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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. [ABSTRACT FROM AUTHOR]

Peptidoglycan comprises repeating units of N‐acetylmuramic acid, N‐acetylglucosamine and short cross‐linking peptides. After the conversion of UDP‐N‐acetylglucosamine (UNAG) to UDP‐N‐acetylmuramic acid (UNAM) by the MurA and MurB enzymes, an amino acid is added to UNAM by UDP‐N‐acetylmuramic acid l‐alanine ligase (MurC). As peptidoglycan is an essential component of the bacterial cell wall, the enzymes involved in its biosynthesis represent promising targets for the development of novel antibacterial drugs. Here, the crystal structure of Mycobacterium bovis MurC (MbMurC) is reported, which exhibits a three‐domain architecture for the binding of UNAM, ATP and an amino acid as substrates, with a nickel ion at the domain interface. The ATP‐binding loop adopts a conformation that is not seen in other MurCs. In the UNAG‐bound structure of MbMurC, the substrate mimic interacts with the UDP‐binding domain of MbMurC, which does not invoke rearrangement of the three domains. Interestingly, the glycine‐rich loop of the UDP‐binding domain of MbMurC interacts through hydrogen bonds with the glucose moiety of the ligand, but not with the pyrophosphate moiety. These findings suggest that UNAG analogs might serve as potential candidates for neutralizing the catalytic activity of bacterial MurC. [ABSTRACT FROM AUTHOR]

The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) is extremely versatile, translocating ~300 effector proteins into host cells. This specialized secretion system employs the Dot/Icm type IVB coupling protein (T4CP) complex, which includes IcmS, IcmW and LvgA, that are known to selectively assist the export of a subclass of effectors. Herein, the crystal structure of a four-subunit T4CP subcomplex bound to the effector protein VpdB reveals an interaction between LvgA and a linear motif in the C-terminus of VpdB. The same binding interface of LvgA also interacts with the C-terminal region of three additional effectors, SidH, SetA and PieA. Mutational analyses identified a FxxxLxxxK binding motif that is shared by VpdB and SidH, but not by SetA and PieA, showing that LvgA recognizes more than one type of binding motif. Together, this work provides a structural basis for how the Dot/Icm T4CP complex recognizes effectors, and highlights the multiple substrate-binding specificities of its adaptor subunit. The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) translocates effector proteins into host cells, and the recognition of these effectors is mediated by the Dot/Icm type IV coupling protein (T4CP) complex. Here, the authors present the crystal structure of a four-subunit containing T4CP subcomplex bound to the effector protein VpdB, and identify a FxxxLxxxK binding motif that is present in a subset of the effectors and which is recognized by the T4CP adaptor subunit LvgA. [ABSTRACT FROM AUTHOR]

Many organisms have genes to protect themselves from toxic conditions such as high ethanol and/or ammonia concentrations. When a high ethanol condition is induced to Zymomonas mobilis ZM4, a representative ethanologenic organism, this bacterium overexpresses several genes to overcome this ethanol stress. Among them, we characterized a gene product annotated as an arginase (zmARG) from Z. mobilis ZM4. Even though all of the arginase-determining sequence motifs are not strictly conserved in zmARG, this enzyme converts L-arginine to urea and L-ornithine in the presence of a divalent manganese ion. The revealed high-resolution crystal structure of zmARG shows that it has a typical globular α/β arginase fold with a protruded C-terminal helix. Two zinc ions reside in the active site, where one metal ion is penta-coordinated and the other has six ligands, discerning this zmARG from the reported arginases with two hexa-liganded metal ions. zmARG forms a dimeric structure in solution as well as in the crystalline state. The dimeric assembly of zmARG is formed mainly by interaction formed between the C-terminal α-helix of one molecule and the α/β hydrolase fold of another molecule. The presented findings demonstrate the first reported dimeric arginase formed by the C-terminal tail and has two metal ions coordinated by different number of ligands. [ABSTRACT FROM AUTHOR]

Sample delivery using injectors is widely used in serial crystallography (SX) and has significantly contributed to the determination of crystal structures at room temperature. However, sophisticated injector nozzle fabrication methods and sample delivery operations have made it difficult for ordinary users to access the SX research. Herein, a simple and easily accessible sample delivery method for SX experiments is introduced, that uses a viscous medium, commercially available syringe and syringe pump. The syringe containing the lysozyme crystals embedded in lipidic cubic phase (LCP) or polyacrylamide (PAM) delivery media was connected to a needle having an inner diameter of 168 µm, after which it was installed on a syringe pump. By driving the syringe pump, the syringe plunger was pushed and the crystal sample was delivered to the X‐ray beam position in a stable manner. Using this system, the room‐temperature crystal structures of lysozyme embedded in LCP and PAM at 1.56 Å and 1.75 Å, respectively, were determined. This straightforward syringe pump‐based sample delivery system can be utilized in SX. [ABSTRACT FROM AUTHOR]

