A5004035076- Enzyme Structure and Function
- Plant Virus Research Studies
- Bacteriophages and microbial interactions
- Protein Structure and Dynamics
- Advanced Electron Microscopy Techniques and Applications
- Advanced X-ray Imaging Techniques
- Photoreceptor and optogenetics research
- X-ray Diffraction in Crystallography
- Photosynthetic Processes and Mechanisms
- Plant and Fungal Interactions Research
- Crystallization and Solubility Studies
- Electron and X-Ray Spectroscopy Techniques
- Viral gastroenteritis research and epidemiology
- Spectroscopy and Quantum Chemical Studies
- Mass Spectrometry Techniques and Applications
- Animal Virus Infections Studies
- RNA and protein synthesis mechanisms
- Chalcogenide Semiconductor Thin Films
- Click Chemistry and Applications
- Biotin and Related Studies
- Neural dynamics and brain function
- Nonlinear Optical Materials Research
- Neuroscience and Neuropharmacology Research
- Genomics and Phylogenetic Studies
- Coenzyme Q10 studies and effects
SPring-8
2013-2025
RIKEN
2013
Institute of Molecular Biology, Academia Sinica
2008
Osaka University
1999-2008
National Institute of Advanced Industrial Science and Technology
2008
University of British Columbia
2008
Scripps Research Institute
2001-2004
Uppsala University
2002
Protein Research Foundation
2000
Structure analysis of small crystals is important in areas ranging from synthetic organic chemistry to pharmaceutical and material sciences, as many compounds do not yield large crystals. Here we present the detailed characterization structure an molecule, rhodamine-6G, determined at a resolution 0.82 Å by X-ray free-electron laser (XFEL). Direct comparison this with that obtained electron crystallography same sample batch microcrystals shows both methods can accurately distinguish position...
Nanoscale imaging of biological specimens in their native condition is long-standing interest, particular with direct, high resolution views internal structures intact specimens, though as yet progress has been limited. Here we introduce wet coherent x-ray diffraction microscopy capable fully hydrated and unstained specimens. Whole cell morphologies better than 25 nm can be clearly visualized without contrast degradation.
A new cryo-EM system has been investigated for single particle analysis of protein structures. The provides parallel illumination a highly-coherent 300 kV electron beam from cold-field emission gun, and boosts image contrast with an in-column energy filter hole-free phase plate. It includes motorized cryo-sample loading automated liquid-nitrogen filling cooling multiple samples. In this study, we describe gun characteristics, demonstrate the suitability reconstructions. performance is tested...
Photosystem II (PSII) catalyzes light-induced water oxidation through an S i -state cycle, leading to the generation of di-oxygen, protons and electrons. Pump–probe time-resolved serial femtosecond crystallography (TR-SFX) has been used capture structural dynamics light-sensitive proteins. In this approach, it is crucial avoid light contamination in samples when analyzing a particular reaction intermediate. Here, method for determining condition that avoids PSII microcrystals while...
In cryo-electron microscopy (cryo-EM) data collection, locating a target object is error-prone. Here, we present machine learning-based approach with real-time locator named yoneoLocr using YOLO, well-known detection system. Implementation shows its effectiveness in rapidly and precisely carbon holes single particle cryo-EM crystals evaluating electron diffraction (ED) patterns automated crystallography (cryo-EX) collection. The proposed will advance high-throughput accurate collection of...
Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, mechanism by which conformational pump an anion to achieve unidirectional transport, from extracellular side cytoplasmic side, anion-pumping remains enigmatic. We collected...
Keap1 protein acts as a cellular sensor for oxidative stresses and regulates the transcription level of antioxidant genes through ubiquitination corresponding factor, Nrf2. A small molecule capable binding to Nrf2 interaction site could be useful medicine. Here, we report two crystal structures, referred soaking cocrystallization forms, Kelch domain with molecule, Ligand1. In these Ligand1 occupied so mimic ETGE motif Nrf2, although mode differed in forms. Because mediated packing both...
Serial femtosecond crystallography (SFX) using ultrashort pulses from X-ray free- electron lasers (XFELs) enables the determination of crystal structures at room temperature while minimizing radiation damage to samples. This method involves irradiating numerous crystals one by with XFEL pulses, allowing even capture snapshots dynamical in biological macromolecules. To achieve this, an efficient sample delivery system is essential for acquiring a large number diffraction patterns. The most...
