Quantitative characterization Quantitative characterization of - - PowerPoint PPT Presentation
EMBO Global Exchange Lecture Course 3 May 2011 Beijing China Quantitative characterization Quantitative characterization of mixtures and complex fo mation formation Peter Konarev European Molecular Biology Laboratory, Hamburg Outstation
EMBO Global Exchange Lecture Course 3 May 2011 Beijing China
Quantitative characterization Quantitative characterization
fo mation formation
Peter Konarev European Molecular Biology Laboratory, Hamburg Outstation BioSAXS group
Scattering from monodisperse Scattering from mixtures ( h l di it ) g p systems (shape polydispersity)
dr sr r p s I
D
= sin ) ( 4 ) ( π∑
k k
sr
k
The scattering is proportional to that The scattering is proportional to that
which allows
to For equilibrium and non-equilibrium mixtures solution scattering permits to determine size, shape and internal structure of the particle at low (1-10 ) l ti mixtures, solution scattering permits to determine the number of components and, given their scattering intensities nm) resolution. and, given their scattering intensities Ik(s), also the volume fractions
Outlines
Polydisperse & interactive systems in ATSAS Equilibrium oligomeric mixtures (OLIGOMER) Assembly/disassembly processes (SVDPLOT, MIXTURE) Natively unfolded proteins and Natively unfolded proteins and multidomains proteins with flexible linkers (EOM, Weifeng Shang lecture) Applications of ATSAS for biological studies Oligomerization tuned by protein/salt concentration Oligomerization tuned by protein/salt concentration Multiple assembly forms Temperature dependent transitions
Program OLIGOMER for SAXS analysis Program OLIGOMER for SAXS analysis
Input parameters: 1) experimental data file (ASCII file *.dat) 2) form-factor file with the scattering from the components (can be easily prepared by FFMAKER)
k k
(can be easily prepared by FFMAKER)
k k k
Output parameters: 1) the fit to experimental data (* fit file) Output parameters: 1) the fit to experimental data (*.fit file) 2) the volume fractions of the components (in oligomer.log) OLIGOMER can be launched in batch mode for multiple data sets:
Konarev, P. V., Volkov, V. V., Sokolova, A. V., Koch, M. H. J. & Svergun, D. I. (2003)
FFMAKER as pre-tool for OLIGOMER
To quickly create form-factor file from pdb files and/or from scattering data files (either from ASCII *.dat files or g ( from GNOM output files where desmeared curve will be taken for intensity) Batch mode: ffmaker 1.dat 2.dat /undat 2 3.out /unout 2 ffmaker *.pdb m1.dat /smax 0.3 /ns 201 /lmmax 20 ffmaker 6lyz.pdb *.dat /sgrid m2.dat ffmaker ALL ffmaker ALL
all data files with "pdb", "ent", "out" or "dat" extension will be taken,
Oligomer content in mixtures
lg I, relative
(1) (2)
Monomer/dimer equilibrium
(2) (3) (4) Volume fraction
1 0
(4) (5) (6)
1.0
Monomer Dimer
( ) (7) (8)
0.5
s, nm-1 1 2
(8) c, mg/ml
2 4 6 8 10 12 0.0
Kozielski, F ., Svergun, D.I., Zaccai, J. Wade, R.H. & Koch, M.H.J. (2001)
Momomer/dimer equiilbrium in tetanus toxin
Monomeric fraction
Electrophoresis, size exclusion chromatography
fraction Dimeric fraction
chromatography and mass spectrometry reveal t ti
Mixtures
concentration- dependent
binding H(C) domain of tetanus toxin 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
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
Oligomeric state of Tricorn protein in solution
Tricorn protease is a major component in the cleavage of oligopeptides produced by the proteasome. Tricorn appeared to be a multifaceted system in solution. The estimated molecular mass of the particles (380 kDa) was significantly lower than the theoretical value of 720 kDa tricorn hexamer suggesting partial tricorn hexamer, suggesting partial dissociation of the tricorn hexamers in solution. SAXS data were fitted by a linear combination of the scattering from tricorn monomers (53%), dimers (14%) and hexamers (33%) using OLIGOMER hexamers (33%) using OLIGOMER. Goettig, P., Brandstetter, H., Croll, M., Gohring, W., Konarev, P.V., Svergun, D.I, Huber, R., and Kim, J.S. (2005) J Biol Chem. 280, 33387-33396
Studies of adrenodoxin (Adx) : cytochrome c (Cc) complex by SAXS and NMR
Adx is involved in steroid hormone biosynthesis by acting as an Solutions of native (WT) and cross-linked (CL) complex of Cc electron shuttle between adrenodoxin reductase and cytochromes. and Adx were measured by SAXS at different conditions: a) solute concentration range from 2.4 to 24.0 mg/ml; b) 10 mM Hepes / 20mM potassium phosphate (pH 7 4) buffer; b) 10 mM Hepes / 20mM potassium phosphate (pH 7.4) buffer; c) with addition of NaCl (from 0 up to 300 mM). Each protein has Molecular Mass (MM) of about 12.5 kDa. Adx Each protein has Molecular Mass (MM) of about 12.5 kDa. For CL complex CcV28C and AdxL80C mutants were linked by a disulfide bond.
