Programme

 

Wednesday, 17 October

 

Basics of small-angle scattering

 

9:00 - 9:30

Welcome: D.Svergun

9:30 - 11:00

Lecture: X-ray scattering - the basics – M. Roessle

Scattering on macromolecules is a standard technique for non or less ordered systems, such as particles in solution, gels, lipid layers and fibers. The basic physical principle of the scattering process is common in all these materials, however the information content of the recorded scattering data is different. In small angle X-ray scattering (SAXS) scattering intensities a very small angle of a few degrees are recorded and a resolution of ~1 nm can be achieved.
In biological solution SAXS determines the structure of macromolecules (e.g. proteins) in terms of average particle size and shape. Information about the overall protein envelope, the folding state and the protein volume can be analyzed already directly from the initial scattering data.
SAXS stations at synchrotrons are the most powerful devices for scattering experiments, although X-ray tube based devices are advancing and comparable results to synchrotron data are obtained.

In this lecture the scattering process of X-rays on atoms is discussed. The focus will be on the special case of particles in solution, but biological systems such as lipids are presented as well. The technical aspects of X-ray production and X-ray optics will be introduced, explaining the devices and parts of a SAXS station. SAXS on X-ray tube-based lab sources are presented as an option.
The basics of standard X-ray data recording are discussed. 

11:00 - 11:30

Coffee break

11:30 - 12:30

Seminar: Participating students presentations, 10 min each

12:30 - 14:30

Lunch at DESY canteen, building 9

14:30 - 16:30

Seminar: Participating students presentations, 10 min each

16:30 - 17:00

Coffee break

17:00 - 18:00

Lecture: Neutron scattering - the basics – J.Trewhella

The scattering of neutrons or X-rays by macromolecules in solution gives rise to small-angle scattering patterns that provide information about the size and shape of the scattering molecule. While there are common underlying principals and methods for extracting this information from neutron or X-ray scattering data, there are important differences in the way X-rays and neutron are produced and in how they interact with matter that can be capitalized on. X-ray sources are inherently more intense than neutron sources and hence require smaller samples and offer greater potential in time-resolved studies. On the other hand, there can be large isotope effects in neutron scattering resulting from the fact that they are scattered by the nuclei in the sample. Isotopic substitution can therefore provide a means to manipulate the neutron scattering signal from an object (contrast variation) without changing its elemental composition. This presentation will describe the basics of neutron scattering, emphasizing how the unique properties of neutrons can be capitalized on to study the structures of the components within macromolecular complexes and assemblies. Contrast variation and solvent matching experiments will be described as well as the methods for analyzing these data sets along with the limitations and strengths of different approaches (1). Examples of how neutron scattering complements and extends what is learned from high resolution techniques and from X-ray scattering will be drawn from our studies of bio-molecular signalling and regulation (2-4).

  1. Whitten, A. E., Cai, S., and Trewhella, J. (2008) MULCh: ModULes for the Analysis of Small-angle Neutron Contrast Variation Data from Biomolecular Complexes. J. Appl. Cryst. 41, 222-226.
  2. Jacques, D. A., Langley, D. B., Hynson, R. M. G., Whitten, A. E., Kwan, A., Guss, J. M., and Trewhella, J. (2011) A Novel Structure of an Antikinase and its Inhibitor. J. Mol. Biol. 405, 214-226.
  3. Jeffries, C. M., Lu, Y., Hynson, R. M. G., Taylor, J. E., Ballesteros, M., Kwan, A. H and Trewhella, J. (2011) Human Cardiac Myosin Binding Protein C: Structural Flexibility within an Extended Modular Architecture. J. Mol. Biol. 414, 735-748.
  4. Lu, Y., Kwan, A., Trewhella, J., Jeffries, C. M. (2011) The C0C1 Fragment of Human Cardiac Myosin-Binding Protein C has Common Binding Determinants for Actin and Myosin. J. Mol. Biol. 413, 908-913.

