Cocktailed fragment screening by X-ray crystallography of the antibacterial target undecaprenyl pyrophosphate synthase from Acinetobacter baumannii
Direct soaking of protein crystals with small-molecule fragments grouped into complementary clusters is a useful technique when assessing the potential of a new crystal system to support structure-guided drug discovery. It provides a robustness check prior to any extensive crystal screening, a double check for assay binding cutoffs and structural data for binding pockets that may or may not be picked out in assay measurements. The structural output from this technique for three novel fragment molecules identified to bind to the antibacterial target Acinetobacter baumannii undecaprenyl pyrophosphate synthase are reported, a...
Source: Acta Crystallographica Section F - January 1, 2020 Category: Biochemistry Authors: Thorpe, J.H. Wall, I.D. Sinnamon, R.H. Taylor, A.N. Stavenger, R.A. Tags: undecaprenyl pyrophosphate synthase fragment screening Acinetobacter baumannii research communications Source Type: research

The Escherichia coli RnlA – RnlB toxin – antitoxin complex: production, characterization and crystallization
The Escherichia coli rnlAB operon encodes a toxin – antitoxin module that is involved in protection against infection by bacteriophage T4. The full-length RnlA – RnlB toxin – antitoxin complex as well as the toxin RnlA were purified to homogeneity and crystallized. When the affinity tag is placed on RnlA, RnlB is largely lost during purification and the resulting crystals exclusively comprise RnlA. A homogeneous preparation of RnlA – RnlB containing stoichiometric amounts of both proteins could only be obtained using a His tag placed C-terminal to RnlB. Native mass spectrometry and SAXS indicate a 1...
Source: Acta Crystallographica Section F - January 1, 2020 Category: Biochemistry Authors: Garcia Rodriguez, G. Talavera Perez, A. Konijnenberg, A. Sobott, F. Michiels, J. Loris, R. Tags: toxin – antitoxin bacterial stress response macromolecular complex Escherichia coli research communications Source Type: research

The RING domain of mitochondrial E3 ubiquitin ligase 1 and its complex with Ube2D2: crystallization and X-ray diffraction
Mitochondrial E3 ubiquitin ligase 1 (MUL1) is located in the mitochondrial outer membrane and regulates various biological processes, including apoptosis, cell growth, mitophagy and mitochondrial dynamics. The C-terminal region of MUL1 faces the cytoplasm and contains the RING domain (MUL1-RING) where the Ub~E2 thioester binds. Unlike most RING-type E3 enzymes, MUL1-RING alone does not have an additional region that recruits a substrate protein, yet is still able to ubiquitylate the substrate, the p53 protein. Nevertheless, the exact mechanism of the ubiquitylation of p53 by MUL1-RING has not yet been elucidated. In order ...
Source: Acta Crystallographica Section F - January 1, 2020 Category: Biochemistry Authors: Lee, S.-O. Lee, C.-K. Ryu, K.-S. Chi, S.-W. Tags: mitochondrial E3 ubiquitin ligase 1 MUL1 RING domain MUL1-RING Ube2D2 ubiquitylation crystallization research communications Source Type: research

Complexation of the nickel and cobalt transcriptional regulator RcnR with DNA
RcnR is a transcription factor that regulates the homeostasis of cobalt and nickel in bacterial cells. Escherichia coli RcnR was crystallized with DNA that encompasses the DNA-binding site. X-ray diffraction data were collected to 2.9   Å resolution. The crystal belonged to space group P6122 or P6522, with unit-cell parameters a = b = 73.59, c = 157.66   Å , α = β = 90, γ = 120 ° . (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - December 24, 2019 Category: Biochemistry Authors: Li, C. Vavra, J.W. Carr, C.E. Huang, H.-T. Maroney, M.J. Wilmot, C.M. Tags: RcnR transcription factor nickel homeostasis cobalt homeostasis research communications Source Type: research

Crystallographic analysis of Eisenia hydrolysis-enhancing protein using a long wavelength for native-SAD phasing
In this study, EHEP was purified from the natural digestive fluid of A. kurodai and was crystallized using the sitting-drop vapor-diffusion method. Native and SAD (single-wavelength anomalous diffraction) data sets were successfully collected at resolutions of 1.20 and 2.48   Å using wavelengths of 1.0 and 2.1   Å , respectively, from crystals obtained in initial screening. The crystals belonged to space group P212121 and contained one EHEP molecule in the asymmetric unit. All 20 S-atom sites in EHEP were located and the phases were determined by the SAD method using the S atoms in the natural protein as an...
Source: Acta Crystallographica Section F - December 24, 2019 Category: Biochemistry Authors: Sun, X. Ye, Y. Sakurai, N. Kato, K. Yuasa, K. Tsuji, A. Yao, M. Tags: EHEP phlorotannin binding solutionless crystal mount native-SAD biofuel research communications Source Type: research

Crystal structure of an oxidized mutant of human mitochondrial branched-chain aminotransferase
This study presents the crystal structure of a thiol variant of the human mitochondrial branched-chain aminotransferase protein. Human branched-chain aminotransferase (hBCAT) catalyzes the transamination of the branched-chain amino acids leucine, valine and isoleucine and α -ketoglutarate to their respective α -keto acids and glutamate. hBCAT activity is regulated by a CXXC center located approximately 10   Å from the active site. This redox-active center facilitates recycling between the reduced and oxidized states, representing hBCAT in its active and inactive forms, respectively. Site-directed mutage...
Source: Acta Crystallographica Section F - December 24, 2019 Category: Biochemistry Authors: Herbert, D. Gibbs, S. Riddick, A. Conway, M. Dong, M. Tags: human mitochondrial branched-chain aminotransferase redox regulation CXXC center N-terminal loop interdomain loop transaminases research communications Source Type: research

