Biological Applications of Small Angle X-ray Scattering (BioSAXS)

Abstracts

J. Günter Grossmann

Solution X-ray scattering of biological molecules

Knowledge of the size and shape of a biological macromolecule is one of the key stages of getting the picture of its functioning. There are a variety of techniques available that provide information at various degrees of reliability and detail. Protein crystallography and multidimensional NMR in solution are the main experimental methods that provide models of biological macromolecules on atomic scale. Three dimensional image reconstructions from electron micrographs is a valuable tool applied routinely in structural biology, particularly for examining large, mostly highly symmetrical molecular assemblies. Small-angle X-ray scattering (or solution X-ray scattering) is an additional promising method to investigate the structure of biological macromolecules in solution ranging from small proteins (from 5-10 kD) to multi-domain and oligomeric proteins and complexes (up to MD). Owing to its dependence on geometric shape, scattering data are sensitive to domain orientations and therefore to conformational changes and/or flexibility as well as to molecular associations in solution. The technique is often most powerful when used as a complementary tool with other structural techniques and may gain in interest due to the major challenges in the post-genome era.

Recent developments of shape restoration from scattering profiles allow the extraction of more meaningful biological information (contrary to only calculating Guinier radii or distance distribution functions). This talk will try to explain some of the technical aspects of the scattering method and its usefulness when combining data interpretation with results from other techniques (CD, EXAFS, NMR, PX and computational tools) so as to enhance the structural information. Several examples will be discussed.

Lecture Notes

Hiro Tsuruta

Structural studies on large biological assemblies by small-angle diffraction and time-resolved scattering

Several groups have recently explored the use of very low-resolution single crystal diffraction data to help solve the diffraction phase problem. Using a modified version of small-angle x-ray scattering instrument at SSRL, we obtained the data set covering (100) reflection (271 Å) to 14 Å from a single crystal of the synthetic Flock House Virus-like particle (sFHV). This data set allowed us to determine the 14 Å structure of sFHV ab initio, starting from a hard sphere as the initial phase model. This structure also revealed the additional density of bulk RNA inside the virus capsid that had not seen by the use of high-resolution data alone.

This talk will also describe time-resolved studies on virus/phage maturation. Many virus/phage particles undergo maturation processes triggered by pH variation in host cells. In our studies a pH-jump is usually accomplished by mixing a virus/solution with a slightly acidic buffer solution, followed by a series of x-ray scattering measurements. Recent studies on NwV, an insect virus and HK97 bacteriophage will be reviewed.

Lecture Notes

Paul G. Scott. and J. Günter Grossmann

Characterization of the solution structure of decorin by small-angle X-ray scattering.

Decorin is a member of the extracellular matrix small leucine-rich repeat glycoprotein/proteoglycan (SLRRP) family. Its functions may include regulation of the formation and/or organization of collagen fibrils, inhibition of the calcification of soft connective tissues, and effects on cell proliferation and behaviour, including the ability to suppress tumour growth. Decorin consists of a protein core of 329 amino acids carrying a single glycosaminoglycan chain and three N-linked oligosaccharides. Previous attempts to characterize this proteoglycan in solution by physical methods had been frustrated by a marked but poorly understood tendency to self-aggregate. Using multi-angle laser light-scattering analysis of the continuously flowing effluent from a size-exclusion column that efficiently separated the small amounts of high molecular weight aggregate, we recently determined that the predominant form of decorin in solution is a stable dimer. Solution X-ray scattering was used to gain insight into the shape of this dimer. X-ray scattering data on samples of intact decorin and its glycoprotein core were collected at station 2.1 of the Synchrotron Radiation Source (SRS), Daresbury Laboratory, U.K. The radius of gyration, the forward scattering intensity, and the intra-particle distance distribution function p(r) were calculated using the indirect Fourier transform method as implemented in the program GNOM. Particle shapes were restored from the experimental scattering profiles using the ab initio procedure based on simulated annealing of a set of dummy spheres representing the amino acid chain of the protein. The program CRYSOL was used to simulate scattering curves from models based on the crystal structures of YopM, the leucine-rich effector protein from Yersinia pestis (PDB codes 1G9U and 1JL5). This particular protein was chosen as a model for decorin based on its overall similarity in size, in length and number of leucine-rich repeats, and in secondary structure. It was concluded that the decorin core dimer approximates the shape of 2 intertwined C's with the parallel, concave, b-sheet faces of the monomers opposed. Other arrangements, such as "horseshoe", "donut" or "S"- shapes could be excluded with confidence. The high-resolution structure subsequently determined for the decorin glycoprotein core, revealed the presence in the crystal of a dimer closely resembling that predicted by solution X-ray scattering. The demonstration that decorin is a dimer in solution, and more specifically that dimerization is mediated by the concave b-sheet face, has important consequences for the understanding of its biological activities which all depend on its ability to interact with other proteins.

Lecture Notes

Hans Vogel, Craig Shepherd, Aalim Weljie, Hidenori Yoshino, and Peter Tieleman

Studying large scale conformational changes in proteins: the case of calmodulin

Calmodulin has a unique dumbbell structure in the crystal structure, but the linker region between the two domains is flexible in solution. Upon binding to a target protein calmodulin often collapses into a more globular structure. These dramatic changes in the overall shape of the protein can be studied by NMR diffusion, dynamic light scattering and small angle xray scattering studies for example. In this presentation I will show that theoretical Molecular Dynamics calculations in combination with SAXS measurements provides unique opportunities to investigate the conformational flexibilty of calmodulin and related proteins.

Bob He

Small Angle X-ray Scattering with the NanoSTAR for Biological Materials

The nanostructure analysis of anisotropic materials such as many polymers, fibrous materials, single crystals and bio-materials by SAXS becomes possible by use of a strong collimated point beam and high speed area detector. The core of the NanoStar system is the Bruker Hi-Star Area Detector. The area detector has high sensitivity and low noise, and the most important, the large dynamic range and simultaneous collection of 2D image.

Our discussion will cover some hardware considerations of X-ray detectors, X-ray collimation and sample stage in laboratory and synchrotron environments for small angle x-ray scattering experiments with biological materials. We will explore results from orientation of the mineral particles in the bone section, with consideration of scanning SAXS. Results from Picea abies (sprucewood) and collagen fibrils (retina) will be elaborated.

Lecture Notes

Biological Applications of Small-Angle X-ray Scattering (SAXS)

Lecture Notes

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