Deriving and refining atomic models in crystallography and cryo-EM: the latest Phenix tools to facilitate structure analysis
(Source: Acta Crystallographica Section D)
Source: Acta Crystallographica Section D - October 1, 2019 Category: Biochemistry Authors: Klaholz, B.P. Tags: structural biology atomic model refinement crystallography cryo electron microscopy Phenix scientific commentaries Source Type: research

Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix
Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these tec...
Source: Acta Crystallographica Section D - October 1, 2019 Category: Biochemistry Authors: Liebschner, D. Afonine, P.V. Baker, M.L. Bunk ó czi, G. Chen, V.B. Croll, T.I. Hintze, B. Hung, L.-W. Jain, S. McCoy, A.J. Moriarty, N.W. Oeffner, R.D. Poon, B.K. Prisant, M.G. Read, R.J. Richardson, J.S. Richardson, D.C. Sammito, M.D. Sobolev, O.V. Stoc Tags: Phenix automation macromolecular crystallography cryo-EM X-rays neutrons diffraction Python cctbx feature articles Source Type: research

Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments
Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can b...
Source: Acta Crystallographica Section D - October 1, 2019 Category: Biochemistry Authors: Andersson, R. Safari, C. B å th, P. Bosman, R. Shilova, A. Dahl, P. Ghosh, S. Dunge, A. Kjeldsen-Jensen, R. Nan, J. Shoeman, R.L. Kloos, M. Doak, R.B. Mueller, U. Neutze, R. Br ä nd é n, G. Tags: serial crystallography lipidic cubic phase membrane proteins protein crystallization research papers Source Type: research

Flexible workflows for on-the-fly electron-microscopy single-particle image processing using Scipion
Electron microscopy of macromolecular structures is an approach that is in increasing demand in the field of structural biology. The automation of image acquisition has greatly increased the potential throughput of electron microscopy. Here, the focus is on the possibilities in Scipion to implement flexible and robust image-processing workflows that allow the electron-microscope operator and the user to monitor the quality of image acquisition, assessing very simple acquisition measures or obtaining a first estimate of the initial volume, or the data resolution and heterogeneity, without any need for programming skills. Th...
Source: Acta Crystallographica Section D - October 1, 2019 Category: Biochemistry Authors: Maluenda, D. Majtner, T. Horvath, P. Vilas, J.L. Jim é nez-Moreno, A. Mota, J. Ram í rez-Aportela, E. S á nchez-Garc í a, R. Conesa, P. del Ca ñ o, L. Rancel, Y. Fonseca, Y. Mart í nez, M. Sharov, G. Garc í a, C.A. Strelak, D. Melero, R. Marabini, Tags: Scipion image processing electron microscopy single-particle analysis stream processing electron-microscopy facilities research papers Source Type: research

Structure determination of the nucleosome core particle by selenium SAD phasing
The eukaryotic genome is compacted inside the nucleus of the cell in the form called chromatin. The fundamental unit of chromatin is the nucleosome, which contains four types of histones (H3, H4, H2A and H2B) and approximately 150 base pairs of DNA wrapped around the histone complex. The structure of the nucleosome is highly conserved across several eukaryotic species, and molecular replacement has been the primary phasing method used to solve nucleosome structures by X-ray crystallography. However, there is currently no simple, widely applicable experimental phasing method for the nucleosome. In the present study, it is d...
Source: Acta Crystallographica Section D - September 24, 2019 Category: Biochemistry Authors: Saotome, M. Horikoshi, N. Urano, K. Kujirai, T. Yuzurihara, H. Kurumizaka, H. Kagawa, W. Tags: nucleosome chromatin histone selenomethionine SAD phasing research papers Source Type: research

The flavin mononucleotide cofactor in α -hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level
The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution c...
Source: Acta Crystallographica Section D - September 24, 2019 Category: Biochemistry Authors: Lyu, S.-Y. Lin, K.-H. Yeh, H.-W. Li, Y.-S. Huang, C.-M. Wang, Y.-L. Shih, H.-W. Hsu, N.-S. Wu, C.-J. Li, T.-L. Tags: electrophilic/nucleophilic duality α -hydroxyacid oxidases flavin mononucleotide oxidative decarboxylation monooxygenase p-hydroxymandelate oxidase research papers Source Type: research