18:00 - 19:00

Practical work: Visiting the beamlines at Doris and Petra

20:00

Welcome dinner at PETRA III, building 48e

 

Thursday, 18 October

 

From sample to data

 

9:00 - 10:00

Lecture: Validation of biomacromolecular structures - what have we learned? – G.Kleywegt

In the past two decades, many methods have been developed for validating crystallographic models of biomacromolecules. We now have a good idea of how such models should be validated, what the relative merits of various statistics and checks are, and what they tell us about any given model and its ability to explain the experimental data. I will explain why validation is important (for X-ray and other methods), what validation should entail, and how it can be carried out in practice. Hopefully, some of the lessons can be applied to the field of Small-Angle Scattering as well. I will also discuss what wwPDB is doing to address the issue of validation for X-ray, NMR and 3DEM structures in its future Deposition & Annotation software system.

10:00 - 11:00

Seminar: Sample preparation and characterisation – R.Meijers

The quality of the sample used in SAXS very much determines the results of the measurements, and it is important to measure aggregation state and purity. Ideally, this should be done just before the SAXS measurement, and the latest developments in this area will be presented. The quality control measures not only help to check the state of the sample, but can also contribute to the interpretation of the SAXS data. The talk will hopefully trigger a discussion on the types of quality control that are necessary and which complementary techniques are most useful.

11:00 - 11:30

Coffee break

11:30 - 12:30

Lecture: Data reduction and processing – A.Kikhney

The elastic scattering of randomly oriented particles in solution results in an isotropic scattering pattern which is usually recorded on a 2D detector. After scaling against the transmitted beam intensity and exposure time the normalized 2D scattering pattern is transformed into a 1D array of scattering intensities I(s) as a function of the modulus of the scattering vector s. The scattering pattern of the macromolecular solute is obtained by subtracting the scattering of the buffer that is measured separately in addition to the macromolecule solution. To monitor possible radiation damages two or more exposures of the same sample are recorded. The concentration of the macromolecular solute must be accurately determined to achieve correct normalization of the subtracted pattern. Several overall parameters (invariants) can be evaluated directly from the SAXS patterns of sufficient quality: molecular mass, radius of gyration (Rg) and hydrated volume. The shape of the distance distribution function p(r) provides information about the main features of the shape and size of the solute particles, including the maximum particle diameter Dmax.

12:30 - 14:30

Lunch at DESY canteen, building 9

14:30 - 16:00

Seminar: Participating students presentations, 10 min each

16:00 - 16:30

Coffee break

16:30 - 18:30

Practical demonstration: Data reduction and processing tutorial – P.Konarev, A.Kikhney

18:30 - 20:30

Practical work: Data analysis practical / SAXS measurements

 

Dinner at Don Quichotte

 

 

Friday, 19 October

 

From data to shape

 

9:00 - 10:00

Lecture: Ab initio methods: how they work – D.Svergun

Principles of ab initio shape determination in small-angle scattering are explained and different approached proposed to reconstruct three-dimensional models out of one-dimensional scattering data are presented. The question of informational content in the small-angle scattering data is related to the possibility of ab initio shape analysis. Several methods are discussed including the angular envelope function based on spherical harmonics (Stuhrmann, 1970; Svergun & Stuhrmann, 1991; Svergun et al., 1996), and Monte-Carlo type approaches using models consisting of densely packed fixed beads (Chacon et al., 1998; Svergun, 1999; Walther, Cohen & Doniach, 2000) or condensation of a gas of dummy residues (Svergun, Petoukhov & Koch, 2001). The possibilities (and necessity) of the use of a priori information are discussed and advantages and limitations of ab initio methods are illustrated in practical applications.

Models used in ab initio methods [image +]
All the methods minimize Discrepancy[Data] + Penalty[Additional info]