The adhesive PitA pilus protein from the early dental plaque colonizer Streptococcus oralis: expression, purification, crystallization and X-ray diffraction analysis
PitA is the putative tip adhesin of the pilus islet 2 (PI-2)-encoded sortase-dependent pilus in the Gram-positive Streptococcus oralis, an opportunistic pathogen that often flourishes within the diseased human oral cavity. Early colonization by S. oralis and its interaction with Actinomyces oris seeds the development of oral biofilm or dental plaque. Here, the PI-2 pilus plays a vital role in mediating adherence to host surfaces and other bacteria. A recombinant form of the PitA adhesin has now been produced and crystallized. Owing to the large size ( ∼ 100   kDa), flexibility and complicated folding of PitA, obtaini...
Source: Acta Crystallographica Section F - December 23, 2019 Category: Biochemistry Authors: Kumari Yadav, R. Krishnan, V. Tags: adhesins PitA PI-2 sortase-dependent pilus Streptococcus oralis proteolysis terbium crystallophore research communications Source Type: research

Intermediate-resolution crystal structure of the human adenovirus B serotype 3 fibre knob in complex with the EC2-EC3 fragment of desmoglein   2
The cryo-electron microscopy (cryo-EM) structure of the complex between the trimeric human adenovirus B serotype 3 fibre knob and human desmoglein 2 fragments containing cadherin domains EC2 and EC3 has been published, showing 3:1 and 3:2 complexes. Here, the crystal structure determined at 4.5   Å resolution is presented with one EC2-EC3 desmoglein fragment bound per fibre knob monomer in the asymmetric unit, leading to an apparent 3:3 stoichiometry. However, in concentrated solution the 3:2 complex is predominant, as shown by small-angle X-ray scattering (SAXS), while cryo-EM at lower concentrations showed a majo...
Source: Acta Crystallographica Section F - November 27, 2019 Category: Biochemistry Authors: Vassal-Stermann, E. Hutin, S. Fender, P. Burmeister, W.P. Tags: virus receptors extracellular domains cell-surface glycoproteins desmosomes viral proteins research communications Source Type: research

Crystal structure of the Wheat dwarf virus Rep domain
The Rep domain of Wheat dwarf virus (WDV Rep) is an HUH endonuclease involved in rolling-circle replication. HUH endonucleases coordinate a metal ion to enable the nicking of a specific ssDNA sequence and the subsequent formation of an intermediate phosphotyrosine bond. This covalent protein – ssDNA adduct makes HUH endonucleases attractive fusion tags (HUH-tags) in a diverse number of biotechnological applications. Solving the structure of an HUH endonuclease in complex with ssDNA will provide critical information about ssDNA recognition and sequence specificity, thus enabling rationally engineered protein – D...
Source: Acta Crystallographica Section F - November 27, 2019 Category: Biochemistry Authors: Everett, B.A. Litzau, L.A. Tompkins, K. Shi, K. Nelson, A. Aihara, H. Evans III, R.L. Gordon, W.R. Tags: crystal structure Wheat dwarf virus Rep domain HUH motif HUH-tag ssDNA engineered protein – ssDNA complexes research communications Source Type: research

Crystal structure of the MIF4G domain of the Trypanosoma cruzi translation initiation factor EIF4G5
Kinetoplastida, a class of early-diverging eukaryotes that includes pathogenic Trypanosoma and Leishmania species, display key differences in their translation machinery compared with multicellular eukaryotes. One of these differences involves a larger number of genes encoding eIF4E and eIF4G homologs and the interaction pattern between the translation initiation factors. eIF4G is a scaffold protein which interacts with the mRNA cap-binding factor eIF4E, the poly(A)-binding protein, the RNA helicase eIF4A and the eIF3 complex. It contains the so-called middle domain of eIF4G (MIF4G), a multipurpose adaptor involved in diff...
Source: Acta Crystallographica Section F - November 20, 2019 Category: Biochemistry Authors: Camillo dos Santos, L.P. de Matos, B.M. de Maman Ribeiro, B.C. Zanchin, N.I.T. Guimar ã es, B.G. Tags: MIF4G domain translation initiation factor EIF4G5 Trypanosoma cruzi research communications Source Type: research

SpaB, an atypically adhesive basal pilin from the lactobacillar SpaCBA pilus: crystallization and X-ray diffraction analysis
The SpaB pilin is recognized as the basal subunit of the sortase-dependent SpaCBA pilus, which is known to be produced by the Gram-positive Lactobacillus rhamnosus GG, a gut-adapted commensal advocated to have health benefits. Despite seeming to function as an archetypal basal pilin by serving as the terminal subunit in pilus assembly, SpaB also assumes an atypical role as a mucoadhesive protein. To shed light on the structural factors that contribute to this dual functional behaviour, a recombinant form of the L. rhamnosus GG SpaB pilin was produced and purified for crystallization and X-ray diffraction experiments. The c...
Source: Acta Crystallographica Section F - November 20, 2019 Category: Biochemistry Authors: Kumar Megta, A. Palva, A. von Ossowski, I. Krishnan, V. Tags: adhesion probiotics basal pilin host – microbe interaction sortase-dependent pili Lactobacillus rhamnosus GG SpaB research communications Source Type: research

An extracellular domain of the EsaA membrane component of the type VIIb secretion system: expression, purification and crystallization
The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48   kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α -helices with a melting point of 34.5 ° C. Size-exclusion chromatography combined with multi-angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedra...
Source: Acta Crystallographica Section F - November 20, 2019 Category: Biochemistry Authors: Mietrach, N. Schlosser, A. Geibel, S. Tags: ESAT-6-like secretion system ESS type VII secretion system EsaA extracellular domain Staphylococcus aureus USA300 research communications Source Type: research

