[en] Synchrotron beamlines are now required to provide an experimental environment that supports digital transformation. Many of the beamlines for macromolecular crystals around the world now employ the systems for automated data acquisition and remote access. At SPring-8, both systems have been newly established with brilliant undulator beamlines to provide rapid and accurate measurements. Here, we will introduce these systems that can be used without visiting the site. (author)

[en] A new crystal-mounting method has been developed that involves a combination of controlled humid air and polymer glue for crystal coating. This method is particularly useful when applied to fragile protein crystals that are known to be sensitive to subtle changes in their physicochemical environment. Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection

[en] Recently, 'S-SAD' phasing in protein crystallography that uses anomalous signal from sulfur atoms included in cysteine and methionine residues in native proteins has been widely noticed. It is effective in phasing to collect diffraction data in a long wavelength near the X-ray absorption edge of sulfur. We did a preliminary X-ray diffraction experiment with protein crystals in several wavelength conditions (>2.3 A) at the BL 15 in the SAGA Light Source, and analyzed the data using S-SAD method. The increase of the anomalous dispersion effect in a longer wavelength was superior to the rise of the measurement errors originating from augmentation of the scatter and attenuation of the diffraction by air and water. The advantage for S-SAD phasing in a longer wavelength was ascertained. (author)

[en] Especially high flux and short pulse-width of XFEL enables us to perform the pump-probe time resolved structural analysis in combination with the method of inducing enzymatic reaction by light irradiation. By using this pump-probe time resolved method, we successfully revealed that the open/closed mechanism of proton pumping pathway of cytochrome c oxidase (CcO), terminal oxidase in mitochondrial respiratory chain. In this topic, we want to discuss the future of XFEL time resolved analysis. (author)

[en] The PAS domain of RsbP, a stress-response protein from B. subtilis, was crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to space group P2₁ and diffraction data were collected to a resolution of 1.6 Å. RsbP, a regulator of RNA polymerase σ^B activity in Bacillus subtilis, is a phosphatase containing a Per–Arnt–Sim (PAS) domain in its N-terminal region that is expected to sense energy stresses such as carbon, phosphate or oxygen starvation. Energy-stress signals are transmitted to the PAS domain and activate the C-terminal phosphatase domain of RsbP, leading to activation of the downstream anti-anti-σ^B factor RsbV. Finally, the general stress response is induced to protect the cells against further stresses. The recombinant PAS domain of RsbP was crystallized by the sitting-drop vapour-diffusion technique using 40% PEG 400 as a precipitant. The crystals belonged to space group P2₁, with unit-cell parameters a = 55.2, b = 71.7, c = 60.2 Å, β = 92.1°. Diffraction data were collected to a resolution of 1.6 Å

[en] Seven beamlines are operated for macromolecular crystallography (MX) at SPring-8. The three undulator beamlines are developed for cutting edge target and four bending-magnet beamlines are developed for high throughput MX. The undulator beamline, BL41XU that provides the most brilliant beam, is dedicated to obtain high quality data even from small-size and weakly-diffracting crystals. The minimum beam size at sample position is achieved to 10 μm diameter using a pin-hole collimator. Its photon flux at wavelength λ = 1.0 A is 2.8x10¹¹ photons/sec. This small beam coupled with irradiation point scanning method is quite useful to take diffraction dataset from small crystals by suppressing the radiation damage. These advanced technologies made a number of difficult protein structure analysis possible, (i.e. Sodium-potassium ATPase). The bending-magnet beamlines BL26B1/B2 and BL38B1 provide automatic data collection exploiting the high mobility of the beam. The beamline operation software 'BSS', sample auto-changer 'SPACE' and web-based data management software 'D-Cha' have made the automatic data collection possible. The 'Mail-in data collection system' that accepts distant users samples via courier service have made users possible to collect diffraction data without visiting SPring-8. The structural genomics research is promoted by these beamlines.