Chacon, P., Moran, F., Diaz, J. F., Pantos, E. & Andreu, J. M. (1998). Low-resolution structures of proteins in solution retrieved from X-ray scattering with a genetic algorithm. Biophys J 74, 2760-75.
Stuhrmann, H. B. (1970). Ein neues Verfahren zur Bestimmung der Oberflaechenform und der inneren Struktur von geloesten globularen Proteinen aus Roentgenkleinwinkelmessungen. Zeitschr. Physik. Chem. Neue Folge 72, 177-198.
Svergun, D. I. (1999). Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys J 76, 2879-86.
Svergun, D. I., Petoukhov, M. V. & Koch, M. H. J. (2001). Determination of domain structure of proteins from X-ray solution scattering. Biophys J 80, 2946-53.
Svergun, D. I. & Stuhrmann, H. B. (1991). New developments in direct shape determination from small-angle scattering 1. Theory and model calculations. Acta Crystallogr. A47, 736-744.
Svergun, D. I., Volkov, V. V., Kozin, M. B. & Stuhrmann, H. B. (1996). New developments in direct shape determination from small-angle scattering 2. Uniqueness. Acta Crystallogr. A52, 419-426.
Walther, D., Cohen, F. E. & Doniach, S. (2000). Reconstruction of low-resolution three-dimensional density maps from one-dimensional small-angle X-ray solution scattering data for biomolecules. J. Appl. Crystallogr.33, 350-363.

10:00 - 10:30

Coffee break

10:30 - 12:30

Practical demonstration: Ab initio programs tutorial - D.Franke, M.Petoukhov

12:30 - 14:30

Lunch at DESY canteen, building 9

14:30 - 20:00

Practical work: Data analysis practical / SAXS measurements

 

Dinner at PETRA III, building 48e

 

 

Saturday, 20 October

 

Hybrid modelling

 

9:00 - 10:00

Lecture: Rigid body refinement (basics) – D.Svergun

The use of high resolution models in small-angle scattering data analysis is considered. The methods to compute X-ray and neutron scattering patterns from the atomic models of particles in solution are presented (Svergun et al., 1995; Svergun et al., 1998). Possibilities for validation of the crystallographic structures in solution (Svergun et al., 2000) are demonstrated. The main idea of rigid body refinement in terms of subunits, domains of structural fragments is given (Svergun, 1991) and the methods for automated and interactive refinement are reviewed (Konarev et al., 2001; Kozin & Svergun, 2000; Petoukhov & Svergun, 2005), including the simultaneous use of SAXS and SANS with the information from other methods (Petoukhov & Svergun, 2006) and also the possibility to add missing linkers (Petoukhov et al., 2002; Petoukhov & Svergun, 2005). The presented methods are illustrated by practical applications.

[image +]

Konarev, P. V., Petoukhov, M. V., & Svergun, D. I. (2001). J. Appl. Crystallogr., 34, 527-532.
Kozin, M. B., & Svergun, D. I. (2000). J. Appl. Crystallogr., 33, 775-777.
Petoukhov, M. V., Eady, N. A., Brown, K. A., & Svergun, D. I. (2002). Biophys J, 83, 3113-3125.
Petoukhov, M. V., & Svergun, D. I. (2005). Biophys J, 89, 1237-1250.
Petoukhov, M. V., & Svergun, D. I. (2006). Eur Biophys J, 35, 567-576.
Svergun, D. I. (1991). J. Appl. Crystallogr., 24, 485-492.
Svergun, D. I., Barberato, C., & Koch, M. H. J. (1995). J. Appl. Crystallogr., 28, 768-773.
Svergun, D. I., Petoukhov, M. V., Koch, M. H. J., & Koenig, S. (2000). J. Biol. Chem., 275, 297-302.
Svergun, D. I., Richard, S., Koch, M. H. J., Sayers, Z., Kuprin, S., & Zaccai, G. (1998). Proc Natl Acad Sci U S A, 95, 2267-72.

10:00 - 10:30

Coffee break

10:30 - 12:30

Practical demonstration: Rigid body refinement tutorial – P.Konarev, M.Petoukhov

 

Free afternoon / Social event

 

 

Sunday, 21 October

 

Mixtures and interacting systems

 

9:30 - 11:00

Lecture: Form and structure factor, interactions, modelling – J.S.Pedersen

The lecture will deal with the classical modeling approach for analysis of small-angle scattering data. In the first part, analytical models are considered, and in a second part, effects of inter-particle interactions are described.

An overview over the form factors of various simple geometrical objects like spheres, ellipsoids, cylinders, discs etc will be given. A few examples are treated in greater detail. In addition, the form factors of polymer models with many internal degrees of freedoms are given, also with a few examples treated in detail. It will be shown how form factors of object consisting of spherical subunits can be derived and similarly how form factors of polymer-like branched structures can be calculated for pre-defined structures. Also models consisting of both spherical and polymer chain subunits will be discussed. It will be described how one can use of Monte Carlo simulations for generating form factors of complex structures for which an analytical calculation is not possible.