Comparison of a retroviral protease in monomeric and dimeric states
Retroviral proteases (RPs) are of high interest owing to their crucial role in the maturation process of retroviral particles. RPs are obligatory homodimers, with a pepsin-like active site built around two aspartates (in DTG triads) that activate a water molecule, as the nucleophile, under two flap loops. Mason – Pfizer monkey virus (M-PMV) is unique among retroviruses as its protease is also stable in the monomeric form, as confirmed by an existing crystal structure of a 13   kDa variant of the protein (M-PMV PR) and its previous biochemical characterization. In the present work, two mutants of M-PMV PR, D26N and ...
Source: Acta Crystallographica Section D - September 20, 2019 Category: Biochemistry Authors: Wosicki, S. Gilski, M. Zabranska, H. Pichova, I. Jaskolski, M. Tags: Mason – Pfizer monkey virus M-PMV retroviruses retropepsin aspartic protease dimerization inhibitor flap structure research papers Source Type: research

Supercell refinement: a cautionary tale
Theoretically, crystals with supercells exist at a unique crossroads where they can be considered as either a large unit cell with closely spaced reflections in reciprocal space or a higher dimensional superspace with a modulation that is commensurate with the supercell. In the latter case, the structure would be defined as an average structure with functions representing a modulation to determine the atomic location in 3D space. Here, a model protein structure and simulated diffraction data were used to investigate the possibility of solving a real incommensurately modulated protein crystal using a supercell approximation...
Source: Acta Crystallographica Section D - August 28, 2019 Category: Biochemistry Authors: Lovelace, J. Petr í cek, V. Murshudov, G. Borgstahl, G.E.O. Tags: aperiodic crystallography supercell crystallographic refinement modulated protein crystals superspace group research papers Source Type: research

X-ray structure of the direct electron transfer-type FAD glucose dehydrogenase catalytic subunit complexed with a hitchhiker protein
In this study, the X-ray structure of BcGDH γ α , the catalytic subunit ( α -subunit) of BcGDH complexed with a hitchhiker protein ( γ -subunit), was determined. The most prominent feature of this enzyme is the presence of the 3Fe – 4S cluster, which is located at the surface of the catalytic subunit and functions in intramolecular and intermolecular electron transfer from FAD to the electron-transfer subunit. The structure of the complex revealed that these two molecules are connected through disulfide bonds and hydrophobic interactions, and that the formation of disulfide bonds is required t...
Source: Acta Crystallographica Section D - August 28, 2019 Category: Biochemistry Authors: Yoshida, H. Kojima, K. Shiota, M. Yoshimatsu, K. Yamazaki, T. Ferri, S. Tsugawa, W. Kamitori, S. Sode, K. Tags: direct electron transfer flavin adenine dinucleotide-dependent dehydrogenase complex glucose dehydrogenase iron – sulfur cluster hitchhiker protein glucose sensors Burkholderia cepacia research papers Source Type: research

Methods for merging data sets in electron cryo-microscopy
Recent developments have resulted in electron cryo-microscopy (cryo-EM) becoming a useful tool for the structure determination of biological macromolecules. For samples containing inherent flexibility, heterogeneity or preferred orientation, the collection of extensive cryo-EM data using several conditions and microscopes is often required. In such a scenario, merging cryo-EM data sets is advantageous because it allows improved three-dimensional reconstructions to be obtained. Since data sets are not always collected with the same pixel size, merging data can be challenging. Here, two methods to combine cryo-EM data are de...
Source: Acta Crystallographica Section D - August 23, 2019 Category: Biochemistry Authors: Wilkinson, M.E. Kumar, A. Casa ñ al, A. Tags: cryo-EM merging of data single-particle processing RELION research papers Source Type: research

