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Protein Crystallography

The special properties of the X-ray beams provided by synchrotron radiation sources (high intensity, low divergence, tunability) are now essential for collecting high-quality X-ray diffraction data from single crystals of macromolecules and their complexes. In recent years a great deal of effort has been devoted to automate the ESRF macromolecular crystallography (MX) beamlines and to provide robotic sample changers, making the beamlines more user-friendly, more reliable and much faster. Additionally, the availability of large-area CCD detectors coupled with the wide energy range tunability of some of these beamlines allows the elucidation of new structures of very large macromolecular systems. Moreover, the recent commissioning of a microfocus beamline dedicated to MX means that we can now routinely collect diffraction data from crystals with a volume as small as 5μm³. Full details of the characteristices of the ESRF MX beamlines can be found at http://www.esrf.fr/UsersAndScience/Experiments/MX/How_to_use_our_beamlines

X-ray diffraction studies of macromolecular crystals are used to reveal the atomic arrangements of proteins, nucleic acids and viruses. The structural knowledge gained using this technique is a powerful tool in the drug design process.

The ESRF offers the industrial community rapid access to its MX beamlines. A data collection service (MXpress) has also been implemented to cater for those clients who prefer not to come to the ESRF to perform experiments: frozen samples sent via courier are analysed by experienced ESRF scientists who provide clients of the MXpress service with both raw diffraction images and processed data, as well as a full report on the data collections carried out.

highlights 2006 p60 – GSK – crystal structure of human cytochrome P450 2D6

3D picture of protein at atomic scale

For Vincent Mikol, head of structural biology with Aventis Pharma in Paris, Mxpress has freed his staff from the travel and unsociable hours associated with working at a synchrotron facility. “We really wanted to avoid spending time, specifically so many nights, at the synchrotron”, he says. So a year ago, Aventis entered into an agreement with Gordon and Monaco (our MX scientists) that has so far provided data on more than 200 biological samples. Mikol is pleased with the results, but acknowledges that staff unused to the idea of trusting a third party to handle their precious samples had to overcome some mental roadblocks. “At first people were reluctant to send new crystals, but after a while, they saw that the ESRF group was responsive and capable”, says Mikol.

SAXS-WAXS

Small and wide-angle X-ray scattering (SAXS and WAXS) are used intensively to study condensed matter samples whether they are in liquid or solid form, and with a spacial resolution ranging from angstroms to hundreds of nanometers. Some examples where these techniques are very useful include phase diagram of colloids, study of the structure of a drug function of the process, understanding of the behaviour of liquid crystals under magnetic field or study of the manufacture of catalytic nanoparticules. The advantage of using SAXS and WAXS compared to traditional techniques like AFM, TEM or dynamic light scattering is that they offer an analysis deep in the bulk, even on turbid solutions. A Combination of techniques like rheology and light scattering is possible, and provides a better understanding of the behaviour of a sample in a short time delay.

Areas of Interest are:

• Colloids & suspensions
• Composite materials
• Crystallisation
• Fiber structure and processing
• Polymers
• Self-assembly of nanoparticules

Microtomography

Microtomography is an imaging technique based on the radiography technique. It is similar to the medical scanner (500 microns) but with a much higher resolution: up to 0.28 micron pixel size. At the hospital, the X-ray source and detector turn around the patient. The synchrotron cannot turn, so the source and detector are fixed and the sample turns from 0 to 180°. A half turn is sufficient due to parallel beam configuration. About 1500 radiographs are recorded during this half turn and the filtered back projection algorithm is used to obtain 3D images. A complete scan takes about 15 minutes, but in certain conditions and for in situ experiments, scan time can be reduced to 20 seconds. Microstructure of various matters can be studied: qualitative analysis (porosity, permeablity, …), but also quantitative ones benefiting from the monochromaticity of synchrotron light. Samples are scanned in usual temperature and pressure conditions, but sample environment devices have been developed in order to perform in situ experiments. Tensile/compression and fatigue stages are available. A cryostat allows temperatures as low as -60°C and a furnace up to 1600°C.

Example of a microtomography experiment

Thanks to recent development in 3D microtomography (ultra-fast acquisition), it is now possible to perform scans with spatial resolution of a few microns in less than 20s. This allows the study of 3D solidification of aluminium alloys which up to now was studied in 2D radiography. The images presented here are 2D sections of 3D data showing the solidification of an Al-Cu-Si alloy solidified at 0.1°C/s. One can see first the dendrites moving in the liquid up to a specific solid fraction. The solidification proceeds up to the transformation of the liquid in eutectic leading to pore formation at the end of solidification.

For the text of this paragraph, a special thanks to:

Oyvind Nielsen SINTEF (Norway)

Nathalie Limodin, Michel Suéry, Luc Salvo (SIMAP - GPM2, France)

E. Boller, P. Cloetens, R. Mosko (ESRF)

S. Gailliègue, K. Madi, S. Forest (Centre des Matériaux, France)

Microscopy RX

The X-ray microscopy technique enables new investigations to study thick specimens and to access K absorption edges of elements of major interest in biological and material sciences. Micro-mapping and quantification of elements in the bulk of your samples, and the detection of traces of this element by X-ray fluorescence is possible. No special sample preparation or vacuum environment is required. This technique is particularly suited for the detection of traces (ppm) of elements like Sulfur, Chromium or Iron in an organic or a mineral matrix and to analyse the oxydation state of this element Cr III or Cr IV in order to differentiate the origin of this element. Such a quantitative and qualitative analysis allows a very precise cartography of a section of your sample to be produced.