In the last part of the lectures, the effects of inter-particle interactions are discussed. The main analytical results will be given for spherical particles and polymer-like particles; approximations valid for particles with some anisotropy are also described.

Throughout the lectures, examples of applications to experimental data are included.

J. Skov Pedersen (1997) Analysis of Small-angle Scattering Data from Polymeric and Colloidal Systems: Modelling and Least-squares Fitting. Advances in Colloid and Interface Science 70, 171-201.
J. Skov Pedersen (2002) Monte Carlo Simulation Techniques Applied in the Analysis of Small-Angle Scattering Data from Colloids and Polymer Systems. Editors: Peter Lindner and Thomas Zemb. Neutrons, X-Rays and Light, 381-389. Elsevier.
J. Skov Pedersen (2002) Modelling of Small-Angle Scattering Data from Colloids and Polymer Systems. Editors: Peter Lindner and Thomas Zemb. Neutrons, X-Rays and Light, 391-420. Elsevier.
K.K. Andersen, C.L.P. Oliveira, K.L. Larsen, F.M. Poulsen, T.H. Callisen, P. Westh, J. Skov Pedersen, D.E. Otzen (2009) The Role of Decorated SDS Micelles in Sub-CMC Protein Denaturation and Association. Journal of Molecular Biology, 391(1), 207-226.

11:00 - 11:30

Coffee break

11:30 - 13:00

Practical demonstration: Analysis of mixtures and fexible systems: tutorial – P.Konarev, G.Tria

13:00 - 15:00

Lunch at EMBL, building 25a

15:00 - 16:00

Seminar: Joint use of SAXS and NMR – A.Pastore

A common tool of Structural Biology is the use of a 'cut-and-paste' approach which consists in dissecting proteins in individual domains, study them separately and eventually combine the information to reconstruct the overall shape of a molecule. Nuclear magnetic resonance (NMR) and small angle X-ray scattering (SAXS) are complementary techniques which have individually proven their own merits. NMR gives structural information of relatively small portions of a molecule, while SAXS provides a description of the overall shape of even large molecules/molecular complexes. Here, I shall discuss a number of examples in which the two techniques may be used in combination. I shall discuss the successes as well as the still open problems and limitations of this approach. I shall for instance show how we have reconstructed the shape of a ternary complex between CyaY, the bacterial ortholog of the frataxin protein that is implicated in a hereditary ataxia and IscS/IscU, the two main components of the iron-sulfur cluster biogenesis. We fit the X-ray structures of the individual components into the SAXS density, while using NMR for mapping the surfaces of interaction. The resulting model was validated by specific mutations. Despite the evident success of our work, much more effort needs to be put to push the application of this approach further.

16:00 - 16:30

Coffee break

16:30 - 20:00

Practical work: Processing and analysis of obtained data / SAXS measurements

 

Dinner at Mahlzeit

 

 

Monday, 22 October

 

Applications and related methods

 

9:00 - 10:00

Lecture: Joint use of SAS and AUC – O.Byron

Analytical ultracentrifugation (AUC) and small-angle x-ray and neutron scattering (SAXS, SANS or SAS overall) are fantastically complementary methods for characterising macromolecules in their near physiological, solution state 1; 2.  Each has its strengths and limitations, but used together, the combination is greater than the sum of its parts.  In this lecture I will present the principles underlying AUC, describe how AUC experiments are typically performed, the types of systems that can be analysed and the data that result.  I will focus on the interpretation of those data using hydrodynamic modelling (e.g. 3), at which point I will stress the synergy between SAS and AUC (e.g. 4; 5; 6; 7 ).  The lecture will close by touching on new developments in hydrodynamic modelling of flexible systems and how these interface with SAS data and modelling.

