Upgrade of a high flux MX beamline BL41XU at SPring-8

BL41XU is the oldest macromolecular crystallography (MX) beamline at SPring-8 [1]. Although it has been contributing to the structure determination of difficult samples since its start of operation in 1997, the targets for the structural study is still getting more challenging and the crystal quality brought to the beamline is getting worse. Therefore, we have upgraded the focusing optics and diffractometer of BL41XU to cope with these targets. Our goal is to achieve an environment which can offer a stable beam with a photon flux of >10¹³ photons/s in the beam size range of 5 ~ 50μm. It is a complementary specification with our micro-focus beamline BL32XU [2], and allows both micro-crystallography and data collection using crystal volume. The new optics adopts a two-step focusing with elliptical figured mirrors: the first optics is a single horizontal mirror and the second one adopts Kirkpatrick–Baez (KB) configuration. At the middle of the two focusing optics, a high precision horizontal slit is installed to define secondary source size. The beam size can be changed either by changing the secondary source size, by offsetting the sample position, or by tilting the vertical mirror. For the stable use of small beam, both KB mirror and diffractometer were equipped on the granite stage, and enclosed in a booth in which the temperature is keep stable. On the new diffractometer, we equipped PILATUS3 6M that enables rapid data collection combining with high flux beam. Together with the upgrade of hardware, software tools, which support diffraction based centering and determination of measurement condition, have been implemented in order to make full use of the renewed beamline. The upgrade was conducted in the long shut-down period between January and March of this year, and the beamline was opened for users in the middle of May after commissioning of one month. The result of commissioning and initial results will be presented. This study was supported by the MEXT of Japan.

Current status of protein micro-crystallography at SPring-8

Protein micro-crystallography is one of the most advanced technologies for protein structure analysis. In order to realize this, an undulator beamline, named BL32XU, was constructed at SPring-8. The beamline can provide beam with size of 0.9 x 0.9 µm and photon flux of 6E10 photons/s. The beam size can be easily changed by users from 1 to 10 µm square with the same flux density. Through three years user operation, we have established several key systems for efficient protein micro-crystallography. One of them is the software for precise positioning of micro-crystals in `raster scan'. SHIKA is a program with GUI which searches diffraction spots in a plenty of low dose diffraction images obtained in raster scan. Finally, it generates 2D map of crystal positions based on the number of spots or spot intensities. Parameters and thresholds in peak search have been empirically optimized for LCP crystals and it provides robust results. Another system is for the data collection strategy. Almost all successful data collections were conducted via `helical data collection' on BL32XU using the line-focused beam. The GUI software, named KUMA, enables estimation of an accumulated dose and suggests suitable experimental conditions for helical data collection. The system is proven to be useful for experimental phasing using tiny LCP crystals of membrane proteins[1-3]. Based on them, the rapid and automatic data collection system using protein micro-crystals is under development. The new CCD detector, Rayonix MX225HS, was installed for faster data acquisition in 10 Hz with the pixel size of 78 µm square. The new SHIKA using GPUs is under development for faster and more accurate crystal alignment. Following this step, KUMA system can suggest experimental conditions for each crystal found on the loop. We also report about the effects of higher dose rate in protein crystallography up to the order of 100 MGy/s. This work was supported by Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Processing of Protein Crystals using by Deep-UV Pulsed Laser

The absorption of X-rays which pass through the protein crystal is possibly the largest source of systematic errors in macromolecular crystallography. Therefore we are developing protein crystal processing system using Pulsed UV Laser Soft Ablation (PULSA) technique [1] to reduce the systematic error as well as background scattering from cryoprotectant agents. For high-quality diffraction data collection from organic material, crystals are usually processed to spherical shape in order to keep X-ray path length in crystal constant. This dramatically reduces systematic errors caused by `absorption of X-rays'. Although shaping crystal was thought to be effective for protein crystallography, there was no usual technique to achieve this because protein crystals are exceedingly fragile against mechanical stress. We are developing protein crystal processing system using PULSA technique. In this system, short pulsed UV-laser (maximum power: 1.0 μJ/pulse, wavelength: 193.4 nm, duration: less than 1.3 nsec) is raised by NSL-193L (Nikon Corporation) and focused on 4 μmφ (FWHM). The focused laser is controlled by galvanomic mirror system and irradiates a sample. Combining this mirror system with four-axis goniometer enables to process crystal to arbitrary shape that is easily defined on GUI. Several protein crystals have been successfully processed into spherical, column and square pole shape, etc. In the case of crystal processed into column shape (diameter is 50 μm), in addition to reducing absorption effects, signal-noise ratio of diffraction data can be increased by removing cryoprotectant agent surrounding the crystal. This work was supported by "Platform for Drug Discovery, Informatics, and Structural Life Science" from MEXT, Japan.

