Joint use of SAXS o o with MX and EM Peter Konarev European - - PowerPoint PPT Presentation

EMBO Global Exchange Lecture Course 2 May 2011 Beijing China

Joint use of SAXS

• with MX and EM

Peter Konarev European Molecular Biology Laboratory, Hamburg Outstation BioSAXS group

Information content in SAXS

In SAXS, the molecule’s rotationally averaged scattering pattern is measured as a function of spatial frequency, typically t 1 3 l ti to 1–3-nm resolution Because of rotational averaging, the information content of a SAXS spectrum is dramatically reduced compared to a SAXS spectrum is dramatically reduced compared to a diffraction pattern in X-ray crystallography or even a density map from EM.

S l Detector Monochromatic beam Sample 2θ

Log (Intensity)

Scattering vector s=4π sinθ/λ 2θ

s=4π sinθ/λ, nm-1

• 1

X-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/analysis size and speed of experiment/analysis

Time to answer Months

NMR (high) EM Cryo-EM (low)

Weeks

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

Days Hours

SAXS/ WAXS ( g )

Hours Minutes

SAXS/ WAXS SANS (low) 100 101 102 103 104 105 106 MM, kDa (kDa) (MDa) (GDa)

Information content in SAXS

∑

∞

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

k k k k

s s D s s D s I s s I

Information content in SAXS

A solution scattering curve f ti l ith i

∑

=

⎥ ⎦ ⎢ ⎣ + −

1

) ( ) ( ) ( ) (

k k k k k

s s D s s D s I s s I

2 4 6 8 10 12

Ns

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)

10

2

I(s)

2 4 6 8 10 12 s 10

2

I(s)

2 4 6 8 10 12 s

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

10

1

10

1

In a typical SAS experiment, Ns ≈ 5-15

• C. E. Shannon & W. Weaver (1949).

10 10

( ) The mathematical theory of

• communication. University of Illinois

Press, Urbana.

0.00 0.05 0.10 0.15 0.20

s, A

• 1

0.00 0.05 0.10 0.15 0.20

s, A

• 1

Information content in SAXS

SAXS spectrum can be transformed into a radial distribution function which is essentially a histogram of all pairwise

Information content in SAXS

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

dr sr r p s I

D

∫

= sin ) ( 4 ) ( π

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

dr sr r p s I

∫

) ( 4 ) ( π

y uninformative about the details of molecular structure.

The recent renaissance of SAXS is to a large extent the result of The 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 i information

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

s I s I X E α χ )] ( ), ( [( }) ({

exp 2

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 and

molecular 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 (flexible

region 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 Ribosome Solution Structure of the E. coli 70S Ribosome at 11.5 A° Resolution

In the Cryo-EM density map reconstruction In 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 i were 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 techniques Radiation sources: Shape determination EM Search volum e 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 Atom ic m odels Incident beam Sample Wave

2θ

Missing fragments NMR Biochem istry Orientations Solvent Resolution nm: Wave vector k, k= 2π/ λ Scattered beam, k 1

I, relative

2 3

Oligomeric mixtures FRET I nterface Resolution, nm: 3.1 1.6 1.0 0.8

lg

1 2

mixtures m apping Bioinform atics Scattering curve I(s)

s, nm -1 2 4 6 8

Flexible systems Secondary structure prediction Scattering vector s= k 1-k, s= 4π sinθ/ λ

Ab initio modelling (DAMMIN/F) g ( )

Using simulated annealing, finds a compact dummy atoms configuration 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 and where χ is the discrepancy between the experimental and calculated curves, P(X) is the penalty to ensure compactness and connectivity, α>0 its weight.

compact compact compact compact loose loose disconnected disconnected

Bead (dummy atoms) model

A sphere of radius Dmax is filled by densely packed beads of radius r0<< Dmax Vector of model parameters: Solvent Particle r0 Dmax Vector of model parameters: Position ( j ) = x( j ) = ( h i t ) ⎩ ⎨ ⎧ solvent if particle if 1 (phase assignments)