Structures of the substrate-binding protein YfeA in apo and zinc-reconstituted holo forms
In this report, structures are presented of substrate-free (apo) and reconstituted substrate-bound (holo) YfeA, a polyspecific cluster A-I SBP from Yersinia pestis. It is demonstrated that an apo cluster A-I SBP can be purified by fractionation when co-expressed with its cognate transporter, adding an alternative strategy to the mutagenesis or biochemical treatment used to generate other apo cluster A-I SBPs. The apo YfeA structure contains 111 disordered protein atoms in a mobile helix located in the flexible carboxy-terminal lobe. Metal binding triggers a 15-fold reduction in the solvent-accessible surface area of the me...
Source: Acta Crystallographica Section D - August 22, 2019 Category: Biochemistry Authors: Radka, C.D. Labiuk, S.L. DeLucas, L.J. Aller, S.G. Tags: substrate-binding protein cluster A-I YfeA Yersinia pestis substrate transfer X-ray crystallography research papers Source Type: research

Relationship between the induced-fit loop and the activity of Klebsiella pneumoniae pullulanase
Klebsiella pneumoniae pullulanase (KPP) belongs to glycoside hydrolase family 13 subfamily 13 (GH13_13) and is the only enzyme that is reported to perform an induced-fit motion of the active-site loop (residues 706 – 710). Comparison of pullulanase structures indicated that only KPP has Leu680 present behind the loop, in contrast to the glycine found in other GH13_13 members. Analysis of the structure and activity of recombinant pullulanase from K. pneumoniae ATCC 9621 (rKPP) and its mutant (rKPP-G680L) indicated that the side chain of residue 680 is important for the induced-fit motion of the loop 706 – 710 an...
Source: Acta Crystallographica Section D - August 22, 2019 Category: Biochemistry Authors: Saka, N. Malle, D. Iwamoto, H. Takahashi, N. Mizutani, K. Mikami, B. Tags: crystal structure pullulanase Klebsiella pneumoniae induced fit mutagenesis research papers Source Type: research

Crystal structure of the N-terminal domain of the major virulence factor BB0323 from the Lyme disease agent Borrelia burgdorferi
Lyme disease is an infection caused by the spirochete Borrelia burgdorferi after it is transmitted to a mammalian organism during a tick blood meal. B.   burgdorferi encodes at least 140 lipoproteins located on the outer or inner membrane, thus facing the surroundings or the periplasmic space, respectively. However, most of the predicted lipoproteins are of unknown function, and only a few proteins are known to be essential for the persistence and virulence of the pathogen. One such protein is the periplasmic BB0323, which is indispensable for B. burgdorferi to cause Lyme disease and the function of which is associate...
Source: Acta Crystallographica Section D - August 22, 2019 Category: Biochemistry Authors: Brangulis, K. Akopjana, I. Kazaks, A. Tars, K. Tags: lipoprotein spectrin superfamily cell fission Lyme disease X-ray crystallography Borrelia burgdorferi research papers Source Type: research

Calcium-ligand variants of the myocilin olfactomedin propeller selected from invertebrate phyla reveal cross-talk with N-terminal blade and surface helices
Olfactomedins are a family of modular proteins found in multicellular organisms that all contain five-bladed β -propeller olfactomedin (OLF) domains. In support of differential functions for the OLF propeller, the available crystal structures reveal that only some OLF domains harbor an internal calcium-binding site with ligands derived from a triad of residues. For the myocilin OLF domain (myoc-OLF), ablation of the ion-binding site (triad Asp, Asn, Asp) by altering the coordinating residues affects the stability and overall structure, in one case leading to misfolding and glaucoma. Bioinformatics analysis reveals a v...
Source: Acta Crystallographica Section D - August 22, 2019 Category: Biochemistry Authors: Hill, S.E. Cho, H. Raut, P. Lieberman, R.L. Tags: olfactomedins helix unwinding calcium ions propeller research papers Source Type: research