ABSTRACT Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on-resin N(τ)-alkylation of histidine residues already bearing a N(π)- alkyl group. This resulted in cationic imidazolium-containing pThr peptides, several of which exhibit single-digit nanomolar PBD-binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio-reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD-binding peptides and peptide mimetics. © 2014 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 444-455, 2014. [ABSTRACT FROM AUTHOR]

Highlights: [•] The crystal structure of one cbbF from Zymomonas mobilis was revealed. [•] Scores of residues form two secondary structures with a non-polar protruded residue. [•] It exists as a dimeric form in solution. [ABSTRACT FROM AUTHOR]

Abstract: The bacterial acquired immune system consists of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRIPSR-associated (Cas) genes, which include Cas-module repeat-associated mysterious proteins (Cmr). The six Cmr proteins of Pyrococcus furiosus (pfCmr1–pfCmr6) form a Cmr effector complex that functions against exogenous nucleic acid. Among the Cmr proteins, the role of pfCmr5 and its involvement in the complex’s cleavage activity have been obscure. The elucidated pfCmr5 structure has two inserted α-helices compared with the other trimeric Cmr5 structure. However, pfCmr5 exists as a monomeric protein both in the crystalline state and in solution. In vitro assays indicate that pfCmr5 interacts with pfCmr4. These structural and biophysical data might help in understanding the complicated and ill-characterized Cmr effector complex. Structured summary of protein interactions: pfCmr4 and pfCmr5 bind by molecular sieving (View interaction) pfCmr4 and pfCmr4 bind by molecular sieving (View interaction) pfCmr5 and pfCmr4 bind by ion exchange chromatography (View interaction) [Copyright &y& Elsevier]

In multifunctional type I restriction enzymes, active methyltransferases (MTases) are constituted of methylation (HsdM) and specificity (HsdS) subunits. In this study, the crystal structure of a putative HsdM subunit from Vibrio vulnificus YJ016 (vvHsdM) was elucidated at a resolution of 1.80 Å. A cofactor-binding site for S-adenosyl- l-methionine (SAM, a methyl-group donor) is formed within the C-terminal domain of an α/β-fold, in which a number of residues are conserved, including the G xGG and (N/D)PP(F/Y) motifs, which are likely to interact with several functional moieties of the SAM methyl-group donor. Comparison with the N6 DNA MTase of Thermus aquaticus and other HsdM structures suggests that two aromatic rings (Phe199 and Phe312) in the motifs that are conserved among the HsdMs may sandwich both sides of the adenine ring of the recognition sequence so that a conserved Asn residue (Asn309) can interact with the N6 atom of the target adenine base (a methyl-group acceptor) and locate the target adenine base close to the transferred SAM methyl group. [ABSTRACT FROM AUTHOR]

Abstract: HlyU is a transcription factor of the ArsR/SmtB family and activates the expression of the pathogenic Vibrio vulnificus RTX toxin. In contrast to the other metal-responding ArsR/SmtB proteins, HlyU does not sense metal ions. To provide its structural information, we elucidated the crystal structure of HlyU from V. vulnificus CMCP6 (HlyU_Vv). The monomeric HlyU_Vv architecture of five α-helices and two β-strands, some of which constitute a typical DNA-binding winged helix-turn-helix (wHTH) motif, is very similar to that of other transcription regulators. Nonetheless, the homo-dimeric HlyU_Vv structure shows several different, three-dimensional features in the spatial position and the detailed dimeric interaction, which were not observed in the modeling study based on the same protein family and sequence similarity. Structured summary: MINT-7710072, MINT-7710086: HlyU_Vv (uniprotkb:Q8DES3) and HlyU_Vv (uniprotkb:Q8DES3) bind (MI:0407) by X-ray crystallography (MI:0114) [Copyright &y& Elsevier]

Abstract: Esterases are one of the most common enzymes and are involved in diverse cellular functions. ybfF protein from Escherichia coli (Ec_ybfF) belongs to the esterase family for the large substrates, palmitoyl coenzyme A and malonyl coenzyme A, which are important cellular intermediates for energy conversion and biomolecular synthesis. To obtain molecular information on ybfF esterase, which is found in a wide range of microorganisms, we elucidated the crystal structures of Ec_ybfF in complexes with small molecules at resolutions of 1.1 and 1.68 Å, respectively. The structure of Ec_ybfF is composed of a globular α/β hydrolase domain with a three-helical bundle cap, which is linked by a kinked helix to the α/β hydrolase domain. It contains a catalytic tetrad of Ser-His-Asp-Ser with the first Ser acting as a nucleophile. The unique spatial arrangement and orientation of the helical cap with respect to the α/β hydrolase domain form a substrate-binding crevice for large substrates. The helical cap is also directly involved in catalysis by providing a substrate anchor, viz., the conserved residues of Arg123 and Tyr208. The high-resolution structure of Ec_ybfF shows that the inserted helical bundle structure and its spatial orientation with respect to the α/β hydrolase domain are critical for creating a large inner space and constituting a specific active site, thereby providing the broad substrate spectrum toward large biomolecules. [Copyright &y& Elsevier]