[en] The RNA-binding protein Hfq from B. subtilis was crystallized using the hanging-drop vapour-diffusion method in two crystal forms that belonged to space groups I422 and F222; diffraction data were collected to 2.2 Å resolution from both forms. The Hfq protein is a hexameric RNA-binding protein which regulates gene expression by binding to RNA under the influence of diverse environmental stresses. Its ring structure binds various types of RNA, including mRNA and sRNA. RNA-bound structures of Hfq from Escherichia coli and Staphylococcus aureus have been revealed to have poly(A) RNA at the distal site and U-rich RNA at the proximal site, respectively. Here, crystals of a complex of the Bacillus subtilis Hfq protein with an A/G-repeat 7-mer RNA (Hfq–RNA) that were prepared using the hanging-drop vapour-diffusion technique are reported. The type 1 Hfq–RNA crystals belonged to space group I422, with unit-cell parameters a = b = 123.70, c = 119.13 Å, while the type 2 Hfq–RNA crystals belonged to space group F222, with unit-cell parameters a = 91.92, b = 92.50, c = 114.92 Å. Diffraction data were collected to a resolution of 2.20 Å from both crystal forms. The hexameric structure of the Hfq protein was clearly shown by self-rotation analysis

[en] Isopentenyl diphosphate isomerase from M. jannaschii has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.08 Å resolution. Type 2 isopentenyl diphosphate isomerase (IDI-2) is a flavoprotein. Recently, flavin has been proposed to play a role as a general acid–base catalyst with no redox role during the enzyme reaction. To clarify the detailed enzyme reaction mechanism of IDI-2 and the unusual role of flavin, structural analysis of IDI-2 from Methanocaldococcus jannaschii (MjIDI) was performed. Recombinant MjIDI was crystallized at 293 K using calcium acetate as a precipitant. The diffraction of the crystal extended to 2.08 Å resolution at 100 K. The crystal belonged to the tetragonal space group I422, with unit-cell parameters a = 126.46, c = 120.03 Å. The presence of one monomer per asymmetric unit gives a crystal volume per protein weight (V_M) of 3.0 ų Da⁻¹ and a solvent constant of 59.0% by volume

[en] A thermophilic bacterial homologue of mitoNEET (a mammalian mitochondrial outer membrane protein) from T. thermophilus HB8 (open reading frame TTHA0026; Tth-NEET0026) has been identified as a water-soluble prototypal [2Fe–2S] protein and crystallized. The bipyramidal crystals of the recombinant Tth-NEET0026 diffracted to 1.80 Å resolution using synchrotron radiation. MitoNEET (a mammalian mitochondrial outer membrane protein) is a potential pharmacological and clinical target of the insulin-sensitizer pioglitazone. The thermophilic homologue of mitoNEET (TTHA0026) from Thermus thermophilus HB8 has been heterologously overproduced in Escherichia coli and purified as a water-soluble prototypal protein containing the mitoNEET-like [2Fe–2S] cluster. The resultant recombinant protein, named Tth-NEET0026, has been crystallized in its oxidized form by the hanging-drop vapour-diffusion method using 17%(w/v) polyethylene glycol 4000, 8.5%(v/v) 2-propanol, 15%(v/v) glycerol and 0.085 M HEPES–NaOH pH 7.2. The dark reddish crystals diffracted to 1.80 Å resolution and belonged to the tetragonal space group P4₃2₁2, with unit-cell parameters a = 45.51, c = 84.26 Å. The asymmetric unit contains one protein molecule

[en] An online UV–visible microspectrophotometer has been developed for the macromolecular crystallography beamline at SPring-8. Details of this spectrophotometer are reported. Measurement of the UV–visible absorption spectrum is a convenient technique for detecting chemical changes of proteins, and it is therefore useful to combine spectroscopy and diffraction studies. An online microspectrophotometer for the UV–visible region was developed and installed on the macromolecular crystallography beamline, BL38B1, at SPring-8. This spectrophotometer is equipped with a difference dispersive double monochromator, a mercury–xenon lamp as the light source, and a photomultiplier as the detector. The optical path is mostly constructed using mirrors, in order to obtain high brightness in the UV region, and the confocal optics are assembled using a cross-slit diaphragm like an iris to eliminate stray light. This system can measure optical densities up to a maximum of 4.0. To study the effect of radiation damage, preliminary measurements of glucose isomerase and thaumatin crystals were conducted in the UV region. Spectral changes dependent on X-ray dose were observed at around 280 nm, suggesting that structural changes involving Trp or Tyr residues occurred in the protein crystal. In the case of the thaumatin crystal, a broad peak around 400 nm was also generated after X-ray irradiation, suggesting the cleavage of a disulfide bond. Dose-dependent spectral changes were also observed in cryo-solutions alone, and these changes differed with the composition of the cryo-solution. These responses in the UV region are informative regarding the state of the sample; consequently, this device might be useful for X-ray crystallography