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Synchrotron serial crystallography (SSX) is an emerging method for determining crystal structure at room temperature using synchrotron radiation facilities. Despite the various approaches available, reducing sample consumption, removing mother liquid from crystal solution, soaking small molecules for protein-ligand complex structure and prevention of sample dehydration are still challenging problems to be overcome in SSX. Therefore, we have developed a new flip-type fixed-target SSX sample holder for conventional protein crystallography beamlines based on nylon mesh and kapton film. The potential of the flip-holder was evaluated in an SSX experiment with lysozyme crystals at room temperature. About 19600 diffraction images were collected during 40 minutes using a repetition rate of 10 Hz at the 11C beamline of the Pohang Accelerator Laboratory, and the crystal structure of lysozyme was determined at 1.89 Å resolution. This straightforward flip-holder can be used in synchrotron beamlines for routine crystallography., (open access.)

In serial crystallography, large numbers of microcrystals are sequentially delivered to an X-ray beam and a diffraction pattern is obtained from each crystal. This serial approach was developed primarily for X-ray Free Electron Lasers (XFELs) where crystals are destroyed by the beam but is increasingly used in synchrotron experiments. The combination of XFEL and synchrotron-based serial crystallography enables time-resolved experiments over an extremely wide range of time domains - from femtoseconds to seconds - and allows intact or pristine structures free of the effects of radiation damage to be obtained. Several approaches have been developed for sample delivery with varying levels of sample efficiency and ease of use. In the fixed target approach, microcrystals are loaded onto a solid support which is then rastered through the X-ray beam. The key advantages of fixed targets are that every crystal loaded can be used for data collection, and that precise control of when crystals are moved into the beam allows for time-resolved experiments over a very wide range of time domains as well as multi-shot experiments characterising the effects of the X-ray beam on the sample. We describe the application of fixed targets for serial crystallography as implemented at beamline I24 at Diamond Light Source and at the SACLA XFEL. We discuss methodologies for time-resolved serial crystallography in fixed targets and describe best practices for obtaining high-quality structures covering sample preparation, data collection strategies and data analysis pipelines., (Copyright © 2024. Published by Elsevier Inc.)

Clustered regularly interspaced short palindromic repeats (CRISPR) in many prokaryotes functions as an adaptive immune system against mobile genetic elements. A heterologous ribonucleoprotein silencing complex composed of CRISPR-associated (Cas) proteins and a CRISPR RNA (crRNA) neutralizes the incoming mobile genetic elements. The type I and III silencing complexes commonly include a protein-helical backbone of several copies of identical subunits, for example, Cas7 in the type I silencing complex. In this study, we structurally characterized type I-B Cas7 (Csh2 from Thermobaculum terrenum; TterCsh2). The revealed crystal structure of TterCsh2 shows a typical glove-like architecture of Cas7, which consists of a palm, a thumb, and a finger domain. Csh2 proteins have 5 conserved sequence motifs that are arranged to form a presumable crRNA-binding site in the TterCsh2 structure. This crRNA binding site of TterCsh2 is structurally and potentially comparable to those observed in helix-forming Cas7 structures in other sub-types. Analysis of the reported Cas7 structures and their sequences suggests that Cas7s can be divided into at least two sub-classes. These data will broaden our understanding on the Cascade complex of CRISPR/Cas systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

BL-11C, a new protein crystallography beamline, is an in-vacuum undulator-based microfocus beamline used for macromolecular crystallography at the Pohang Accelerator Laboratory and it was made available to users in June 2017. The beamline is energy tunable in the range 5.0-20 keV to support conventional single- and multi-wavelength anomalous-dispersion experiments against a wide range of heavy metals. At the standard working energy of 12.659 keV, the monochromated beam is focused to 4.1 µm (V) × 8.5 µm (H) full width at half-maximum at the sample position and the measured photon flux is 1.3 × 10 12  photons s -1 . The experimental station is equipped with a Pilatus3 6M detector, a micro-diffractometer (MD2S) incorporating a multi-axis goniometer, and a robotic sample exchanger (CATS) with a dewar capacity of 90 samples. This beamline is suitable for structural determination of weakly diffracting crystalline substances, such as biomaterials, including protein, nucleic acids and their complexes. In addition, serial crystallography experiments for determining crystal structures at room temperature are possible. Herein, the current beamline characteristics, technical information for users and some recent scientific highlights are described., (open access.)