Conformational heterogeneity in apo and drug-bound structures of Toxoplasma gondii prolyl-tRNA synthetase
In this study, structures of apo and FF-bound T. gondii (TgPRS) are revealed and the dynamic nature of the conformational changes that occur upon FF binding is unraveled. In addition, this study highlights significant conformational plasticity within two different crystal structures of apo PRSs but not within drug-bound PRSs. The apo PRSs exist in multi-conformational states and manifest pseudo-dimeric structures. In contrast, when FF is bound the PRS dimer adopts a highly symmetrical architecture. It is shown that TgPRS does not display extant fold switching, in contrast to P.   falciparum PRS, despite having over 65...
Source: Acta Crystallographica Section F - November 7, 2019 Category: Biochemistry Authors: Mishra, S. Malhotra, N. Kumari, S. Sato, M. Kikuchi, H. Yogavel, M. Sharma, A. Tags: toxoplasmosis prolyl-tRNA synthetase enzyme – inhibitor complexes comparative crystallography drug design r.m.s.d. per residue profile apo holo transistions research communications Source Type: research

The non-swapped monomeric structure of the arginine-binding protein from Thermotoga maritima
Domain swapping is a widespread oligomerization process that is observed in a large variety of protein families. In the large superfamily of substrate-binding proteins, non-monomeric members have rarely been reported. The arginine-binding protein from Thermotoga maritima (TmArgBP), a protein endowed with a number of unusual properties, presents a domain-swapped structure in its dimeric native state in which the two polypeptide chains mutually exchange their C-terminal helices. It has previously been shown that mutations in the region connecting the last two helices of the TmArgBP structure lead to the formation of a variet...
Source: Acta Crystallographica Section F - November 5, 2019 Category: Biochemistry Authors: Smaldone, G. Ruggiero, A. Balasco, N. Abuhammad, A. Autiero, I. Caruso, D. Esposito, D. Ferraro, G. Gelardi, E.L.M. Moreira, M. Quareshy, M. Romano, M. Saaret, A. Selvam, I. Squeglia, F. Troisi, R. Kroon-Batenburg, L.M.J. Esposito, L. Berisio, R. Vitaglia Tags: domain swapping protein oligomerization protein structure dynamics – stability proline residue arginine-binding protein Thermotoga maritima research communications Source Type: research

Structural analysis of free and liganded forms of the Fab fragment of a high-affinity anti-cocaine antibody, h2E2
A high-affinity anti-cocaine monoclonal antibody, designated h2E2, is entering phase 1 clinical trials for cocaine abuse therapy. To gain insight into the molecular details of its structure that are important for binding cocaine and cocaine metabolites, the Fab fragment was generated and crystallized with and without ligand. Structures of the unliganded Fab and the Fab fragment bound to benzoylecgonine were determined, and were compared with each other and with other crystallized anti-cocaine antibodies. The affinity of the h2E2 antibody for cocaine is 4   nM, while that of the cocaine metabolite benzoylecgonine is 20 ...
Source: Acta Crystallographica Section F - November 5, 2019 Category: Biochemistry Authors: Tan, K. Zhou, M. Ahrendt, A.J. Duke, N.E.C. Tabaja, N. Ball, W.J. Kirley, T.L. Norman, A.B. Joachimiak, A. Schiffer, M. Wilton, R. Pokkuluri, P.R. Tags: high-affinity anti-cocaine antibody antigen-binding fragment crystal structure benzoylecgonine complex h2E2 Fab fragment research communications Source Type: research

Major conformational changes in the structure of lysozyme obtained from a crystal with a very low solvent content
A new crystal form of lysozyme with a very low solvent content (26.35%) has been obtained in the orthorhombic space group P212121 (with unit-cell parameters a = 30.04, b = 51.68, c = 61.53   Å ). The lysozyme structure obtained from these crystals does not show the typical overall fold. Instead, major conformational changes take place in some elements of the secondary structure and in the hydrophobic core of the protein. At the end of the central α -helix ( α 2), Glu35 is usually buried in the catalytic site and shows an abnormally high pKa value, which is key to the activity of the enzyme. The high pKa...
Source: Acta Crystallographica Section F - November 5, 2019 Category: Biochemistry Authors: Salinas-Garcia, M.C. Plaza-Garrido, M. Alba-Elena, D. Camara-Artigas, A. Tags: low solvent content lysozyme folding intermediate research communications Source Type: research

Inexpensive robotic system for standard and fluorescent imaging of protein crystals
Protein-crystallization imaging and classification is a labor-intensive process typically performed either by humans or by instruments that currently cost well over $100   000. This cost puts the use of crystallization-trial imaging outside the reach of most academic laboratories, and also start-up biotechnology firms, where resources are scarce. An imaging system has been designed and prototyped which automatically captures images from multi-well protein-crystallization experiments using both standard and fluorescent imaging techniques at a cost 28 times lower than current market rates. The machine uses a Panowin F1 3D ...
Source: Acta Crystallographica Section F - November 5, 2019 Category: Biochemistry Authors: Handzlik, D. Larson, E.T. Munschy, E. Obmolova, G. Collin, D. Craig, T.K. Tags: fluorescent imaging automated microscope imaging system open source robotics methods communications Source Type: research

Automatic annotation of protein residues in published papers
This work presents an annotation tool that automatically locates mentions of particular amino-acid residues in published papers and identifies the protein concerned. These matches can be provided in context or in a searchable format in order for researchers to better use the existing and future literature. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - November 5, 2019 Category: Biochemistry Authors: Firth, R. Talo, F. Venkatesan, A. Mukhopadhyay, A. McEntyre, J. Velankar, S. Morris, C. Tags: NLP natural language processing named entity recognizer residue methods communications Source Type: research

Taking biological structure communications into the third dimension
(Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - November 1, 2019 Category: Biochemistry Authors: van Raaij, M. Newman, J. Tags: annotation online structure visualization augmented reality integrated multimedia editorial Source Type: research