Serial crystallography (SX) technique using synchrotron X-ray allows the visualization of room-temperature crystal structures with low-dose data collection as well as time-resolved molecular dynamics. In an SX experiment, delivery of numerous crystals for X-ray interaction, in a serial manner, is very important. Fixed-target scanning approach has the advantage of dramatically minimizing sample consumption as well as any physical damage to crystal sample, compared to other sample delivery methods. Here, we introduce the simple approach of fixed-target serial synchrotron crystallography (FT-SSX) using nylon mesh and enclosed film (NAM)-based sample holder. The NAM-based sample holder consisted of X-ray-transparent nylon-mesh and polyimide film, attached to a magnetic base. This sample holder was mounted to a goniometer head on macromolecular crystallography beamline, and translated along vertical and horizontal directions for raster scanning by the goniometer. Diffraction data were collected in two raster scanning approaches: (i) 100 ms X-ray exposure and 0.011° oscillation at each scan point and (ii) 500 ms X-ray exposure and 0.222° oscillation at each scan point. Using this approach, we determined the room-temperature crystal structures of lysozyme and glucose isomerase at 1.5–2.0 Å resolution. The sample holder produced negligible X-ray background scattering for data processing. Therefore, the new approach provided an opportunity to perform FT-SSX with high accessibility using macromolecular crystallography beamlines at synchrotron without any special equipment. [ABSTRACT FROM AUTHOR]

Type I restriction-modification enzymes are oligomeric proteins composed of methylation (M), DNA sequence-recognition (S), and restriction (R) subunits. The different bipartite DNA sequences of 2–4 consecutive bases are recognized by two discerned target recognition domains (TRDs) located at the two-helix bundle of the two conserved regions (CRs). Two M-subunits and a single S-subunit form an oligomeric protein that functions as a methyltransferase (M 2 S 1 MTase). Here, we present the crystal structure of the intact MTase from Vibrio vulnificus YJ016 in complex with the DNA-mimicking Ocr protein and the S-adenosyl-L-homocysteine (SAH). This MTase includes the M-domain with a helix tail (M-tail helix) and the S 1/2 -domain of a TRD and a CR α-helix. The Ocr binds to the cleft of the TRD surface and SAH is located in the pocket within the M-domain. The solution- and negative-staining electron microscopy-based reconstructed (M 1 S 1/2) 2 structure reveals a symmetric (S 1/2) 2 assembly using two CR-helices and two M-tail helices as a pivot, which is plausible for recognizing two DNA regions of same sequence. The conformational flexibility of the minimal M 1 S 1/2 MTase dimer indicates a particular state resembling the structure of M 2 S 1 MTases. [ABSTRACT FROM AUTHOR]

Abstract: The ethanologenic bacterium Zymomonas mobilis ZM4 is of special interest because it has a high ethanol yield. This is made possible by the two alcohol dehydrogenases (ADHs) present in Z. mobilis ZM4 (zmADHs), which shift the equilibrium of the reaction toward the synthesis of ethanol. They are metal-dependent enzymes: zinc for zmADH1 and iron for zmADH2. However, zmADH2 is inactivated by oxygen, thus implicating zmADH2 as the component of the cytosolic respiratory system in Z. mobilis. Here, we show crystal structures of zmADH2 in the form of an apo-enzyme and an NAD+–cofactor complex. The overall folding of the monomeric structure is very similar to those of other functionally related ADHs with structural variations around the probable substrate and NAD+ cofactor binding region. A dimeric structure is formed by the limited interactions between the two subunits with the bound NAD+ at the cleft formed along the domain interface. The catalytic iron ion binds near to the nicotinamide ring of NAD+, which is likely to restrict and locate the ethanol to the active site together with the oxidized Cys residue and several nonpolar bulky residues. The structures of the zmADH2 from the proficient ethanologenic bacterium Z. mobilis, with and without NAD+ cofactor, and modeling ethanol in the active site imply that there is a typical metal-dependent catalytic mechanism. [Copyright &y& Elsevier]

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. • Crystal structure of Cascade backbone-forming type I-B Cas7 (Csh2 from Thermobaculum terrenum ; TterCsh2) was determined. • TterCsh2 adopts a typical folding of Cas7. • TterCsh2 is structurally and sequentially more similar to type I-C Cas7 than to those of type I-E and I-F. • Comparative analysis of TterCsh2 with reported Cas7s suggests that Cas7 can be divided into at least two sub-classes. [ABSTRACT FROM AUTHOR]