The subatomic resolution study of laccase inhibition by chloride and fluoride anions using single-crystal serial crystallography: insights into the enzymatic reaction mechanism
Laccases are enzymes that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of molecular oxygen to water. Here, a subatomic resolution X-ray crystallographic study of the mechanism of inhibition of the laccase from the basidiomycete fungus Steccherinum murashkinskyi by chloride and fluoride ions is presented. Three series of X-ray diffraction data sets were collected with increasing doses of absorbed X-ray radiation from a native S. murashkinskyi laccase crystal and from crystals of complexes of the laccase with chloride and fluoride ions. The data for the native laccas...
Source: Acta Crystallographica Section D - August 22, 2019 Category: Biochemistry Authors: Polyakov, K.M. Gavryushov, S. Fedorova, T.V. Glazunova, O.A. Popov, A.N. Tags: laccase inhibition single-crystal serial crystallography enzymatic oxygen reduction reaction mechanisms research papers Source Type: research

Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX
Human carbonic anhydrase IX (CA IX) expression is upregulated in hypoxic solid tumours, promoting cell survival and metastasis. This observation has made CA IX a target for the development of CA isoform-selective inhibitors. To enable structural studies of CA IX – inhibitor complexes using X-ray and neutron crystallography, a CA IX surface variant (CA IXSV; the catalytic domain with six surface amino-acid substitutions) has been developed that can be routinely crystallized. Here, the preparation of protiated (H/H), H/D-exchanged (H/D) and deuterated (D/D) CA IXSV for crystallographic studies and their structural comp...
Source: Acta Crystallographica Section D - August 22, 2019 Category: Biochemistry Authors: Koruza, K. Lafumat, B. Nyblom, M. Mahon, B.P. Knecht, W. McKenna, R. Fisher, S.Z. Tags: carbonic anhydrase proton transfer X-ray crystallography carbonic anhydrase IX neutron protein crystallography perdeuteration research papers Source Type: research

Structure determination of the CAMP factor of Streptococcus agalactiae with the aid of an MBP tag and insights into membrane-surface attachment
This study provides novel insights into the mechanism of the membrane-permeabilizing activity of CAMP factor. (Source: Acta Crystallographica Section D)
Source: Acta Crystallographica Section D - July 31, 2019 Category: Biochemistry Authors: Li, Y. Zeng, W. Li, Y. Fan, W. Ma, H. Fan, X. Jiang, J. Brefo-Mensah, E. Zhang, Y. Yang, M. Dong, Z. Palmer, M. Jin, T. Tags: group B streptococcus CAMP factor hemolytic mechanism MBP tag structure determination Streptococcus agalactiae research papers Source Type: research

Location-specific quantification of protein-bound metal ions by X-ray anomalous dispersion: Q-XAD
Here, a method is described which exploits X-ray anomalous dispersion (XAD) to quantify mixtures of metal ions in the binding sites of proteins and can be applied to metalloprotein crystals of average quality. This method has successfully been used to study site-specific metal binding in a protein from the R2-like ligand-binding oxidase family which assembles a heterodinuclear Mn/Fe cofactor. While previously only the relative contents of Fe and Mn in each metal-binding site have been assessed, here it is shown that the method can be extended to quantify the relative occupancies of at least three different transition metal...
Source: Acta Crystallographica Section D - July 31, 2019 Category: Biochemistry Authors: Griese, J.J. H ö gbom, M. Tags: metal quantification quantitative X-ray anomalous dispersion R2-like ligand-binding oxidase Q-XAD X-ray crystallography research papers Source Type: research

Sequence assignment for low-resolution modelling of protein crystal structures
The performance of automated model building in crystal structure determination usually decreases with the resolution of the experimental data, and may result in fragmented models and incorrect side-chain assignment. Presented here are new methods for machine-learning-based docking of main-chain fragments to the sequence and for their sequence-independent connection using a dedicated library of protein fragments. The combined use of these new methods noticeably increases sequence coverage and reduces fragmentation of the protein models automatically built with ARP/wARP. (Source: Acta Crystallographica Section D)
Source: Acta Crystallographica Section D - July 31, 2019 Category: Biochemistry Authors: Chojnowski, G. Pereira, J. Lamzin, V.S. Tags: model building sequence assignment ARP/wARP macromolecular crystallography loop building low-resolution modelling research papers Source Type: research