Crystal structure of the N-terminal domain of the fibronectin-binding protein PavA from Streptococcus pneumoniae
In this study, the crystal structure of the N-terminal domain of PavA (SpPavA-N) determined at a resolution of 2.39   Å is reported. Each monomer of the dimeric protein consists of two domains (domains I and II) and a short α -helix ( α 6) at the C-terminus that are connected by elongated loops. Comparison of the SpPavA-N structure with that of its homolog from Streptococcus suis (FBPS-N) revealed differences in α 5, α 6 and the domain II/ α 6 inter-loop region within domain II. The α 5 helix of FBPS-N folds back toward domain I, whereas in SpPavA-N it adopts an elongated rod sha...
Source: Acta Crystallographica Section F - September 24, 2019 Category: Biochemistry Authors: Manne, K. Narayana, S. Chattopadhyay, D. Tags: fibronectin-binding protein Streptococcus pneumoniae X-ray crystallography pneumococcal adherence and virulence factor A PavA crystal structure research communications Source Type: research

Structure of an RNA helix with pyrimidine mismatches and cross-strand stacking
The structure of a 22-base-pair RNA helix with mismatched pyrimidine base pairs is reported. The helix contains two symmetry-related CUG sequences: a triplet-repeat motif implicated in myotonic dystrophy type 1. The CUG repeat contains a U – U mismatch sandwiched between Watson – Crick pairs. Additionally, the center of the helix contains a dimerized UUCG motif with tandem pyrimidine (U – C/C – U) mismatches flanked by U – G wobble pairs. This region of the structure is significantly different from previously observed structures that share the same sequence and neighboring base pairs. The tand...
Source: Acta Crystallographica Section F - September 24, 2019 Category: Biochemistry Authors: Montemayor, E.J. Virta, J.M. Hagler, L.D. Zimmerman, S.C. Butcher, S.E. Tags: RNA myotonic dystrophy type 1 pyrimidine mismatches research communications Source Type: research

Rv0100, a proposed acyl carrier protein in Mycobacterium tuberculosis: expression, purification and crystallization
Acyl carrier proteins (ACPs) are important components in fatty-acid biosynthesis in prokaryotes. Rv0100 is predicted to be an essential ACP in Mycobacterium tuberculosis, the pathogen that is the causative agent of tuberculosis, and therefore has the potential to be a novel antituberculosis drug target. Here, the successful cloning and purification of Rv0100 using Mycobacterium smegmatis as a host is reported. Crystals of the purified protein were obtained that diffracted to a resolution of 1.9   Å . Overall, this work lays the foundation for the future pursuit of drug discovery and development against this potenti...
Source: Acta Crystallographica Section F - September 24, 2019 Category: Biochemistry Authors: Bondoc, J.M.G. Gutka, H.J. Almutairi, M.M. Patwell, R. Rutter, M.W. Wolf, N.M. Samudrala, R. Mehboob, S. Movahedzadeh, F. Tags: acyl carrier protein Mycobacterium tuberculosis Mycobacterium smegmatis Rv0100 research communications Source Type: research

The structure of bovine β -lactoglobulin in crystals grown at pH 3.8 exhibiting novel threefold twinning
Bovine β -lactoglobulin was crystallized from 3   M NaCl buffered at pH 3.8 with sodium citrate as thick hexagonal prisms of greater than 1   mm in edge length. Analyses of the X-ray diffraction intensities using three different current algorithms were unanimous in specifying the space group to be P6322, with unit-cell dimensions a = b = 75.47, c = 140.79   Å . No progress could be made, however, towards an acceptable solution by molecular replacement using this symmetry. In the end, it was found that the true space group was C2221, a subgroup of P6322, with a = 65.89, b = 114.12, c = 140.51   Å , ...
Source: Acta Crystallographica Section F - September 20, 2019 Category: Biochemistry Authors: Yeates, T.O. McPherson, A. Tags: β -lactoglobulin milk tritohedral twinning symmetry whey proteins space group determination research communications Source Type: research

Crystal structure of the Fab region of a neutralizing antibody against granulocyte-macrophage colony-stimulating factor
An increased level of granulocyte-macrophage colony-stimulating factor has a potential role in the development of autoimmune diseases, and the neutralization of its activity by monoclonal antibodies is a promising therapy for some diseases. Here, the crystal structure of the Fab region of EV1007, a fully human antibody expressed in Chinese hamster ovary cells that was developed from human peripheral blood mononuclear cells, is described. The structure closely resembles that of MB007, which is the Fab region of the same antibody expressed in Escherichia coli [Blech et al. (2012), Biochem. J. 447, 205 – 215], except at...
Source: Acta Crystallographica Section F - September 20, 2019 Category: Biochemistry Authors: Angkawidjaja, C. Torashima, T. Tags: monoclonal antibodies Fab region granulocyte-macrophage colony-stimulating factor anti-GM-CSF EV1007 research communications Source Type: research

Structural characterization of a prolyl aminodipeptidase (PepX) from Lactobacillus helveticus
Prolyl aminodipeptidase (PepX) is an enzyme that hydrolyzes peptide bonds from the N-terminus of substrates when the penultimate amino-acid residue is a proline. Prolyl peptidases are of particular interest owing to their ability to hydrolyze food allergens that contain a high percentage of proline residues. PepX from Lactobacillus helveticus was cloned and expressed in Escherichia coli as an N-terminally His-tagged recombinant construct and was crystallized by hanging-drop vapor diffusion in a phosphate buffer using PEG 3350 as a precipitant. The structure was determined at 2.0   Å resolution by molecular replacem...
Source: Acta Crystallographica Section F - September 20, 2019 Category: Biochemistry Authors: Ojennus, D.D. Bratt, N.J. Jones, K.L. Juers, D.H. Tags: prolyl aminodipeptidase PepX α / β -hydrolases protein structure calcium binding calcium-blade zone gluten Lactobacillus helveticus research communications Source Type: research