Abstract We have designed and evaluated a cryo-electron microscopy (cryo-EM) system for higher-resolution single particle analysis high-precision electron 3D crystallography. The comprises JEOL CRYO ARM 300 microscope—the first machine of this model—and direct detection device camera, scintillator-coupled GPU clusters connected with camera control computer software automated-data collection efficient accurate operation. microscope provides parallel illumination highly coherent 300-kV beam to...
Cytochrome c oxidase (C O) is part of the respiratory chain and contributes to electrochemical membrane gradient in mitochondria as well many bacteria, it uses energy released reduction oxygen pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography study structural response active site upon flash photolysis carbon monoxide (CO) from reduced heme a 3 ba -type C O. In contrast with aa enzyme, our data show how CO stabilized...
The single segment, double-stranded RNA genome of the L-A virus (L-A) yeast encodes two proteins: major coat protein Gag (76 kDa) and Gag-Pol fusion (180 kDa). icosahedral capsid is formed by 120 copies has architecture similar to that seen in reovirus, blue tongue rice dwarf inner shells. chemically removes m7GMP caps from host cellular mRNAs. Previously we identified a trench on outer surface included His154, which are covalently attached. Here report refined coordinates at 3.4 angstroms...
It was essential for the structural genomics of Thermus thermophilus HB8 to efficiently crystallize a number proteins. To this end, three conventional robots, an HTS-80 (sitting-drop vapour diffusion), Crystal Finder (hanging-drop diffusion) and TERA (modified microbatch) robot, were subjected crystallization condition screening test involving 18 proteins from T. HB8. In addition, TOPAZ (microfluidic free-interface designed specifically initial also briefly examined. The diffraction-quality...
The major capsid protein VP1 encoded by genome segment S1 of Bombyx mori cypovirus 1 was expressed in a baculovirus system. In the absence any other proteins, found to assemble into single-shelled virus-like particles. particles were more sensitive acidic conditions than intact
The core protein P3 of Rice dwarf virus constructs asymmetric dimers, one which is inserted by the amino-terminal region another protein. proteins with serial deletions, expressed in a baculovirus system, formed particles gradually decreasing stability. capacity for self-assembly disappeared when 52 amino acids had been deleted. These results demonstrated that insertion arm into appears to play an important role stabilizing particles.
Serial femtosecond crystallography (SFX) with an X-ray free-electron laser is used for the structural determination of proteins from a large number microcrystals at room temperature. To examine feasibility pharmaceutical applications SFX, ligand-soaking experiment using thermolysin has been performed SFX. The results were compared those conventional synchrotron radiation (SR) 100 K. A protein-ligand complex structure was successfully obtained SFX soaked small-molecule ligand; both oil-based...
In order to elucidate features of the denatured state ensembles that exist in equilibrium with native under physiological conditions, we performed 1.4-μs molecular dynamics (MD) simulations at 400 K and 450 using monomer subunits three CutA1 mutants from Escherichia coli: an SH-free mutant (Ec0SH) denaturation temperature (Td) = 85.6 °C, a hydrophobic (Ec0VV) Td 113.3 ionic (Ec0VV_6) 136.8 °C. The occupancy salt bridges by six substituted charged residues Ec0VV_6 was 140.1% 300 89.5% K,...
Phosphoketolase and transketolase are thiamine diphosphate-dependent enzymes play a central role in the primary metabolism of bifidobacteria: bifid shunt. The both catalyze phosphorolytic cleavage xylulose 5-phosphate or fructose 6-phosphate first reaction step, but possess different substrate specificity second where phosphoketolase utilize inorganic phosphate (P i ) D-ribose 5-phosphate, respectively, as acceptor substrate. Structures Bifidobacterium longum holoenzyme its complex with...
Abstract Picorna-like plant viruses are non-enveloped RNA spherical of ~30 nm. Part the survival these depends on their capsid being stable enough to harbour viral genome and yet malleable allow its release. However, molecular mechanisms remain obscure. Here, we report a structure picorna-like virus, apple latent at 2.87 Å resolution by single-particle cryo-electron microscopy (cryo-EM) with cold-field emission beam. The cryo-EM map reveals unique composed three proteins Vp25, Vp20, Vp24....
In order to elucidate the contribution of charged residues protein stabilization at temperatures over 100 °C, we constructed many mutants CutA1 ( EcCutA1) from Escherichia coli. The goal was see if one can achieve same stability as for a hyperthermophile Pyrococcus horikoshii that has denaturation temperature near 150 °C. hydrophobic mutant EcCutA1 Ec0VV) with Td) 113.2 °C used template mutations. highest Td Ec0VV substituted by single residue 118.4 Multiple ion were also combination and...