A novel structurally characterized haloacid dehalogenase superfamily phosphatase from Thermococcus thioreducens with diverse substrate specificity
This study focuses on a new hypothetical HAD phosphatase from Thermococcus thioreducens. The protein crystallized in space group P21212, with unit-cell parameters a = 66.3, b = 117.0, c = 33.8   Å , and the crystals contained one molecule in the asymmetric unit. The protein structure was determined by X-ray crystallography and was refined to 1.75   Å resolution. The structure revealed a putative active site common to all HAD members. Computational docking into the crystal structure was used to propose substrates of the enzyme. The activity of this thermophilic enzyme towards several of the selected substrat...
Source: Acta Crystallographica Section D - July 30, 2019 Category: Biochemistry Authors: Havlickova, P. Brinsa, V. Brynda, J. Pachl, P. Prudnikova, T. Mesters, J.R. Kascakova, B. Kuty, M. Pusey, M.L. Ng, J.D. Rezacova, P. Kuta Smatanova, I. Tags: HAD superfamily hypothetical phosphatase crystal structure docking phosphatase assay research papers Source Type: research

Biochemical and structural explorations of α -hydroxyacid oxidases reveal a four-electron oxidative decarboxylation reaction
p-Hydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes mandelate to benzoylformate. How the FMN-dependent oxidation is executed by Hmo remains unclear at the molecular level. A continuum of snapshots from crystal structures of Hmo and its mutants in complex with physiological/nonphysiological substrates, products and inhibitors provides a rationale for its substrate enantioselectivity/promiscuity, its active-site geometry/reactivity and its direct hydride-transfer mechanism. A single mutant, Y128F, that extends the two-electron oxidation reaction to a four-electron oxidative decarb...
Source: Acta Crystallographica Section D - July 30, 2019 Category: Biochemistry Authors: Yeh, H.-W. Lin, K.-H. Lyu, S.-Y. Li, Y.-S. Huang, C.-M. Wang, Y.-L. Shih, H.-W. Hsu, N.-S. Wu, C.-J. Li, T.-L. Tags: mandelate oxidase flavin mononucleotide oxidative decarboxylation hydride transfer α -hydroxyacids research papers Source Type: research

Structures of major pilins in Clostridium perfringens demonstrate dynamic conformational change
Pili in Gram-positive bacteria are flexible rod proteins associated with the bacterial cell surface, and they play important roles in the initial adhesion to host tissues and colonization. The pilus shaft is formed by the covalent polymerization of major pilins, catalyzed by sortases, a family of cysteine transpeptidases. Here, X-ray structures of the major pilins from Clostridium perfringens strains 13 and SM101 and of sortase from strain SM101 are presented with biochemical analysis to detect the formation of pili in vivo. The major pilin from strain 13 adopts an elongated structure to form noncovalently linked polymeric...
Source: Acta Crystallographica Section D - July 30, 2019 Category: Biochemistry Authors: Tamai, E. Katayama, S. Sekiya, H. Nariya, H. Kamitori, S. Tags: Clostridium perfringens conformational change major pilin sortase-mediated polymerization research papers Source Type: research

Prediction of models for ordered solvent in macromolecular structures by a classifier based upon resolution-independent projections of local feature data
Current software tools for the automated building of models for macromolecular X-ray crystal structures are capable of assembling high-quality models for ordered macromolecule and small-molecule scattering components with minimal or no user supervision. Many of these tools also incorporate robust functionality for modelling the ordered water molecules that are found in nearly all macromolecular crystal structures. However, no current tools focus on differentiating these ubiquitous water molecules from other frequently occurring multi-atom solvent species, such as sulfate, or the automated building of models for such specie...
Source: Acta Crystallographica Section D - July 30, 2019 Category: Biochemistry Authors: Jones, L. Tynes, M. Smith, P. Tags: PeakProbe solvent modelling electron-density analysis supervised learning decorrelation resolution data mining research papers Source Type: research