SHIKA: a fast and accurate spot finder for raster scan on microfocus beamlines

On BL32XU, a microfocus beamline at SPring-8, oscillation data are collected with typical horizontal beam size of 1 μm. Hence it requires very accurate crystal centering, which is difficult especially for invisible crystals e.g. LCP crystals. Therefore, we perform raster diffraction scan to find crystal positions based on their diffractivity using low-dose exposure. It had been time consuming process due to two reasons; i) slow readout time of CCD, ii) manual inspection of hundreds of diffraction images. To tackle this problem, we installed new fast-readout CCD detector, MX225HS (Rayonix, L.L.C.), and developed support tool for raster scan based crystal centering. The tool visually shows possible crystal position on 2D map based on spot populations, and therefore it is named SHIKA (Spot-wo Hirotte Ichiwo Kimeru Application; a Japanese abbreviation which means the application for crystal positioning by picking up spots). SHIKA automatically detects new images when raster scan started and finishes just after raster scan ends. On GUI, user can find and pass the crystal position information to KUMA (a tool suggesting helical data collection strategy with predicted radiation damage) to start data collection immediately. User can also see picked spots on diffraction images with GUI. SHIKA has been developed based on DISTL [1] and modified to be faster and more accurate, especially for LCP crystal which is an important target on BL32XU. SHIKA picks up spots after subtracting smoothed pseudo-background which is a key for better separation of spots and ring-like diffuse background of lipids. Smoothing is time-consuming, but SHIKA now uses GPUs for almost all process including high-speed median filter [2] so that it can be done within ~100 msec. Further development is under way for faster processing. Now SHIKA can be also used on BL41XU, a high flux beamline at SPring-8 with some adjustment for PILATUS3 (Detectris Ltd.) detector.

Kinetic Crystallography on MutT and its homolog

Oxidized deoxynucleotides cause replicational errors because of their misincorporations into DNA. The MutT and related proteins prevent transversion mutations by hydrolyzing mutagenic oxidized nucleotides such as 8-oxo-dGTP and 2-oxo-dATP, and there is a difference in substrate specificities between them. E. coli MutT hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP with extremely high substrate specificity. On the other hand, its human homolog has broad substrate specificity for oxidized nucleotides and hydrolyzes 8-oxo-dGTP as well as 2-oxo-dATP. In order to understand mechanisms of their substrate specificities, we solved the crystal structures of MutT and its homolog complexed with their substrates and revealed structural basis of the high substrate specificity of E. coli MutT for 8-oxoguanine nucleotide and the broad substrate specificity of its human honolog for oxidized nucleotides. In this paper, we report the hydrolysis mechanisms of both enzymes revealed by kinetic protein crystallography. Both hydrolysis reactions were initiated by soaking the enzyme-substrate complex crystals in divalent metal solution. After incubation under various conditions, the reactions were terminated by freezing the crystals at 100K. X-ray diffraction data were collected at Spring-8 and Photon Factory. In the MutT crystals, the structures of sequential catalytic intermediates showed the activation mechanism of the nucleophilic water molecule synchronized with the coordination of metal ions. Now by using the crystals of its human homolog, the trial of the catching the intermediate state of catalysis is in progress.

Intact structure determination of a highly radiation sensitive protein at SACLA

X-ray irradiation on a protein crystal can cause some subtle structural modification on the protein structure even if the radiation dose is much smaller than a dose used for a common crystal structure determination. In some case such structural modification increases ambiguity of structural inspection, and eventually could be an obstacle on the elucidation of structure basis of protein function. Bovine heart cytochrome c oxidase (CcO) is one of such proteins having some problem caused by the radiation damage. The proton pumping of CcO is coupled with O2 reduction at the O2 reduction site, thus accurate structure determination of bound ligand as well as CcO itself is very important. Whereas accurate structure determination was impeded by the immediate photolysis of the peroxide ligand upon X-ray irradiation even at a cryogenic temperature[1]. We developed a goniometer based data collection system for the femtosecond crystallography at SACLA (SPring-8 Angstrom Compact free-electron LAser). The femtosecond crystallography is expected to have an advantage in high-resolution and radiation damage free structure determination of very large protein by combined usage of large crystal and femtosecond intense X-ray pulse. In this presentation we are going to show the result of the femtosecond crystallography on the crystal of CcO having large unit cell dimensions. The close inspection of the electron density map calculated at 1.9 Å resolution showed the femtosecond crystallography worked fine for the high resolution and radiation damage free crystal structure determination of CcO.