Structure of GTP cyclohydrolase I from Listeria monocytogenes, a potential anti-infective drug target
A putative open reading frame encoding GTP cyclohydrolase I from Listeria monocytogenes was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified and was confirmed to convert GTP to dihydroneopterin triphosphate (Km = 53   µ M; vmax = 180   nmol   mg − 1   min − 1). The   protein was crystallized from 1.3   M sodium citrate pH 7.3 and the crystal structure was solved at a resolution of 2.4   Å (Rfree = 0.226) by molecular replacement using human GTP cyclohydrolase I as a template. The protein is a D5-symmetric decamer with ten topologically equival...
Source: Acta Crystallographica Section F - August 30, 2019 Category: Biochemistry Authors: Sch ü ssler, S. Haase, I. Perbandt, M. Illarionov, B. Siemens, A. Richter, K. Bacher, A. Fischer, M. Gr ä wert, T. Tags: GTP cyclohydrolase I Listeria monocytogenes crystal structure high-throughput screening tetrahydrofolate biosynthesis listeriosis research communications Source Type: research

Structure of the archaeal chemotaxis protein CheY in a domain-swapped dimeric conformation
In this study, two new crystal forms and protein structures of CheY are reported. In both crystal forms, CheY is arranged in a domain-swapped conformation. CheF, the protein bridging the chemotaxis signal transduction system and the motility apparatus, was recombinantly expressed, purified and subjected to X-ray data collection. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - August 30, 2019 Category: Biochemistry Authors: Paithankar, K.S. Enderle, M. Wirthensohn, D.C. Miller, A. Schlesner, M. Pfeiffer, F. Rittner, A. Grininger, M. Oesterhelt, D. Tags: chemotaxis signal transduction response regulator CheY CheF archaellum protein evolution research communications Source Type: research

Structure of the dihydrolipoamide succinyltransferase catalytic domain from Escherichia coli in a novel crystal form: a tale of a common protein crystallization contaminant
The crystallization of amidase, the ultimate enzyme in the Trp-dependent auxin-biosynthesis pathway, from Arabidopsis thaliana was attempted using protein samples with at least 95% purity. Cube-shaped crystals that were assumed to be amidase crystals that belonged to space group I4 (unit-cell parameters a = b = 128.6, c = 249.7   Å ) were obtained and diffracted to 3.0   Å resolution. Molecular replacement using structures from the PDB containing the amidase signature fold as search models was unsuccessful in yielding a convincing solution. Using the Sequence-Independent Molecular replacement Based on Avail...
Source: Acta Crystallographica Section F - August 29, 2019 Category: Biochemistry Authors: Andi, B. Soares, A.S. Shi, W. Fuchs, M.R. McSweeney, S. Liu, Q. Tags: dihydrolipoamide succinyltransferase auxin amidase contaminant crystallization protein crystallography molecular replacement research communications Source Type: research

Crystal structure of UDP-glucose pyrophosphorylase from Yersinia pestis, a potential therapeutic target against plague
Yersinia pestis, the causative agent of bubonic plague, is one of the most lethal pathogens in recorded human history. Today, the concern is the possible misuse of Y. pestis as an agent in bioweapons and bioterrorism. Current therapies for the treatment of plague include the use of a small number of antibiotics, but clinical cases of antibiotic resistance have been reported in some areas of the world. Therefore, the discovery of new drugs is required to combat potential Y.   pestis infection. Here, the crystal structure of the Y. pestis UDP-glucose pyrophosphorylase (UGP), a metabolic enzyme implicated in the survival...
Source: Acta Crystallographica Section F - August 28, 2019 Category: Biochemistry Authors: Gibbs, M.E. Lountos, G.T. Gumpena, R. Waugh, D.S. Tags: Yersinia pestis galU UDP-glucose pyrophosphorylase plague drug targets research communications Source Type: research

Structural basis for the substrate recognition of aminoglycoside 7 ′ ′ -phosphotransferase-Ia from Streptomyces hygroscopicus
Hygromycin B (HygB) is one of the aminoglycoside antibiotics, and it is widely used as a reagent in molecular-biology experiments. Two kinases are known to inactivate HygB through phosphorylation: aminoglycoside 7 ′ ′ -phosphotransferase-Ia [APH(7 ′ ′ )-Ia] from Streptomyces hygroscopicus and aminoglycoside 4-phosphotransferase-Ia [APH(4)-Ia] from Escherichia coli. They phosphorylate the hydroxyl groups at positions 7 ′ ′ and 4 of the HygB molecule, respectively. Previously, the crystal structure of APH(4)-Ia was reported as a ternary complex with HygB and 5 ′ -adenylyl- β , &...
Source: Acta Crystallographica Section F - August 28, 2019 Category: Biochemistry Authors: Takenoya, M. Shimamura, T. Yamanaka, R. Adachi, Y. Ito, S. Sasaki, Y. Nakamura, A. Yajima, S. Tags: aminoglycoside antibiotics enzyme-mediated resistance kinases SAD phasing selenomethionine hygromycin B Streptomyces hygroscopicus research communications Source Type: research

No magnesium is needed for binding of the stimulator of interferon genes to cyclic dinucleotides
Stimulator of interferon genes (STING) binds cyclic dinucleotides (CDNs), which induce a large conformational change of the protein. The structural basis of activation of STING by CDNs is rather well understood. Unliganded STING forms an open dimer that undergoes a large conformational change ( ∼ 10   Å ) to a closed conformation upon the binding of a CDN molecule. This event activates downstream effectors of STING and subsequently leads to activation of the type 1 interferon response. However, a previously solved structure of STING with 3 ′ ,3 ′ -c-di-GMP shows Mg atoms mediating the interaction of...
Source: Acta Crystallographica Section F - August 28, 2019 Category: Biochemistry Authors: Smola, M. Birkus, G. Boura, E. Tags: STING cGAS CDN 3 ′ ,3 -c-di-GMP crystal structure research communications Source Type: research