  1. Lebowitz, J., Lewis, M. S. & Schuck, P. (2002). Modern analytical ultracentrifugation in protein science: A tutorial review. Protein Science 11, 2067-2079.
  2. Ralston, G. (1993). Introduction to Analytical Ultracentrifugation, Beckman Instruments, Inc., Palo Alto, California, USA.
  3. Byron, O. (2008). Hydrodynamic modeling: The solution conformation of macromolecules and their complexes. In Methods in Cell Biology (Correia, J. J. & Detrich, H. W., eds.), Vol. 84, pp. 327-373. Elsevier.
  4. Vijayakrishnan, S., Kelly, S. M., Gilbert, R. J. C., Callow, P., Bhella, D., Forsyth, T., Lindsay, J. G. & Byron, O. (2010). Solution structure and characterisation of the human pyruvate dehydrogenase complex core assembly. Journal of Molecular Biology 399, 71-93.
  5. Vijayakrishnan, S., Callow, P., Nutley, M. A., McGow, D., Gilbert, D., Kropholler, P., Cooper, A., Byron, O. & Lindsay, J. G. (2011). Variation in the organisation and subunit composition of the mammalian pyruvate dehydrogenase complex E2/E3BP core assembly. Biochemical Journal 437, 565-574.
  6. Gabrielsen, M., Beckham, K. S. H., Cogdell, R. J., Byron, O. & Roe, A. J. (2012). FolX from Pseudomonas aeruginosa is octameric in both crystal and solution. Febs Letters 586, 1160-1165.
  7. Gabrielsen, M., Beckham, K. S. H., Feher, V. A., Zetterstrom, C. E., Wang, D., Müller, S., Elofsson, M., Amaro, R. E., Byron, O. & Roe, A. J. (2012). Structural characterisation of Tpx from Yersinia pseudotuberculosis reveals insights into the binding of salicylidene acylhydrazide compounds. Plos One 7.

10:00 - 11:00

Lecture: Maximum occurrence of conformations from average data – C.Luchinat

In many biologically relevant cases, protein-ligand or protein-protein recognition occurs thanks to the availability of multiple conformational states of at least one of the partners. In this talk I will demonstrate that Paramagnetic NMR is a powerful tool to address the conformational freedom of proteins. We have developed a rigorous theoretical interpretation of the average data that permits the characterization of biologically relevant protein-protein interactions in solution. Besides Paramagnetic NMR data, other average data such as SAXS can be used, separately or together, in the analysis, further increasing the power of the method. By our method one can calculate what we call "Maximum Occurrence", or MO, of any conformation in an ensemble. It can be empirically shown that the MO of a conformation correlates with the weight that this conformation has in the ensemble. Two-domain metalloproteins such as calmodulin or two-domain metalloenzymes such as matrix metalloproteinases (MMP) are paradigmatic examples. In the case of MMPs, conformational freedom has been demonstrated recently. Interdomain flexibility of MMPs can solve the paradox of the ability of MMPs to "devour" prays such as collagen that are much bigger than MMPs themselves. The program "MaxOcc" has been developed under the WeNMR grid infrastructure to allow researchers from outside NMR or SAXS laboratories to extract this type of information from their own average data.

11:00 - 11:30

Coffee break

11:30 - 12:30

Seminar: Following the process of ubiquitin conjugation and deconjugation in DNA regulation – T.Sixma

DNA repair processes are intricately regulated. Ubiquitin conjugation processes have emerged as a critical signalling system that is essential for many different processes in the cell. The attachment of this 76 amino-acid protein changes the target fate, resulting in relocalization or degradation by the proteasome.
We are interested in the regulation of ubiquitin conjugation and deconjugation processes, studying how the enzyme reactions involved are regulated. SAXS analysis has given insights into conformational states in conjugating and deconjugating enzymes as well as the ubiquitinated target. Together with structural biology and biochemical analysis this has helped us gain new insights in these essential processes.

12:30 - 14:30

Lunch at DESY canteen, building 9

14:30 - 15:30

Seminar: Structural studies of lipid systems – R.Willumeit

Lipids contribute up to 40% of the components of cell membranes and it became evident in recent years that they exhibit very specific functions and interactions with membrane proteins or membrane active substances such as peptide antibiotics. Therefore a significant interest grew in the understanding of the structure of lipid systems and their functionality within the cells. In this presentation an overview of possible structures an ensemble of lipids can build is given and examples will show how structure and function of lipids systems are related.