Chacón, P. et al. (1998) Biophys. J. 74, 2760-2775. S D I (1999) Bi h J 76 2r0 Svergun, D.I. (1999) Biophys. J. 76, 2879-2886 2r0

Dmax

Validation of Electron Microscopy Models py

EM2DAM EM2DAM

Contour level level DENSITY MAP (MRC format) from EMDB BEAD MODEL

13

Validation of Electron Microscopy Models py

CRYSOL

BEAD MODEL EM2DAM: surface layer th h ld + SAXS EXPERIMENTAL DATA threshold

14

Refinement with DAMMIN

Ab initio modelling (GASBOR)

Using simulated annealing, finds a spatial distribution of K dummy residues within a

g ( )

y sphere with diameter Dmax to fit the scattering data by minimizing

[ ]

}) ({ }) { , ( ), ( }) ({

exp 2 i i DR i

r P r s I s I r f α χ + =

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

Number of neighbours 4 5 6

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

0.2 0.4 0.6 0.8 1.0 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 g in SAXS

• Validation of theoretically predicted models
• Analysis of similarities between macromolecules

in solution and in the crystal

• Modelling
• f

the quaternary structure

• f

multisubunit particles/complexes by rigid body p / p y g y refinement

Scattering from a Macromolecule in Solution g

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

Ω Ω

The use of multipole expansion If the intensity is represented using spherical ♦ As(s): scattering from the excluded l If the intensity is represented using spherical harmonics the average is performed analytically:

2 2 2

) ( 2 ) ( ) ( s A A s I

l

∑ ∑

∞

π s

volume

) ( 2 ) ( ) ( s A A s I

lm l m l

∑ ∑

− = = Ω =

= π s

♦ 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 rapid algorithms 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

CRYSOL CRYSOL and and CRYSON CRYSON: X d t tt i f l l d t tt i f l l X-ray and neutron scattering from macromolecules ray and neutron scattering from macromolecules

∑ ∑

+

L l

s B s E s A s I

2 2

) ( ) ( ) ( 2 ) ( δρ ρ π

• The programs:

∑ ∑

= − =

+ − =

l l m lm lm lm

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

either fit the experimental data by varying the density

• f the hydration layer δρ (affects the third term) and

y y ρ ( ) the total excluded volume (affects the second term)

• r predict the scattering from the atomic structure

(particle is surrounded by an angular envelope and (particle is surrounded by an angular envelope and 0.3 nm thick border layer is built around the envelope) provide output files (scattering amplitudes) for rigid b d fi t ti body refinement routines compute particle envelope function F(ω)

Scattering components (lysozyme) Scattering components (lysozyme) 1) Atomic 1) Atomic 2) Shape 3) B d 3) Border 4) Difference

Effect of the hydration shell, X-rays

lg I, relative

Effect of the hydration shell, X rays

3 Experimental data Fit with shell Fit without shell 2

Lysozyme Hexokinase

1

EPT

• 1

PPase

s, nm-1

1 2 3 4

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

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

lg I, relative

2 SAXS experiment Fit by 1yzb Fit by 2aga 1 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.

Identification of biologically active

• ligomers
• ligomers

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

Solution structure of eucaryotic release factor RF1

• For Release factor 1 (RF1), responsible for termination
• f 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 i

• bserved by X-ray crystallography compared with that observed in

complex with the ribosome by electron microscopy (EM).

• Small-angle X-ray scattering was able to resolve these differences

S a a g e ay sca e g as ab e o eso e ese d e e ces by demonstrating that the more extended (EM) form was present in solution, 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 peptidyl decoding and peptidyl transferase centers

• Crystal: compact, does

not span this distance

lg I, relative

2

p

Red: cryo-EM Orange: Xtal

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

A

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.

s

0.0 0.1 0.2 0.3

Domain closure of 3-phosphoglycerate kinase b d b SAXS

• bserved 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.