Cc
Studies of (Adx) : (Cc) complex formation CL Complex CL Complex
DAMMIN and SASREF models
The experimental scattering from the CL complex does not depend on the solute concentration and addition of NaCl. It is compatible with 1:1 complex.
Studies of (Adx) : (Cc) complex formation Native Complex
Conc=4.8 mg/ml, 200 mM NaCl DAMMIN and SASREF models
Native Complex
Conc=24 mg/ml No salt
The native complex strongly depends on the sample concentration and on the amount of NaCl in the buffer.
At high protein concentration it forms heterotetramer with 2:2 stoichiometry, whereas at high salt concentration it dissociates into two individual proteins.
proteins.
Studies of (Adx) : (Cc) complex formation Native Complex
lgI, relative
4 (1)
Native complex, no salt CL complex
Native Complex
1 2 3 (1) (2) (3)
c,mg/ml 24 12 6 2.4 3-12 Rg, Å 28.3±0.7 28.3±0.7 26.5±0.5 24.4±0.7 21.4±0.5
2
(4) (5)
Dmax, Å 90±5 90±5 90±5 80±5 80±5 Vp, 103 Å3 63±6 52±5 43±5 35±4 42±5 MM kD 44 5 42 5 35 4 25 4 22 3
0.1 0.2 0.3 0.4
MM, kDa 44±5 42±5 35±4 25±4 22±3 Vmon,% 6±5 24±5 V 8±5 25±5 24±5 100
s, A-1
8±5 25±5 24±5 100 Vtri,% 48±5 47±5 54±5 52±5 Vtet,% 52±5 45±5 15±5
OLIGOMER fits
tet,%
Studies of adrenodoxin (Adx) : cytochrome c (Cc) complex by SAXS and NMR
NMR structure of CL complex NMR structure of CL complex
The ensemble of native Adx:Cc complex structures from the PCS simulation. Oligomerization behavior of the native complex in solution indicates a stochastic nature of complex formation. The native Adx/Cc is entirely dynamic and can be considered as a pure encounter complex.
Solution structure of human Pex5/Pex14/PTS1
protein complexes obtained by SAXS protein complexes obtained by SAXS
The Pex5p import receptor recognizes peroxisomal matrix DAMMIN and BUNCH models of Pex5p g p proteins with C-terminal peroxisomal targeting signal (PTS). After docking to protein complexes on the membrane these proteins are translocated across the membrane. The interaction of the cargo- loaded Pex5p receptor and the loaded Pex5p receptor and the peroxisomal membrane protein Pex14p is the essential primary docking step. The free full length human Pex5p is monomeric in solution, with an elongated, partially unfolded N-terminal domain. Shiozawa, K., Konarev, P.V., Neufeld, C., Wilmanns, M., Svergun, D.I. (2009) J Biol Chem. 284, 25334-25342 p y
Solution structure of human Pex5/Pex14/PTS1
protein complexes obtained by SAXS protein complexes obtained by SAXS
Titration studies yielded Titration studies yielded a 1:6 stoichiometry for the Pex5p/Pex14p complex Shiozawa, K., Konarev, P.V., Neufeld, C., Wilmanns, M., Svergun, D.I. (2009) J Biol Chem. 284, 25334-25342
Solution structure of human Pex5/Pex14/PTS1
protein complexes obtained by SAXS protein complexes obtained by SAXS
DAMMIF and SASREF models of ternary complex Inter subunit contacts were imposed for Pex14p(N) interactions with the WxxxY/F motifs of Pex5p(F) based on NMR data Shiozawa, K., Konarev, P.V., Neufeld, C., Wilmanns, M., Svergun, D.I. (2009) J Biol Chem. 284, 25334-25342 based on NMR data
Solution structure of human Pex5/Pex14/PTS1
protein complexes obtained by SAXS protein complexes obtained by SAXS
Ab initio MONSA models of ternary complex The model of the complex reveals that the The model of the complex reveals that the N-terminus of Pex5p remains extended in the presence of cargo and Pex14p, the latter proteins being significantly intermingled with th P 5 i t Shiozawa, K., Konarev, P.V., Neufeld, C., Wilmanns, M., Svergun, D.I. (2009) J Biol Chem. 284, 25334-25342 the Pex5p moiety.