15:30 - 16:00

Coffee break

16:00 - 20:00

Practical work: Processing and analysis of obtained data

 

Dinner at PETRA III, building 48e

 

 

Tuesday, 23 October

 

Future outlook

 

9:00 - 12:30

Practical work: Processing and analysis of obtained data

12:30 - 14:30

Lunch at DESY canteen, building 9

14:30 - 15:30

Lecture: High brilliance synchrotrons for SAXS and WAXS – R.Fischetti

Synchrotron based, X-ray crystallography has emerged as a very powerful tool for the study of proteins and macromolecular complexes.  Recent advances in crystallization techniques have allowed the “crystallization bottleneck” to be overcome for certain classes of molecules, but still remains a significant challenge for large complexes. 
However the periodic boundary conditions of a three-dimensional crystal often limit which conformers of a molecule will crystal and severely limit one’s ability to study dynamics.

SAXS/WAXS is a very complementary technique to X-ray crystallography; and over the past decade, it has become the tool of choice for many researchers wanted to study biological molecules in a more natural solution state.  Thus one can explore the static ensemble average structure, and more importantly, the time-resolved dynamics of molecules initiated via external perturbations.  In addition, SAXS can be used to determine the arrangement of the components of a macromolecular complex.

In my lecture, I will preset recent and planned developments for SAXS/WAXS at the APS, and will summarize the current and planned state of beamlines at other third-generation synchrotron facilities around the world.  Micro-crystallographic techniques are often needed to study the most challenging macromolecular complexes because they yield tiny crystals. I will compare and contrast the needs of theses complementary techniques and summarize the state of micro-crystallography beamlines around the world.

15:30 - 16:30

Lecture: Combining microfluidics and Synchrotron SAXS – L.Arleth

Proteins and other macromolecular systems adopt different structural conformations as a direct response to their local environment, as defined both in terms of, e.g., pH, salt concentration, temperature, and by their participation in larger complexes. For this reason, the dependence between structural conformation and the local environment lies at the heart of understanding biomacromolecular function and is the core issue when exploiting and manipulating biomacromolecules with pharmaceutical and biotechnological applications in mind. In the last few years microfluidics based techniques have been proposed from several sides as the answer to the combined request of gaining access to high-throughput screening of proteins under a large number of solution conditions while at the same time minimizing the sample consumption. The combination of microfluidics with SAXS has been investigated by a few pioneering groups and the approach appears particularly promising in combination with the very intense beams at modern synchrotron SAXS facilities. The lecture will give an overview of recent results within the field and pin-point and discuss some of the central challenges.

Proposed literature:

  • The origins and the future of microfluidics, Whitesides GM, Nature, 2006, Vol 442(7101), 368-373.
  • High-throughput Small Angle X-ray Scattering from proteins in solution using a microfluidic front-end, Toft KN, Vestergaard B, Nielsen SS, Snakenborg D, Jeppesen MG, Jacobsen JK, Arleth L, Kutter JP, Analytical Chemistry, 2008, 80 (10), 3648-3654.
  • Automated microfluidic sample-preparation platform for high-throughput structural investigation of proteins by small-angle X-ray scattering, J.P. Lafleur, D. Snakenborg, S.S. Nielsen, M. Moller, K.N.  Toft, A. Menzel, J.K. Jacobsen, B. Vestergaard, L. Arleth, and J.P. Kutter, J. Appl. Cryst., 2011,  44(5), 1090-1099.
  • Conformational changes of calmodulin upon Ca2+ binding studied with a microfluidic mixer, Park HY, Kim SA, Korlach J, Rhoades E, Kwok LW, Zipfell WR, Waxham MN, Webb WW, Pollack L, PNAS, 2008, 105(2), 542-547.

16:30 - 17:00

Coffee break

17:00 - 19:30

Discussion: Results obtained by participating students, 10 min each

20:00

Course dinner at Lambert / X33 farewell party

 

 

Wednesday, 24 October

 

General discussion

 

9:00 - 10:30

Discussion: Results obtained by participating students, 10 min each

10:30 - 11:00

Coffee break

11:00 - 12:00

Discussion: Results obtained by participating students, 10 min each

12:00 - 13:00

Conclusions, SASQuest awards

 

Lunch at EMBL, building 25a

 

Departure