Crystal structure of TchmY from Actinoplanes teichomyceticus
Moenomycin-type antibiotics are phosphoglycolipids that are notable for their unique modes of action and have proven to be useful in animal nutrition. The gene clusters tchm from Actinoplanes teichomyceticus and moe from Streptomyces are among a limited number of known moenomycin-biosynthetic pathways. Most genes in tchm have counterparts in the moe cluster, except for tchmy and tchmz, the functions of which remain unknown. Sequence analysis indicates that TchmY belongs to the isoprenoid enzyme C2-like superfamily and may serve as a prenylcyclase. The enzyme was proposed to be involved in terminal cyclization of the moenoc...
Source: Acta Crystallographica Section F - August 28, 2019 Category: Biochemistry Authors: Yang, Z. Zhang, L. Yu, X. Wu, S. Yang, Y. Hu, Y. Li, Q. Shang, N. Guo, R.-T. Chen, C.-C. Dai, L. Liu, W. Tags: crystal structure prenylcyclases Actinoplanes teichomyceticus research communications Source Type: research

AMi: a GUI-based, open-source system for imaging samples in multi-well plates
Described here are instructions for building and using an inexpensive automated microscope (AMi) that has been specifically designed for viewing and imaging the contents of multi-well plates. The X, Y, Z translation stage is controlled through dedicated software (AMiGUI) that is being made freely available. Movements are controlled by an Arduino-based board running grbl, and the graphical user interface and image acquisition are controlled via a Raspberry Pi microcomputer running Python. Images can be written to the Raspberry Pi or to a remote disk. Plates with multiple sample wells at each row/column position are supporte...
Source: Acta Crystallographica Section F - August 6, 2019 Category: Biochemistry Authors: Bohm, A. Tags: AMi AMiGUI automated microscopy multi-well plates crystallography methods communications Source Type: research

The Rel stringent factor from Thermus thermophilus: crystallization and X-ray analysis
The stringent response, controlled by (p)ppGpp, enables bacteria to trigger a strong phenotypic resetting that is crucial to cope with adverse environmental changes and is required for stress survival and virulence. In the bacterial cell, (p)ppGpp levels are regulated by the concerted opposing activities of RSH (RelA/SpoT homologue) enzymes that can transfer a pyrophosphate group of ATP to the 3 ′ position of GDP (or GTP) or remove the 3 ′ pyrophosphate moiety from (p)ppGpp. Bifunctional Rel enzymes are notoriously difficult to crystallize owing to poor stability and a propensity for aggregation, usually leadin...
Source: Acta Crystallographica Section F - August 2, 2019 Category: Biochemistry Authors: Van Nerom, K. Tamman, H. Takada, H. Hauryliuk, V. Garcia-Pino, A. Tags: stringent response Rel/RelA/SpoT (p)ppGpp bacterial alarmone Thermus thermophilus research communications Source Type: research

Crystal structure of the Schizosaccharomyces pombe U7BR E2-binding region in complex with Ubc7
Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality-control pathway in eukaryotes in which misfolded ER proteins are polyubiquitylated, extracted and ultimately degraded by the proteasome. This process involves ER membrane-embedded ubiquitin E2 and E3 enzymes, as well as a soluble E2 enzyme (Ubc7 in Saccharomyces cerevisiae and UBE2G2 in mammals). E2-binding regions (E2BRs) that recruit these soluble ERAD E2s to the ER have been identified in humans and S. cerevisiae, and structures of E2 – E2BR complexes from both species have been determined. In addition to sequence and structural differen...
Source: Acta Crystallographica Section F - August 2, 2019 Category: Biochemistry Authors: Hann, Z.S. Metzger, M.B. Weissman, A.M. Lima, C.D. Tags: endoplasmic reticulum ERAD ubiquitin protein degradation ubiquitin-conjugating enzyme E2 E2-binding protein Schizosaccharomyces pombe research communications Source Type: research

Co-crystal structure of the iMango-III fluorescent RNA aptamer using an X-ray free-electron laser
Turn-on aptamers are in vitro-selected RNAs that bind to conditionally fluorescent small molecules and enhance their fluorescence. Upon binding TO1-biotin, the iMango-III aptamer achieves the largest fluorescence enhancement reported for turn-on aptamers (over 5000-fold). This aptamer was generated by structure-guided engineering and functional reselection of the parental aptamer Mango-III. Structures of both Mango-III and iMango-III have previously been determined by conventional cryocrystallography using synchrotron X-radiation. Using an X-ray free-electron laser (XFEL), the room-temperature iMango-III – TO1-biotin...
Source: Acta Crystallographica Section F - August 2, 2019 Category: Biochemistry Authors: Trachman, R.J. Stagno, J.R. Conrad, C. Jones, C.P. Fischer, P. Meents, A. Wang, Y.-X. Ferr é -D'Amar é , A.R. Tags: RNA structure fluorescence turn-on aptamers X-ray free-electron lasers iMango-III XFELS room temperature research communications Source Type: research

Introducing Methods Communications, a new category of contributions to Acta Crystallographica F
(Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - August 1, 2019 Category: Biochemistry Authors: Newman, J. van Raaij, M.J. Tags: methods communications topical reviews multimedia editorial Source Type: research

