Go Ueno

Determination of damage-free crystal structure of an X-ray–sensitive protein using an XFEL

We report a method of femtosecond crystallography for solving radiation damage–free crystal structures of large proteins at sub-angstrom spatial resolution, using a large single crystal and the femtosecond pulses of an X-ray free-electron laser (XFEL). We demonstrated the performance of the method by determining a 1.9-Å radiation damage–free structure of bovine cytochrome c oxidase, a large (420-kDa), highly radiation-sensitive membrane protein.

A Norovirus Protease Structure Provides Insights into Active and Substrate Binding Site Integrity

ABSTRACT Norovirus 3C-like proteases are crucial to proteolytic processing of norovirus polyproteins. We determined the crystal structure of the 3C-like protease from Chiba virus, a norovirus, at 2.8-Å resolution. An active site including Cys139 and His30 is present, as is a hydrogen bond network that stabilizes the active site conformation. In the oxyanion hole backbone, a structural difference was observed probably upon substrate binding. A peptide substrate/enzyme model shows that several interactions between the two components are critical for substrate binding and that the S1 and S2 sites appropriately accommodate the substrate P1 and P2 residues, respectively. Knowledge of the structure and a previous mutagenesis study allow us to correlate proteolysis and structure.

Sample management system for a vast amount of frozen crystals at SPring-8

The RIKEN Structural Genomics Beamlines at SPring-8 have been constructed for high-throughput protein crystallography. Beamline operation is automated, cooperating with the sample management system developed along with the beamline facilities. The system consists of a database of a large number of sample crystals, and sample handling robots, named SPACE (SPring-8 Precise Automatic Cryo-sample Exchanger), at the beamline and the laboratory. The system has been working reliably during the commissioning of the automatic beamline operation, started in October 2003.

Dose dependence of radiation damage for protein crystals studied at various X-ray energies.

Radiation damage to protein crystals is the most serious problem in obtaining accurate structures from protein crystallography. In order to examine the photon energy dependence of radiation damage, 12 to 15 data sets from each of nine tetragonal lysozyme crystals were collected at nine different X-ray energies (6.5, 7.1, 8.3, 9.9, 12.4, 16.5, 20.0, 24.8 and 33.0 keV) using beamline BL41XU at SPring-8. All results were compared on the basis of absorbed dose, expressed in Gray (Gy). Crystallographic statistics, such as the values of lattice constants, R(merge) and I/sigma(I), for each data set degraded at all nine energies as the exposure time for each crystal increased. In all data sets, radiation damage was observed after the absorbed dose exceeded 10(6) Gy. However, from the point of view of crystallographic statistics normalized to the absorbed dose, no clear dependence on photon energy was observed in these results. Structural refinement showed that the average B-factor for the last data set was larger than that for the first data set at all energies tested. However, no energy dependence of radiation damage on B-factor was found. Furthermore, disruption of disulfide bonds due to radiation damage was observed in electron density maps even at the highest photon energy (33 keV) used in this study. Therefore, these results suggest that radiation damage in the energy range investigated could be evaluated based on absorbed dose without energy dependence, and that it is important to minimize the absorbed dose in a crystal sample for obtaining an accurate protein structure.

Beamline Scheduling Software: administration software for automatic operation of the RIKEN structural genomics beamlines at SPring-8.

Beamline Scheduling Software (BSS) has been developed for the schedule management and equipment control of the RIKEN Structural Genomics Beamlines I and II (BL26B1 and BL26B2 at SPring-8). The beamline operation is automated with a sample-changer robot and a database system. The experimental schedule is registered to BSS as a list of diffraction measurements for numbers of protein crystals. BSS implements the beamline operation communicating with beamline instruments through the computer network. The RIKEN Structural Genomics Beamline I (BL26B1) opened for users using BSS in October 2002, and the automatic operation was implemented at BL26B2 in October 2003.

Structural basis of leukotriene B4 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase catalytic mechanism and a possible SH3 binding loop

The bifunctional leukotriene B4 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase (LTB4 12-HD/PGR) is an essential enzyme for eicosanoid inactivation. It is involved in the metabolism of the E and F series of 15-oxo-prostaglandins (15-oxo-PGs), leukotriene B4 (LTB4), and 15-oxo-lipoxin A4 (15-oxo-LXA4). Some nonsteroidal anti-inflammatory drugs (NSAIDs), which primarily act as cyclooxygenase inhibitors also inhibit LTB4 12-HD/PGR activity. Here we report the crystal structure of the LTB4 12-HD/PGR, the binary complex structure with NADP+, and the ternary complex structure with NADP+ and 15-oxo-PGE2. In the ternary complex, both in the crystalline form and in solution, the enolate anion intermediate accumulates as a brown chromophore. PGE2 contains two chains, but only the ω-chain of 15-oxo-PGE2 was defined in the electron density map in the ternary complex structure. The ω-chain was identified at the hydrophobic pore on the dimer interface. The structure showed that the 15-oxo group forms hydrogen bonds with the 2′-hydroxyl group of nicotine amide ribose of NADP+ and a bound water molecule to stabilize the enolate intermediate during the reductase reaction. The electron-deficient C13 atom of the conjugated enolate may be directly attacked by a hydride from the NADPH nicotine amide in a stereospecific manner. The moderate recognition of 15-oxo-PGE2 is consistent with a broad substrate specificity of LTB4 12-HD/PGR. The structure also implies that a Src homology domain 3 may interact with the left-handed proline-rich helix at the dimer interface and regulate LTB4 12-HD/PGR activity by disruption of the substrate binding pore to accommodate the ω-chain.

Achievement of protein micro-crystallography at SPring-8 beamline BL32XU

A micro-focused X-ray beam with size ranging from 1 × 1 to 10 × 10 μm has been achieved at beamline BL32XU at SPring-8, Japan. Combining the available micro-beam with newly developed techniques has enabled efficient protein micro-crystallography.

Mail-in data collection at SPring-8 protein crystallography beamlines

A mail-in data collection system at SPring-8, which is a web application with automated beamline operation, has been developed.

The Catalytic Architecture of Leukotriene C4 Synthase with Two Arginine Residues

Leukotriene (LT) C4 and its metabolites, LTD4 and LTE4, are involved in the pathobiology of bronchial asthma. LTC4 synthase is the nuclear membrane-embedded enzyme responsible for LTC4 biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA4 as a derivative of arachidonic acid and a water-soluble glutathione (GSH). A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104. In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA4 binding model. In this study enzymatic assay with mutant enzymes demonstrates that Arg-104 is required for the binding and activation of GSH and that Arg-31 is needed for catalysis probably by activating the epoxide group of LTA4.

New micro-beam beamline at SPring-8, targeting at protein micro-crystallography

A new protein micro‐crystallography beamline BL32XU at SPring‐8 is under construction and scheduled to start operation in 2010. The beamline is designed to provide the stabilized and brilliant micro‐beam to collect high‐quality data from micro‐crystals. The beamline consists of a hybrid in‐vacuum undulator, a liquid‐nitrogen cooled double crystal monochromator, and K‐B focusing mirrors with large magnification factor. Development of data acquisition system and end station consists of high‐precision diffractometer, high‐efficiency area detector, sample auto‐changer etc. are also in progress.