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abstracts
The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallise on much smaller scales and are frequently mounted in opaque or highly refractive materials.[1,2] It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this poster the use of X-ray microradiography and microtomography is reported as a tool for crystal visualisation, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals, and crystals mounted in opaque materials such as lipidic cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to a diffraction grid scan. However, for sample location and shape estimation alone, just a few radiographic projections may be required; hence reducing the dose the crystals will be exposed to prior to the diffraction data collection.[3]
abstracts
Diamond Light Source (DLS) provides a suite of world-leading beamlines for macromolecular crystallography (MX) experiments which are used by scientists from all over the world. Key to their success is an integrated approach to automating the hardware and software environments such that it is only necessary to enter the beamline hutch to change large batches of samples for the robotic sample changers. Alongside investing in automation, all beamlines are also equipped with Pilatus 6M-F detectors (25 - 100 Hz) resulting in fast data collections and high turnover of samples. Rapid downstream processing of the resulting data has been developed since this is essential to drive experimental decisions. The feedback from these pipelines needs to be made readily available to users in a timely manner and a number of tools are available for both local and remote visualization of results. Remote access to these facilities was a design requirement from the outset. Several tools have been integrated and developed to streamline the process of remote access yet give the same software environment remotely as would be experienced if the experimenter was present at the beamline. The advent of remote access for cryogenically frozen samples has led to the implementation of new shift patterns for the user programme, enabling frequent short shifts for the many groups who use DLS. Remote access to MX beamlines is also a prerequisite of many industrial clients of DLS. For the future we are moving forward with the development of remote access for in-situ data collection from crystallization plates following on from the success of this method for screening and collecting data by users at the beamlines. The implementation and impact of remote access at DLS will be presented here.
abstracts
Single-wavelength anomalous dispersion of sulfur atoms (S-SAD) is an elegant phasing method to determine crystal structures that does not require heavy atom incorporation or selenomethionine derivatization. Nevertheless this technique has been limited by the paucity of the signal at usual X-ray wavelengths, requiring very accurate measurement of the anomalous differences. Here we report the data collection and structure solution of the N-terminal domain of the ectodomain of Hepatitis C virus (HCV) E1, from crystals that diffracted very weakly. By combining the data from 32 crystals it was possible to solve the sulfur substructure and calculate initial maps at 7Å resolution, and after density modification and phase extension, using a higher resolution native dataset, to 3.5Å resolution, model building was achievable. The crystal structure of the N-terminal domain of reveals a complex network of covalently linked intertwined homodimers that do not harbor the expected truncated class II fusion protein fold.