Crystal structure of the inhibitor-free form of the serine protease kallikrein-4
In this study, an inhibitor-free form of KLK4 was crystallized. The crystals obtained belonged to space group P1, contained four molecules in the asymmetric unit and diffracted to 1.64   Å resolution. Interestingly, a nonstandard rotamer of the specificity-determining residue Asp189 was observed in all chains. This model will provide a useful unliganded structure for the future structure-guided design of KLK4 inhibitors. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - July 16, 2019 Category: Biochemistry Authors: Riley, B.T. Hoke, D.E. McGowan, S. Buckle, A.M. Tags: kallikrein-related peptidase 4 KLK4 apo structure unliganded research communications Source Type: research

The crystal structure of haemoglobin from Atlantic cod
has been solved to 2.54   Å resolution. The structure consists of two tetramers in the crystallographic asymmetric unit. The structure of haemoglobin obtained from one individual cod suggests polymorphism in the tetrameric assembly. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - July 16, 2019 Category: Biochemistry Authors: Helland, R. Bj ø rkeng, E.K. Rothweiler, U. Sydnes, M.O. Pampanin, D.M. Tags: haemoglobin Gadus morhua Atlantic cod research communications Source Type: research

Structural insights into the substrate specificity of SP_0149, the substrate-binding protein of a methionine ABC transporter from Streptococcus pneumoniae
Successful pathogenesis is a cumulative effect of the virulence factors of a pathogen and its capability to efficiently utilize the available nutrients from the host. Streptococcus pneumoniae, a Gram-positive opportunistic pathogen, may either reside asymptomatically as a nasopharyngeal commensal inside the human host or cause lethal diseases, including pneumonia, meningitis and sepsis. S. pneumoniae is known to acquire methionine (Met) from its host through a Met importer. Here, the crystal structure of the substrate-binding protein (SBP; SP_0149) of an ABC importer with Met bound is reported at a resolution of 1.95   &...
Source: Acta Crystallographica Section F - July 4, 2019 Category: Biochemistry Authors: Jha, B. Vyas, R. Bhushan, J. Sehgal, D. Biswal, B.K. Tags: Streptococcus pneumoniae ABC transporter substrate-binding protein methionine X-ray crystallography research communications Source Type: research

A promiscuous kinase inhibitor delineates the conspicuous structural features of protein kinase CK2a1
Protein kinase CK2a1 is a serine/threonine kinase that plays a crucial role in the growth, proliferation and survival of cells and is a well known target for tumour and glomerulonephritis therapies. Here, the crystal structure of the kinase domain of CK2a1 complexed with 5-iodotubercidin (5IOD), an ATP-mimetic inhibitor, was determined at 1.78   Å resolution. The structure shows distinct structural features and, in combination with a comparison of the crystal structures of five off-target kinases complexed with 5IOD, provides valuable information for the development of highly selective inhibitors. (Source: Acta Cry...
Source: Acta Crystallographica Section F - July 4, 2019 Category: Biochemistry Authors: Tsuyuguchi, M. Nakaniwa, T. Sawa, M. Nakanishi, I. Kinoshita, T. Tags: protein kinase CK2 ATP-analogue inhibitor selectivity off-target kinases structural comparison research communications Source Type: research

Structural comparison of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida with PobA from other Pseudomonas spp. and other monooxygenases
The crystal structure is reported of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida, a possible drug target to combat tetracycline resistance, in complex with flavin adenine dinucleotide (FAD). The structure was refined at 2.2   Å resolution with four polypeptide chains in the asymmetric unit. Based on the results of pairwise structure alignments, PobA from P. putida is structurally very similar to PobA from P. fluorescens and from P. aeruginosa. Key residues in the FAD-binding and substrate-binding sites of PobA are highly conserved spatially across the proteins from all three species. Additionally, ...
Source: Acta Crystallographica Section F - July 2, 2019 Category: Biochemistry Authors: Lazar, J.T. Shuvalova, L. Rosas-Lemus, M. Kiryukhina, O. Satchell, K.J.F. Minasov, G. Tags: Pseudomonas oxygenases antibiotic resistance PobA pHBH research communications Source Type: research

Crystal structure of the glycoside hydrolase PssZ from Listeria monocytogenes
In this study, the crystal structure of PssZ has been determined at 1.6   Å resolution. PssZ belongs to glycoside hydrolase family 8 and adopts a classical ( α / α )6-barrel fold. This architecture forms a deep groove which may serve as the substrate-binding pocket. The conserved catalytic residues (Glu72, Trp110, Asn119, Phe167, Tyr183 and Asp232) are localized at the centre of the groove. This crystal structure will help to improve the understanding of the hydrolytic mechanism of PssZ and its application as a biofilm disrupter. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - June 28, 2019 Category: Biochemistry Authors: Wu, H. Qiao, S. Li, D. Guo, L. Zhu, M. Ma, L.Z. Tags: Listeria monocytogenes glycoside hydrolases SAD phasing PssZ ( α / )6-barrel fold biofilms research communications Source Type: research

Co-crystal structure of the Fusobacterium ulcerans ZTP riboswitch using an X-ray free-electron laser
Riboswitches are conformationally dynamic RNAs that regulate gene expression by binding specific small molecules. ZTP riboswitches bind the purine-biosynthetic intermediate 5-aminoimidazole-4-carboxamide riboside 5 ′ -monophosphate (ZMP) and its triphosphorylated form (ZTP). Ligand binding to this riboswitch ultimately upregulates genes involved in folate and purine metabolism. Using an X-ray free-electron laser (XFEL), the room-temperature structure of the Fusobacterium ulcerans ZTP riboswitch bound to ZMP has now been determined at 4.1   Å resolution. This model, which was refined against a data set from &s...
Source: Acta Crystallographica Section F - June 28, 2019 Category: Biochemistry Authors: Jones, C. Tran, B. Conrad, C. Stagno, J. Trachman, R. Fischer, P. Meents, A. Ferr é -D'Amar é , A. Tags: RNA structure riboswitches ZTP X-ray free-electron lasers research communications Source Type: research

