[What Kind of Measurements Can Be Made with an X-ray Free Electron Laser at SACLA?].

Three-dimensional structural information is indispensable to understand the function of proteins in living organisms and X-ray crystallography plays a major role in determining the three-dimensional structure. X-ray free-electron laser (XFEL), which is intense and femtosecond X-ray pulses, enables us to obtain X-ray diffraction intensity data before the destruction of protein molecules, and is expected to be a technology to obtain dynamic structural information. This year marks the 10th anniversary of SPring-8 Angstrom Compact Free Electron Laser (SACLA), Japan's X-ray free electron laser facility.

The crystal structure of the CmABCB1 G132V mutant, which favors the outward-facing state, reveals the mechanism of the pivotal joint between TM1 and TM3.

CmABCB1 is a homologue of human P-glycoprotein, which extrudes various substrates by iterative cycles of conformational changes between the inward- and outward-facing states. Comparison of the inward- and outward-facing structures of CmABCB1 suggested that pivotal joints in the transmembrane domain regulate the tilt of transmembrane helices. Transmembrane helix 1 (TM1) forms a tight helix-helix contact with TM3 at the TM1-3 joint.

ABCB1/MDR1/P-gp employs an ATP-dependent twist-and-squeeze mechanism to export hydrophobic drugs.

ABCB1, also called MDR1 or P-glycoprotein, exports various hydrophobic compounds and plays an essential role as a protective physiological barrier in several organs, including the brain, testis, and placenta. However, little is known about the structural mechanisms that allow ABCB1 to recognize hydrophobic compounds of diverse structures or the coupling of ATP hydrolysis to uphill substrate export. High-resolution X-ray crystal structures of the pre- and post-transport states and FRET analyses in living cells have revealed that an aromatic hydrophobic network at the top of the inner cavity is key for the conformational change in ABCB1 that is triggered by a hydrophobic substrate.

Improvement of Production and Isolation of Human Neuraminidase-1 Crystals.

crystallization is a developing technique to provide crystals for protein structure determination, particularly for proteins that are difficult to prepare by crystallization. This method has a key advantage: it requires neither a protein purification step nor a crystallization step. However, there is still no systematic strategy for improving the technique of crystallization because the process occurs spontaneously.

Inward- and outward-facing X-ray crystal structures of homodimeric P-glycoprotein CmABCB1.

P-glycoprotein extrudes a large variety of xenobiotics from the cell, thereby protecting tissues from their toxic effects. The machinery underlying unidirectional multidrug pumping remains unknown, largely due to the lack of high-resolution structural information regarding the alternate conformational states of the molecule. Here we report a pair of structures of homodimeric P-glycoprotein: an outward-facing conformational state with bound nucleotide and an inward-facing apo state, at resolutions of 1.

Experimental phase determination with selenomethionine or mercury-derivatization in serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) holds enormous potential for the structure determination of proteins for which it is difficult to produce large and high-quality crystals. SFX has been applied to various systems, but rarely to proteins that have previously unknown structures. Consequently, the majority of previously obtained SFX structures have been solved by the molecular replacement method.

Hydroxyethyl cellulose matrix applied to serial crystallography.

Serial femtosecond crystallography (SFX) allows structures of proteins to be determined at room temperature with minimal radiation damage. A highly viscous matrix acts as a crystal carrier for serial sample loading at a low flow rate that enables the determination of the structure, while requiring consumption of less than 1 mg of the sample. However, a reliable and versatile carrier matrix for a wide variety of protein samples is still elusive.

Development of a dose-limiting data collection strategy for serial synchrotron rotation crystallography.

Serial crystallography, in which single-shot diffraction images are collected, has great potential for protein microcrystallography. Although serial femtosecond crystallography (SFX) has been successfully demonstrated, limited beam time prevents its routine use. Inspired by SFX, serial synchrotron crystallography (SSX) has been investigated at synchrotron macromolecular crystallography beamlines.

Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography.

Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme-substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.

Native sulfur/chlorine SAD phasing for serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.

An isomorphous replacement method for efficient de novo phasing for serial femtosecond crystallography.

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) holds great potential for structure determination of challenging proteins that are not amenable to producing large well diffracting crystals. Efficient de novo phasing methods are highly demanding and as such most SFX structures have been determined by molecular replacement methods. Here we employed single isomorphous replacement with anomalous scattering (SIRAS) for phasing and demonstrate successful application to SFX de novo phasing.

Synthesis and inhibitory activity of substrate-analog fructosyl peptide oxidase inhibitors.

Fructosyl peptide oxidases (FPOXs) play a crucial role in the diagnosis of diabetes. Their main function is to cleave fructosyl amino acids or fructosyl peptides into glucosone and the corresponding amino acids/dipeptides. In this study, the substrate-analog FPOX inhibitors 1a-c were successfully designed and synthesized.

Crystal structures of β-primeverosidase in complex with disaccharide amidine inhibitors.

β-Primeverosidase (PD) is a disaccharide-specific β-glycosidase in tea leaves. This enzyme is involved in aroma formation during the manufacturing process of oolong tea and black tea. PD hydrolyzes β-primeveroside (6-O-β-d-xylopyranosyl-β-d-glucopyranoside) at the β-glycosidic bond of primeverose to aglycone, and releases aromatic alcoholic volatiles of aglycones.

Structural basis for gating mechanisms of a eukaryotic P-glycoprotein homolog.

P-glycoprotein is an ATP-binding cassette multidrug transporter that actively transports chemically diverse substrates across the lipid bilayer. The precise molecular mechanism underlying transport is not fully understood. Here, we present crystal structures of a eukaryotic P-glycoprotein homolog, CmABCB1 from Cyanidioschyzon merolae, in two forms: unbound at 2.

