbiosynthesis.

The enzyme forms icosahedral capsids with a total molecularweight of about 960 kDa.

SAXS measurements were made f

pentamer unit

for native and mutant enzyme species in different solvents and at different pH.

pentamer unitThe formation of mutliple assembly states was observed. They are interconvertable via equilibrium which is sensitive to solvent type and pHwhich is sensitive to solvent type and pH.

X. Zhang, P.V.Konarev, M.V.Petoukhov, D.I.Svergun, L.Xing, R.H.Cheng, I.Haase, M.Fischer, A.Bacher, R. Ladenstein & W. Meining (2006) JMB 362, 753-770

WT

SAXS and EM study of Lymazine synthaseMutant

WT, phosphate buffer

WT, Tris buffer

MIXTURE fitspH 7 WT, Borate bufferpH 7

pH 10


SAXS and EM study of Lymazine synthase

Cryo-EM micrographs

Ab initio models with and withoutwith and without icosahedral symmetry

The data show that multiple assembly forms are a general feature of lumazine synthases.


pH induced virus maturationNudaurelia capensis Omega Virus (NwV)

Cryo-EM Crystallography

Maturation is an important event associated with establishing virus infectivity

It occurs in many complex viruses in order to accommodate the need for weak interactions between subunits t hi lf bl dto achieve proper self-assembly and the requirement for a robust particleto survive the extra cellular environment.

Maturation results from a program encoded in the initial, often fragile, immature particle that directs largeimmature particle that directs large conformational changes (LCC)resulting in a robust infectious virion.

Immature particle mature particle Matsui T, Tsuruta H, Johnson JE.Biophys J. (2010) 98, 1337

pH induced virus maturationNudaurelia capensis Omega Virus (NwV)

Maturation is often triggered by changes

SAXS

Maturation is often triggered by changes in pH or other electrostatic events within the cell allowing in vitro maturation to be controlled by careful adjustment of the pH.

Time resolved SAXS showed that there were three kinetic stages initiated with an

Time-resolved

gincremental drop in pH; (1) a rapid (<10 ms) collapse to an

incrementally smaller particle, (2) a continuous size reduction over SAXS(2) a continuous size reduction over

the next 5 seconds, (3) a smaller final transition

occurring in 2-3 minutes.

Matsui T, Tsuruta H, Johnson JE.Biophys J. (2010) 98, 1337

Encapsulated Magnetic Iron Oxide Nanoparticles (EM and SAXS)Nanoparticles (EM and SAXS)

Highly monodisperse NPs are prepared by thermal decomposition of i d i l diiron compounds including oxygen-containing ligands in boiling surfactants. The NPs are coated by phospholipids with PEG Tailsphospholipids with PEG Tailsto become soluble.

lg I, relative

5shoulder

lg I, relative

5Ab initio analysis: peculiarities of

TEM image, scale bar 100 nm

1

2

3

4

5123

1st minimum

p(R), relative1

2

3

4

5123

shoulder

1st minimum

p(R), relative

Ab initio analysis: peculiarities of organization of different NPs

s nm-10.0 0.5 1.0 1.5 2.0 2.5 3.0

-2

-1

0

1

R, nm0 5 10 15 2002468

1012

-10.0 0.5 1.0 1.5 2.0 2.5 3.0

-2

-1

0

1

R, nm0 2 4 6 8 100.0

0.5

1.0

1.5

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 s, nm 1

DAMMIN fits

Encapsulated Magnetic Iron Oxide Nanoparticles (EM and SAXS)Nanoparticles (EM and SAXS)

Rigid body analysis reveals equilibrium clusters

lg I, relative lg I, relative

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

3

4

512

3

4

512

1

2

3

1

2

3

s, nm-10.0 0.5 1.0 1.5 2.0 2.5 3.0

-1

0

s, nm-10.0 0.5 1.0 1.5 2.0 2.5 3.0

-1

0

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

SASREF fits

Data analysisDetector

SAXS in structural biology (biased)

R l ti

Sh2θ

SampleIncident beam

Wave vector k k=2π/λ g

I,

rela

tive

2

3

Scattering I( )

Resolution, nm:

3.1 1.6 1.0 0.8

Shape determination

Rigid body

Solvent

k, k=2π/λ

Scatteredbeam, k1

l

1

curve I(s)

Missing

Rigid body modellingRadiation sources:

X-ray tube (λ = 0.1 - 0.2 nm)Synchrotron (λ = 0.05 - 0.5 nm)Thermal neutrons (λ = 0.1 - 1 nm)

s, nm -10 2 4 6 8

EM

Complementary Complementary techniquestechniques

Oligomeric

gfragments

Homologymodels

Atomicmodels

MS Distances

Crystallography

NMR

i h i

Bioinformatics

mixturesOrientationsInterfaces

AdditionalAdditionalinformationinformation

Biochemistry

FRET

AUCFlexible systems

EPR

Joint use of SAXS,MX and EM for biological macromolecules:biological macromolecules:

conclusionsN hi k b i i i l l i• Nothing known: ab initio low resolution structure (SAXS and EM)

• Complete high resolution structure known: validation in• Complete high resolution structure known: validation in solution and biologically active oligomers (SAXS and MX)

• Incomplete high resolution structure known: probable p g pconfiguration of missing portions (SAXS, MX and EM)

• High resolution structure of domains/subunits known: t t t b i id b d fi tquaternary structure by rigid body refinement

(SAXS, MX and EM)

Acknowledgements:Collaborative projects

Release factor RF1: B.Vestergaard (University of Copenhagen, Denmark)Tyrosine Kinase: E. Gherardi (Medical Research Council Centre, UK)Myomesin-1: M.Wilmanns (EMBL Hamburg Outstation, Germany)Lymazine synthase: R. Ladenstein (Karolinska Institute, Sweden)PTB S C (I i l C ll UK)PTB: S. Curry (Imperial College, UK)3PGK enzyme: M. Vas (Institute of Enzymology, Hungary)Iron Nanoparticles: L.M. Bronstein (Indiana University, USA)Filamin C: K Djinovic-Carugo (University of Vienna Austria)

EMBL H b

Filamin C: K.Djinovic-Carugo (University of Vienna, Austria)Ataxin-3: A. Pastore (National Institute for Medical Research, UK)

EMBL-HamburgD.I. Svergun, M.W. Roessle, M.V. Petoukhov,D. Franke, A.G. Kikhney, W. Shang, H. Mertens

BioSAXS groupBioSAXS group

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