A case study

Cadmium is a metal of high toxicity for plants. Resolving its distribution and speciation in the Arabidopsis Thaliana, a species of plant which hyperaccumulates metal, has been essential for understanding the mechanism involved in Cd tolerance, trafficking and accumulation. The elemental distribution of Cd, P, S K and Ca was investigated in the roots and the leaves using scanning electron microscopy (SEM) coupled with synchrotron-based micro X-ray fluorescence (µ-XRF). The chemical form of Cadmium has been established by micro X-ray absorption near-edge structure (µ-XANES).

FTIR Microscopy

Synchrotron radiation obtained at the ESRF provides a highly-coherent broadband Infrared Source which covers the whole Infrared spectrum. Its extraordinarily high brightness can provide more than three orders of magnitude improvement in intensity and its almost linear broadband spectral coverage over the whole IR region makes it an ideal IR source. Coupled to an IR microscope, Synchrotron FTIR microscopy is a complementary analytical tool, in particular for micro-imaging with high lateral resolution, high chemical sensitivity and a low detection limit. The signal/noise ration is 1000 times better than with a conventional FTIR microscope. Whereas the X-ray scanning microscope enables identification and location of atoms by µ-X-ray fluorescence and atom environments (oxidation state, geometry) by µ-XANES, FTIR microscopy gives invaluable information on molecular groups and structures. This technique is now extensively used over a wide area of disciplines where µ-mapping is required for the characterization of biological and biomedical systems, in the food industry, surface treatment characterization, polymer industry and cosmetics.

X-ray Absorption Spectroscopy: EXAFS, XANES

X-ray absorption spectroscopy techniques provide information on the atomic organisation and chemical bonding around an absorbing atom in whatever medium it is embedded, ie. solid, liquid or gas. Although many synchrotrons have the ability to perform X-ray absorption spectroscopy, the ESRF has the following advantages:

• Studies in ultra-dilute conditions (down to a few ppm)
• Studies of fast kinetic processes (ms scale)
• High flexibility to perform in situ reactions or real time studies
• Well-defined beam polarisation

These are particularly useful for the study of catalytic reactions in situ and the characterisation of reaction intermediates. Further characterisation is also possible because of the availability on-site of an extensive range of other techniques.

Powder diffraction, High-energy diffraction, Surface diffraction

Diffraction remains the most efficient technique for the determination of the atomic structure in condensed materials. The combination of diffraction with the qualities of the X-ray beams available at the ESRF still extends the possibilities of diffraction techniques.

• Powder diffraction at the ESRF permits the measurement of the utmost high-resolution data for solving crystal structures, for quantitative phase analysis or real-time studies at different stages of development of a new drug in the pharmaceutical industry. Such precision is of prime importance in the study of polymorphism, stability during storage and structural changes that are induced either by temperature, hydration and other physico-chemical parameters, or after industrial processes like milling, drying, grinding and tableting. Important also is the ability to make a highly-accurate comparison of the crystalline forms of a generic drug with that of its original form
• High-energy diffraction at the ESRF offer a unique opportunity to explore thick materials in the bulk. It is broadly used in material science and in metallurgy including studies of crystal perfection and phase transitions, residual stress and strain in alloys, magnetic ordering and spin densities. New areas are the study of the structures of liquids and amorphous solids, imaging by scattering, electronic structure of superconducting materials and in situ strain measurements in alloys as a function of time
• Surface diffraction is a technique dedicated to surfaces and interfaces structural characterizations. It can be used for performing static surface crystallography studies or for studying processes at surfaces in real time. Even if several other techniques allow a structural determination of surfaces, X-ray diffraction offers unique possibilities. X-rays are weakly absorbed by matter. As a consequence surface X-ray diffraction is not limited to free surfaces under UHV conditions but it can be applied with success to study solid interfaces. Solid/liquid interfaces and high pressure gas/solid interfaces are well adapted to the technique. This is of particular importance in the case of heterogeneous catalytic reaction where the role of the catalyser can be studied under real working conditions. In addition to crystallographic studies, surface diffraction is also suited to dynamical studies such as epitaxial growth, ion patterning, surface kinetics and phase transitions. In particular, ion erosion with ion beams in combination with grazing-incidence small-angle scattering has been employed to study the dynamical evolution of medium-range correlations during nanopatterning. The possibility of tuning the energy allows to carry out experiments at resonance energies of particular elements. This is important to investigate surface magnetism in order to determine the depth distribution and magnetisation of the resonant atoms. Resonance can also be used to determine the involvement and role of a particular atomic species in the surface/interface structure

Many types of materials can be studied: minerals, metals alloys, organic and inorganic compounds, polymers, emulsions, colloids, fibres, coatings, thin layers.