BL-11C Micro-MX: a high-flux microfocus macromolecular-crystallography beamline for micrometre-sized protein crystals at Pohang Light Source II.

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 × 1012 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.

Structural basis for the selective addition of an oxygen atom to cyclic ketones by Baeyer-Villiger monooxygenase from Parvibaculum lavamentivorans.

Baeyer-Villiger monooxygenase (BVMO) catalyzes insertion of an oxygen atom into aliphatic or cyclic ketones with high regioselectivity. The BVMOs from Parvibaculum lavamentivorans (BVMOParvi) and Oceanicola batsensis (BVMOOcean) 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 BVMOParvi structure shows that BVMOParvi forms a two-domain structure like other BVMOs. It has two inserted residues, compared with BVMOOcean, 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 BVMOParvi and Ser-Thr in BVMOOcean) allows a large volume in BVMOParvi. 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.

An asymmetric dimeric structure of TrmJ tRNA methyltransferase from Zymomonas mobilis with a flexible C-terminal dimer.

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.

Structural characterization of the type I-B CRISPR Cas7 from Thermobaculum terrenum.

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.

A CRISPR RNA Is Closely Related With the Size of the Cascade Nucleoprotein Complex.

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.

Structures of Zymomonas 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase with and without a Substrate Analog at the Phosphate-Binding Loop.

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.