Nucleoplasmin and its complexes with histones (SAXS and ITC study)
The joint use of SAXS and ITC confirmed that NP pentamer can accommodate five hi t ith H2A H2B di H5 d
( y)
histones, either H2A–H2B dimers or H5, and that NP core and tail domains are involved in the association with histones.
+
Taneva, S.G., Bañuelos, S., Falces, J., Arregi, I., Muga, A., Konarev, P.V., Svergun, D.I., Velázquez-Campoy, A., Urbaneja, M.A. (2009) J Mol Biol. 393, 448-463
Singular value decomposition (SVD)
For model-independent analysis of multiple scattering data sets from polydisperse systems, singular value decomposition (SVD) (Golub & Reinsh 1970) can be applied (Golub & Reinsh, 1970) can be applied. The matrix A = {Aik} = {I(k)(si)},
{
ik}
{ ( )(
i)}
(i = 1, . . . , N, k = 1, . . . , K, where N is number of experimental points in the scattering curve and K is the number of data sets) is represented as and K is the number of data sets) is represented as
A = U*S*VT, where the matrix S is diagonal,
and the columns of the orthogonal matrices U and V are and the columns of the orthogonal matrices U and V are the eigenvectors of the matrices A*AT and AT*A, respectively. p y
Singular value decomposition (SVD)
T T
T T
The matrix U yields a set of so-called l ft i l t i th l b i left singular vectors, i.e. orthonormal basic curves U(k)(si), that spans the range of matrix A, whereas th di l f S t i th i i t d i l l i the diagonal of S contains their associated singular values in descending order (the larger the singular value, the more significant the vector). g )
Singular value decomposition (SVD)
The number of significant singular vectors in SVD (i.e. non-random curves with significant singular values) yields the minimum number of independent curves required to represent the entire data set by their linear combinations (e g for mixtures) their linear combinations (e.g. for mixtures). SVD method has found wide-ranging applications: *Spectrum analysis. *Image processing and compression. *Information Retrieval. *Molecular dynamics. *Analysis of gene expression data. y g p *Small-angle Scattering etc.
Th SVDPLOT t th SVD f th
Program SVDPLOT for SAXS analysis
The program SVDPLOT computes the SVD from the active data sets in the PRIMUS toolbox and displays the singular vectors and singular values. A non-parametric test of randomness due to Wald and Wolfowitz (Larson 1975) is implemented to obtain and Wolfowitz (Larson, 1975) is implemented to obtain the number of significant singular vectors, which provides an estimate of the minimum number of i d d t t i ilib i
j N =
independent components in equilibrium or nonequilibrium mixtures [e.g. number of (un)folding or assembly intermediates].
1
( ) ( ) ( )
i ij j j
I s s V s λ
=
= ∑
( ) ( ) ( ) ( )
j p
δ λ
=
1
( ) ( ) ( ) ( )
i i ij j j
I s I s s V s δ λ
=
= −∑
Program SVDPLOT for SAXS analysis
Svdplot Svdplot
PRIMUS: Number of independent components
p
SVDPLOT SVDPLOT SVDPLOT SVDPLOT
Mixture of monomers and dimers
PRIMUS: Svdplot – singular value decomposition
Ncomp = 2 Ncomp = 2
Mixture of monomers and dimers
Complex mixtures (size and shape polydispersity interactions) polydispersity, interactions)
Δ =
K k k sh k k k k k k
R s S R R s I const s I ) , , , ( ) , , ( ) ( τ η ϕ
= k k k k k k k k k 1
) , , , ( ) , , ( ) ( η ϕ
Main structural task is determination of the volume fractions, average sizes, polydispersities and interactions by simulations or by non-linear fitting simulations or by non linear fitting
Application of the program MIXTURE to AOT
♦ Aim: to quantitatively characterize morphological transitions
g microemulsions
♦ Aim: to quantitatively characterize morphological transitions in the AOT water-in-oil microemulsions caused by temperature and by the composition of the mixture AOT organization in the oil-rich L2 phase ♦ Spherical water droplets, moderately polydisperse, average radius depends on the water/AOT ratio (wo) ♦ Long cylindrical aggregates ♦ Long cylindrical aggregates ♦ Reverse AOT micelles containing bound water only
D.I. Svergun, P .V . Konarev, V .V . Volkov, M.H.J. Koch, W.F .C. Sager,
. 113 , p. 1651-1665
Schematic profile
AOT Water
Oil
Electron
ρAOT
♦ AOT = sodium bis(2- ethylhexyl) sulfosuccinate