Impact of site-directed mutant luciferase on quantitative green and orange/red emission intensities in firefly bioluminescence.

Firefly bioluminescence has attracted great interest because of its high quantum yield and intriguing modifiable colours. Modifications to the structure of the enzyme luciferase can change the emission colour of firefly bioluminescence, and the mechanism of the colour change has been intensively studied by biochemists, structural biologists, optical physicists, and quantum-chemistry theorists. Here, we report on the quantitative spectra of firefly bioluminescence catalysed by wild-type and four site-directed mutant luciferases.

Crystal structure of peroxisomal targeting signal-2 bound to its receptor complex Pex7p-Pex21p.

Appropriate targeting of matrix proteins to peroxisomes is mainly directed by two types of peroxisomal targeting signals, PTS1 and PTS2. Although the basis of PTS1 recognition has been revealed by structural studies, that of PTS2 recognition remains elusive. Here we present the crystal structure of a heterotrimeric PTS2-recognition complex from Saccharomyces cerevisiae, containing Pex7p, the C-terminal region of Pex21p and the PTS2 of the peroxisomal 3-ketoacyl-CoA thiolase.

Crystallization and preliminary crystallographic analysis of two eukaryotic fructosyl peptide oxidases.

Fructosyl peptide oxidase (FPOX) catalyses the oxidation of α-glycated dipeptides such as N(α)-(1-deoxy-D-fructos-1-yl)-L-valyl-L-histidine (Fru-ValHis) and is used in the diagnosis of diabetes mellitus. Here, two thermostable mutants of FPOX, CFP-T7 and EFP-T5M, were crystallized by the sitting-drop vapour-diffusion method. The crystal of CFP-T7 belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 110.

Crystallization and preliminary X-ray crystallographic analysis of Pz peptidase B from Geobacillus collagenovorans MO-1.

Pz peptidase B is an intracellular M3 metallopeptidase that is found together with Pz peptidase A in the thermophile Geobacillus collagenovorans MO-1 and recognizes collagen-specific tripeptide units (-Gly-Pro-X-). These peptidases have low homology in their primary structures; however, their cleavage patterns towards peptide substrates are similar. In this work, Pz peptidase B was crystallized using the counter-diffusion method.

Structural basis for docking of peroxisomal membrane protein carrier Pex19p onto its receptor Pex3p.

Peroxisomes require peroxin (Pex) proteins for their biogenesis. The interaction between Pex3p, which resides on the peroxisomal membrane, and Pex19p, which resides in the cytosol, is crucial for peroxisome formation and the post-translational targeting of peroxisomal membrane proteins (PMPs). It is not known how Pex3p promotes the specific interaction with Pex19p for the purpose of PMP translocation.

The exquisite structure and reaction mechanism of bacterial Pz-peptidase A toward collagenous peptides: X-ray crystallographic structure analysis of PZ-peptidase a reveals differences from mammalian thimet oligopeptidase.

Pz-peptidase A, from the thermophilic bacterium Geobacillus collagenovorans MO-1, hydrolyzes a synthetic peptide substrate, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-PLGPR), which contains a collagen-specific tripeptide sequence, -Gly-Pro-X-, but does not act on collagen proteins themselves. The mammalian enzyme, thimet oligopeptidase (TOP), which has comparable functions with bacterial Pz-peptidases but limited identity at the primary sequence level, has recently been subjected to x-ray crystallographic analysis; however, no crystal structure has yet been reported for complexes of TOP with substrate analogues. Here, we report crystallization of recombinant Pz-peptidase A in complex with two phosphinic peptide inhibitors (PPIs) that also function as inhibitors of TOP and determination of the crystal structure of these complexes at 1.

Deleting two C-terminal alpha-helices is effective to crystallize the bacterial ABC transporter Escherichia coli MsbA complexed with AMP-PNP.

An MsbA deletion mutant DeltaC21 that lacks the two C-terminal alpha-helices was expressed in Escherichia coli strain C41 and purified by metal-affinity and gel-filtration chromatography. Purified DeltaC21 retained 26% of the activity of the wild-type ATPase and had a similar binding affinity to fluorescent nucleotide derivatives. Although crystals of wild-type MsbA complexed with adenosine 5'-(beta,gamma-imido)triphosphate could not be obtained, crystals of DeltaC21 that diffracted to 4.

X-ray crystallographic study of an HIV-1 fusion inhibitor with the gp41 S138A substitution.

The S138A substitution of fusion inhibitory peptides derived from the C-terminal heptad repeat (C-HR) of the human immunodeficiency virus type 1 (HIV-1) gp41 leads to enhanced binding affinity to the N-terminal heptad repeat (N-HR). As such, these peptides exhibit highly potent anti-HIV-1 activity. X-ray crystallographic analysis was performed to understand the effect of the substitution on binding affinity.

Functional role of the linker region in purified human P-glycoprotein.

Human P-glycoprotein (P-gp), which conveys multidrug resistance, is an ATP-dependent drug efflux pump that transports a wide variety of structurally unrelated compounds out of cells. P-gp possesses a 'linker region' of approximately 75 amino acids that connects two homologous halves, each of which contain a transmembrane domain followed by a nucleotide-binding domain. To investigate the role of the linker region, purified human P-gp was cleaved by proteases at the linker region and then compared with native P-gp.

Structural basis for gibberellin recognition by its receptor GID1.

Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. A nuclear GA receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1), has a primary structure similar to that of the hormone-sensitive lipases (HSLs). Here we analyse the crystal structure of Oryza sativa GID1 (OsGID1) bound with GA(4) and GA(3) at 1.