The currently known prokaryotic adaptive immune system against mobile genetic elements is based on clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated (Cas) proteins and the transcribed short CRISPR RNA (crRNA) molecule form a heterologous ribonucleoprotein complex that neutralizes invading foreign nucleic acids, wherein the crRNA molecule base-pairs with the exogenous genetic elements. In the ribonucleoprotein complexes of the type I CRISPR system, a helical backbone of six identical subunits is commonly found. However, it is not clear how this ribonucleoprotein complex is assembled and what is the determinant factor for its size. We elucidated the crystal structure of the Csy3 subunit of the type I-F ribonucleoprotein complex from Zymomonas mobilis (ZmCsy3), in which seven ZmCsy3 protomers in the asymmetric unit form a molecular helix that is part of a filamentous structure in the entire crystal system. This ZmCsy3 helical structure is remarkably similar to the crRNA-bound hexameric Csy3 backbone from Pseudomonas aeruginosa , with conserved interactions between neighboring subunits. The monomeric ZmCsy3 in solution is transformed into different oligomeric states depending on the added crRNAs. These results suggest that a crRNA and Csy3 subunit play a determinant role in the stepwise formation of the functional Cascade ribonucleoprotein complex and the recruitment of other subunits, and crRNA functions as a molecular ruler for determining the size of the Cascade silencing complex., (Copyright © 2019 Gu, Ha and Kim.)

Baeyer-Villiger monooxygenase (BVMO) catalyzes insertion of an oxygen atom into aliphatic or cyclic ketones with high regioselectivity. The BVMOs from Parvibaculum lavamentivorans (BVMO Parvi ) and Oceanicola batsensis (BVMO Ocean ) are interesting because of their homologies, with >40% sequence identity, and reaction with the same cyclic ketones with a methyl moiety to give different products. The revealed BVMO Parvi structure shows that BVMO Parvi forms a two-domain structure like other BVMOs. It has two inserted residues, compared with BVMO Ocean , that form a bulge near the bound flavin adenine dinucleotide in the active site. Furthermore, this bulge is linked to a nearby α-helix via a disulfide bond, probably restricting access of the bulky methyl group of the substrate to this bulge. Another sequence motif at the entrance of the active site (Ala-Ser in BVMO Parvi and Ser-Thr in BVMO Ocean ) allows a large volume in BVMO Parvi . These minute differences may discriminate a substrate orientation in both BVMOs from the initial substrate binding pocket to the final oxygenation site, resulting in the inserted oxygen atom being in different positions of the same substrate., (Copyright © 2019 Elsevier Inc. All rights reserved.)

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes aldol cleavage and condensation reactions, has two distinct substrate-binding sites. The substrate-binding mode at the catalytic site and Schiff-base formation have been well studied. However, structural information on the phosphate-binding loop (P-loop) is limited. Zymomonas mobilis KDPG aldolase is one of the aldolases with a wide substrate spectrum. Its structure in complex with the substrate-mimicking 3-phosphoglycerate (3PG) shows that the phosphate moiety of 3PG interacts with the P-loop and a nearby conserved serine residue. 3PG-binding to the P-loop replaces water molecules aligned from the P-loop to the catalytic site, as observed in the apo-structure. The extra electron density near the P-loop and comparison with other aldolases suggest the diversity and flexibility of the serine-containing loop among KDPG aldolases. These structural data may help to understand the substrate-binding mode and the broad substrate specificity of the Zymomonas KDPG aldolase.

The tRNA methyltransferase J (TrmJ) and D (TrmD) catalyze the transferring reaction of a methyl group to the tRNA anticodon loop. They commonly have the N-terminal domain (NTD) and the C-terminal domain (CTD). Whereas two monomeric CTDs symmetrically interact with a dimeric NTD in TrmD, a CTD dimer has exhibited an asymmetric interaction with the NTD dimer in the presence of a product. The elucidated apo-structure of the full-length TrmJ from Zymomonas mobilis ZM4 shows a dimeric CTD that asymmetrically interacts with the NTD dimer, thereby distributing non-symmetrical potential charge on the both side of the protein surface. Comparison with the product-bound structures reveals a local re-orientation of the two arginine-containing loop at the active site, which interacts with the product. Further, the CTD dimers have diverse orientations compared to the NTD dimers, suggesting their flexibility. These data indicate that an asymmetric interaction between the NTD dimer and the CTD dimer is a common structural feature among TrmJ proteins, regardless of the presence of a substrate or a product., (Copyright © 2017 Elsevier Inc. All rights reserved.)