Domain closure of 3-phosphoglycerate kinase b d b SAXS

• bserved by SAXS

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. Varga, B. Flachner, P.V. Konarev, E. Gráczer, J. Szabó, D.I. Svergun, P.

Závodszky, & M. Vas (2006) FEBS Lett. 580, 2698-2706.

Domain closure of 3-phosphoglycerate kinase b d b SAXS

• bserved by SAXS

A SAXS fingerprint of

• pen/closed conformation

for humang PGK (1) – no ligand (2-4) binary PGK complexes (5-6) ternary PGK complexes

SAXS data supports that the simultaneous binding of both

• A. Varga, B. Flachner, P.V. Konarev, E. Gráczer, J. Szabó, D.I. Svergun, P.

Závodszky, & M. Vas (2006) FEBS Lett. 580, 2698-2706.

pp g substrates to PGK are required for complete domain closure.

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

hPGK can accommodate the mirror image L-enantiomer

• f MgADP into its
• f MgADP into its

nucleotide-binding site and 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 not

Assuming the tertiary structure is not changed by complex formation, arbitrary complex can be constructed by moving and rotating the subunits.

• For

each subunit this

• peration

depends on three orientational and depends on three orientational and three translational parameters.

Scattering from a Complex Particle Scattering from a Complex Particle

Scattering amplitudes from individual subunits in reference positions/orientations are evaluated using CRYSOL/ CRYSON

Shift: x, y, z

positions/orientations are evaluated using CRYSOL/ CRYSON

Rotation: α, β, γ A A0

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

, β, γ

and the six positional parameters (three Euler rotation angles and 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 refinement

Scattering amplitudes of the subunits are pre-computed and positional parameters 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 l Fits (multiple X-ray and neutron) scattering curve(s) from partial constructs or contrast variation using simulated annealing Requires models of subunits, builds interconnected models without steric clashes steric 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-1

0.5 1.0 1.5 2.0 8

SASREF Restraints

2

Set of penalties formulating various restraints

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

• To ensure the interconnectivity of the entire complex

each subunit should have a contact with at least one

• ther subunit.

The contact distance between C atoms of distinct

Interconnectivity and steric clashes

• The contact distance between Cα atoms of distinct

subunits: 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 contacts

data the information on contacting subunits and even individual residues can be available.

• SASREF

allows

• ne

to account for this information by specifying the ranges of residues information by specifying the ranges of residues

• r

nucleotides which can be involved in interactions between the partners.

Building native-like folds of missing fragments

2

Set of penalties formulating various restraints

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

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

Number of neighbours 5 6 1 2 3 4

Excluded volume

Shell radius, nm 0.2 0.4 0.6 0.8 1.0

Neighbors distribution Knowledge-based potentials Bond angles & dihedrals distribution p

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 initio

modelling to find the optimal positions and

• rientations 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 f fitted 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 K137 K134 H133 4 1 3 2 N N

D

R185 K266 K94 R122 K65 K64

C

L255 R185 K238 K271 K266 K259 2 5 3 1 4 C N

B

I187 R254 F216 K218 Q223

NMR: high resolution structures

A

Multiple scattering curves from NMR: high resolution structures

• f 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 protein p ypy g p

Overlap of the ab initio and rigid body models Multiple scattering curves from deletion mutants fitted simultaneously and 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)

• btained by SAXS

Filamins are dimeric actin-binding proteins that contribute to organization of the actin based cytoskeleton and to its remodelling by integrating different the 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 C showed 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 SAXS

• btained by SAXS

DAMMIN and BUNCH models No symmetry 5 nm

• 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 Met 60 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

• f cell division and cell migration
• f 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 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.

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

DAMMIN SASREF

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.

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

• TC HGF/SF - Met

TC 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.

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.