Structural characterization of Treponema pallidum Tp0225 reveals an unexpected leucine-rich repeat architecture
The phylogenetically divergent spirochete bacterium Treponema pallidum subsp. pallidum is the causative agent of syphilis. Central to the capacity of T.   pallidum to establish infection is the ability of the pathogen to attach to a diversity of host cells. Many pathogenic bacteria employ leucine-rich repeat (LRR) domain-containing proteins to mediate protein – protein interactions, including attachment to host components and establishment of infection. Intriguingly, T. pallidum expresses only one putative LRR domain-containing protein (Tp0225) with an unknown function. In an effort to ascribe a function to Tp02...
Source: Acta Crystallographica Section F - June 26, 2019 Category: Biochemistry Authors: Ramaswamy, R. Houston, S. Loveless, B. Cameron, C.E. Boulanger, M.J. Tags: syphilis Treponema pallidum X-ray crystallography leucine rich-repeat domain research communications Source Type: research

Crystal structure of an iron superoxide dismutase from the pathogenic amoeba Acanthamoeba castellanii
The iron superoxide dismutase found in the pathogenic amoeba Acanthamoeba castellanii (AcFeSOD) may play essential roles in the survival of the parasite, not only by protecting it from endogenous oxidative stress but also by detoxifying oxidative killing of the parasite by host immune effector cells. The AcFeSOD protein was expressed in a stable form using an Escherichia coli expression system and was crystallized by the microbatch and hanging-drop vapour-diffusion methods. The structure was determined to 2.33   Å resolution from a single AcFeSOD crystal. The crystal belonged to the hexagonal space group P61 and co...
Source: Acta Crystallographica Section F - June 26, 2019 Category: Biochemistry Authors: Dao, O. Asaithambi, K. Na, B.K. Lee, K.H. Tags: iron superoxide dismutase Acanthamoeba castellanii research communications Source Type: research

Crystal structure of (S)-3-O-geranylgeranylglyceryl phosphate synthase from Thermoplasma acidophilum in complex with the substrate sn-glycerol 1-phosphate
In this study, the crystal structure of the archaeal group II GGGPS from Thermoplasma acidophilum (TaGGGPS) was determined at 2.35   Å resolution. The structure of TaGGGPS showed that it has a TIM-barrel fold, the third helix of which is disordered ( α 3*), and that it forms a homodimer, although a pre-existing structure of an archaeal group II GGGPS (from Methanothermobacter thermautotrophicus) showed a hexameric form. The structure of TaGGGPS showed the precise G1P-recognition mechanism of an archaeal group II GGGPS. The structure of TaGGGPS and molecular-dynamics simulation analysis showed fluctuation of t...
Source: Acta Crystallographica Section F - June 26, 2019 Category: Biochemistry Authors: Nemoto, N. Miyazono, K. Tanokura, M. Yamagishi, A. Tags: geranylgeranylglyceryl phosphate synthase TIM barrel archaea prenyltransferases ether lipid biosynthesis research communications Source Type: research

Changes in the allosteric site of human liver pyruvate kinase upon activator binding include the breakage of an intersubunit cation – π bond
In this study, the X-ray crystal structures of four hLPYK variants were solved to make structural correlations with existing functional data. The variants are D499N, W527H, Δ 529/S531G (called GGG here) and S531E. The results revealed a conformational toggle between the open and closed positions of the allosteric loop. In the absence of Fru-1,6-BP the open position is stabilized, in part, by a cation – π bond between Trp527 and Arg538 ′ (from an adjacent monomer). In the S531E variant glutamate binds in place of the 6 ′ -phosphate of Fru-1,6-BP in the allosteric site, leading to partial allosteri...
Source: Acta Crystallographica Section F - June 10, 2019 Category: Biochemistry Authors: McFarlane, J.S. Ronnebaum, T.A. Meneely, K.M. Chilton, A. Fenton, A.W. Lamb, A.L. Tags: pyruvate kinase allosterism human liver isozyme research communications Source Type: research

Structural analysis of the HIN1 domain of interferon-inducible protein 204
Interferon-inducible protein 204 (p204) binds to microbial DNA to elicit inflammatory responses and induce interferon production. p204 also modulates cell proliferation and differentiation by regulating various transcription factors. The C-terminal HIN domains in p204 are believed to be responsible for DNA binding, but the binding mode is not fully understood. The DNA-binding affinity of the p204 HIN1 domain has been characterized and its crystal structure has been determined, providing insight into its interaction with DNA. Surface-charge distribution together with sequence alignment suggests that the p204 HIN domain uses...
Source: Acta Crystallographica Section F - June 10, 2019 Category: Biochemistry Authors: Tian, Y. Yin, Q. Tags: interferon-inducible protein 204 p204 HIN domain DNA binding research communications Source Type: research

Crystal structure of the flavin-dependent thymidylate synthase Thy1 from Thermus thermophilus with an extra C-terminal domain
The thymidylate synthases ThyA and Thy1 are enzymes that catalyse the formation of thymidine monophosphate from 2 ′ -deoxyuridine monophosphate. Thy1 (or ThyX) requires flavin for catalytic reactions, while ThyA does not. In the present study, the crystal structure of the flavin-dependent thymidylate synthase Thy1 from Thermus thermophilus HB8 (TtThy1, TTHA1096) was determined in complex with FAD and phosphate at 2.5   Å resolution. TtThy1 is a tetrameric molecule like other Thy1 proteins, to which four FAD molecules are bound. In the crystal of TtThy1, two phosphate ions were bound to each dUMP-binding site....
Source: Acta Crystallographica Section F - June 6, 2019 Category: Biochemistry Authors: Ogawa, A. Sampei, G. Kawai, G. Tags: flavin-dependent thymidylate synthase pyrimidine nucleotide biosynthetic pathway C-terminal domain research communications Source Type: research