ρwater
Electron density profile
dh
ρAOT ρoil
ethylhexyl) sulfosuccinate ♦ A water-in-oil (w/o) microemulsion (L2 phase)
ρwater
I t ti ti k
R R
R0
Interaction:sticky hard sphere potential
Rhs
Scattering patterns from AOT microemulsions g p
♦At low temperatures: mostly spherical particles ♦At high temperatures: mostly long aggregates ♦Without water: small reverse micelles
Distribution functions at limiting temperatures g p
A three-component AOT mixture
Influence of the reverse micelles: wo=35 c=5% wo 35, c 5%
♦Without accounting for the reverse micelles it is impossible to fit the it is impossible to fit the
ray scattering data
Influence of the structure factor: wo=25 factor: wo 25, c=20%
♦Without accounting f f for the structure factor it is impossible to fit the experimental data at p lower temperatures
Temperature dependence, wo=25, c=10%
Red: spherical droplets Green: cylinders Yellow: reverse micelles Red: spherical droplets Green: cylinders Yellow: reverse micelles
Stickiness of the droplets, c=20%
♦Attraction between droplets grows with diminishing the droplet diminishing the droplet size and with increasing temperature
Application of the program MIXTURE to AOT i l i AOT microemulsions
♦A general method for non linear fitting of small angle ♦A general method for non-linear fitting of small-angle scattering data from polydisperse mixtures was developed ♦ The method was applied to quantitatively characterise the AOT microemulsions in a wide range of temperatures, water and salt concentrations water and salt concentrations ♦ More than 500 scattering patterns were fitted yielding a consistent picture of morphological transitions in the consistent picture of morphological transitions in the microemulsions
SAXS and EM study of Lymazine synthase
This enzyme catalyzes the formation
6,7-dimethyl-8- ribityllumazine in the penultimate step
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
which is sensitive to solvent type and pH which is sensitive to solvent type and pH.
A.Bacher, R. Ladenstein & W. Meining (2006) JMB 362, 753-770
SAXS data from Lumazine synthase
SVD analysis yielded that the equilibrium mixtures y y q for LSBS and LSAQ data contain five major components.
A.Bacher, R. Ladenstein & W. Meining (2006) JMB 362, 753-770
Lymazine synthase data analysis
WT Mutant WT, phosphate buffer WT, Tris buffer
MIXTURE fits pH 7
WT, Borate buffer
pH 7 pH 10
A.Bacher, R. Ladenstein & W. Meining (2006) JMB 362, 753-770
Lymazine synthase data analysis
The system was successfully described by 5 components: Cryo-EM micrographs by 5 components: complete and incomplete small capsids (T= 1) complete and incomplete big capsids (T= 3,4) free facets free facets.
The data show that multiple assembly forms are a general feature of lumazine synthases. Ab initio models
A.Bacher, R. Ladenstein & W. Meining (2006) JMB 362, 753-770
Conclusions
♦ ATSAS package allows one to quantitatively analyze interacting and
flexible systems and mixtures flexible systems and mixtures. With the present ATSAS 2.4 version it is possible:
♦ to determine volume fractions of oligomers (OLIGOMER) ♦ to make model-independent estimation of significant components f t d t diff t diti f k ti for systems measured at different conditions or for kynetic processes (SVDPLOT) ♦ to quantitatively characterize systems with size and shape polydispersity q y y p p y p y as well as systems with interparticle interactions (MIXTURE) ♦ to quantitatively analyze intrinsically unfolded proteins or multidomain proteins with flexible parts (EOM Weifeng Shang lecture) multidomain proteins with flexible parts (EOM ,Weifeng Shang lecture).
Acknowledgements: g
Collaborative projects Adr:Cc: M Ubbink (Leiden University Belgium) Adr:Cc: M. Ubbink (Leiden University, Belgium) Lymazine synthase: R. Ladenstein (Karolinska Institute, Sweden) Pex5p: M. Wilmanns (EMBL Hamburg Outstation, Germany) AOT microemulsions: W.F.C. Sager (FZ-Juelich, Germany) Hcp: K. Brown (Imperial College, UK) NP:Histones: S.Taneva (University of Bilbao, Spain)
EMBL H b
( y , p )
EMBL-Hamburg D.I. Svergun, M.W. Roessle, M.V. Petoukhov,
BioSAXS group BioSAXS group
EMBO Global Exchange Lecture Course