Combining SAXS, MX and CryoEM data g y

The

active two-chain form of HGF/SF forms a 1:1 complex with Met and displays HGF/SF wrapped around the β-propeller β p p (Sema) domain of MET928.

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.

Hepatocyte growth factor/scatter factor and MET signalling

A

truncated Met ectodomain ( Met5 = Sema +PSI ) builds a 2:2 complex with two-chain HGF/SF with two chain HGF/SF assembled around the dimerization interface seen in the crystal structure of in the crystal structure of the NK1 fragment

• f

HGF/SF, which displays the features

• f

a functional features

• f

a functional, signaling unit.

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.

SAXS and EM study of Lymazine synthase

This enzyme catalyzes the formation

• f

6,7-dimethyl-8- ribityllumazine in the penultimate step

• f

riboflavin biosynthesis biosynthesis. The enzyme forms icosahedral capsids with a total molecular weight 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 unit

The formation of mutliple assembly states was

• bserved. They are interconvertable via equilibrium

which is sensitive to solvent type and pH which 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 synthase

Mutant WT, phosphate buffer WT, Tris buffer

MIXTURE fits pH 7

WT, Borate buffer

pH 7 pH 10

• 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

SAXS and EM study of Lymazine synthase

Cryo-EM micrographs Ab initio models with and without with and without icosahedral symmetry The data show that multiple assembly forms are a general feature of lumazine synthases.

• 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

pH induced virus maturation

Nudaurelia capensis Omega Virus (NwV) Cryo-EM Crystallography Maturation is an important event associated with establishing virus infectivity It occurs in many complex viruses in

• rder to accommodate the need for

weak interactions between subunits t hi lf bl d to achieve proper self-assembly and the requirement for a robust particle to survive the extra cellular environment. Maturation results from a program encoded in the initial, often fragile, immature particle that directs large immature 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 maturation

Nudaurelia 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 g incremental 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

• ccurring 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 di iron compounds including oxygen- containing ligands in boiling

• surfactants. The NPs are coated by

phospholipids with PEG Tails phospholipids with PEG Tails to become soluble.

lg I, relative 5

shoulder

lg I, relative 5

Ab initio analysis: peculiarities of

TEM image, scale bar 100 nm

1 2 3 4 5 1 2 3

1st minimum

p(R), relative

1 2 3 4 5 1 2 3

shoulder 1st minimum

p(R), relative

Ab initio analysis: peculiarities of

• rganization of different NPs

s nm-1 0.0 0.5 1.0 1.5 2.0 2.5 3.0

• 2
• 1

1

R, nm 5 10 15 20 2 4 6 8 10 12

• 1

0.0 0.5 1.0 1.5 2.0 2.5 3.0

• 2
• 1

1

R, nm 2 4 6 8 10 0.0 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

• f the NPs stabilized by magnetic interactions

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

• 1

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

• 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 SASREF fits

Data analysis

Detector

SAXS in structural biology (biased)

R l ti

Sh

2θ Sample Incident beam Wave vector k k=2π/λ

g I, relative

2 3

Scattering I( ) Resolution, nm: 3.1 1.6 1.0 0.8

Shape determination Rigid body

Solvent k, k=2π/λ Scattered beam, k1

l

1

curve I(s)

Missing Rigid body modelling

Radiation 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 -1 2 4 6 8

EM

Complementary Complementary techniques techniques Oligomeric g fragments

Hom ology m odels Atom ic m odels MS Distances Crystallography NMR i h i Bioinform atics

mixtures

Orientations I nterfaces

Additional Additional information information

Biochem istry FRET AUC

Flexible systems

EPR

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

N 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 p configuration of missing portions (SAXS, MX and EM)

• High resolution structure of domains/subunits known:

t t t b i id b d fi t quaternary 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-Hamburg D.I. Svergun, M.W. Roessle, M.V. Petoukhov,

• D. Franke, A.G. Kikhney, W. Shang, H. Mertens

BioSAXS group BioSAXS group

EMBO Global Exchange Lecture Course