Crystal structures of the closed form of Mycobacterium tuberculosis dihydrofolate reductase in complex with dihydrofolate and antifolates
Tuberculosis is a disease caused by Mycobacterium tuberculosis and is the leading cause of death from a single infectious pathogen, with a high prevalence in developing countries in Africa and Asia. There still is a need for the development or repurposing of novel therapies to combat this disease owing to the long-term nature of current therapies and because of the number of reported resistant strains. Here, structures of dihydrofolate reductase from M.   tuberculosis (MtDHFR), which is a key target of the folate pathway, are reported in complex with four antifolates, pyrimethamine, cycloguanil, diaverdine and pemetre...
Source: Acta Crystallographica Section D - July 4, 2019 Category: Biochemistry Authors: Ribeiro, J.A. Chavez-Pacheco, S.M. de Oliveira, G.S. Silva, C.S. Giudice, J.H.P. Libreros-Z ú ñ iga, G.A. Dias, M.V.B. Tags: dihydrofolate reductase Mycobacterium tuberculosis antifolates crystal structure research papers Source Type: research

Combining random microseed matrix screening and the magic triangle for the efficient structure solution of a potential lysin from bacteriophage P68
In this study, the phasing molecule 5-amino-2,4,6-triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high-quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg-white lysozyme and the N-terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion-associated lysin or endolysin. (Sourc...
Source: Acta Crystallographica Section D - July 2, 2019 Category: Biochemistry Authors: Truong, J.Q. Panjikar, S. Shearwin-Whyatt, L. Bruning, J.B. Shearwin, K.E. Tags: crystallography seeding random microseed matrix screening I3C magic triangle phasing lysin research papers Source Type: research

Barbara Wharton Low (1920 – 2019)
(Source: Acta Crystallographica Section D)
Source: Acta Crystallographica Section D - June 28, 2019 Category: Biochemistry Authors: Glusker, J.P. Tags: obituaries Barbara Wharton Low Source Type: research

LAT1 (SLC7A5) and CD98hc (SLC3A2) complex dynamics revealed by single-particle cryo-EM
Solute carriers are a large class of transporters that play key roles in normal and disease physiology. Among the solute carriers, heteromeric amino-acid transporters (HATs) are unique in their quaternary structure. LAT1 – CD98hc, a HAT, transports essential amino acids and drugs across the blood – brain barrier and into cancer cells. It is therefore an important target both biologically and therapeutically. During the course of this work, cryo-EM structures of LAT1 – CD98hc in the inward-facing conformation and in either the substrate-bound or apo states were reported to 3.3 – 3.5   Å resol...
Source: Acta Crystallographica Section D - June 28, 2019 Category: Biochemistry Authors: Chiduza, G.N. Johnson, R.M. Wright, G.S.A. Antonyuk, S.V. Muench, S.P. Hasnain, S.S. Tags: transporters cryo-EM amino-acid transporters solute carriers LAT1 CD98hc SLC7A5 SLC3A2 research papers Source Type: research

ATP-specificity of succinyl-CoA synthetase from Blastocystis hominis
Succinyl-CoA synthetase (SCS) catalyzes the only step of the tricarboxylic acid cycle that leads to substrate-level phosphorylation. Some forms of SCS are specific for ADP/ATP or for GDP/GTP, while others can bind all of these nucleotides, generally with different affinities. The theory of `gatekeeper' residues has been proposed to explain the nucleotide-specificity. Gatekeeper residues lie outside the binding site and create specific electrostatic interactions with incoming nucleotides to determine whether the nucleotides can enter the binding site. To test this theory, the crystal structure of the nucleotide-binding doma...
Source: Acta Crystallographica Section D - June 26, 2019 Category: Biochemistry Authors: Huang, J. Nguyen, V.H. Hamblin, K.A. Maytum, R. van der Giezen, M. Fraser, M.E. Tags: nucleotide specificity ATP-grasp fold thermal stability acyl-CoA synthetase (ADP-forming) differential scanning fluorimetry research papers Source Type: research

Structures of the transcriptional regulator BgaR, a lactose sensor
The structure of BgaR, a transcriptional regulator of the lactose operon in Clostridium perfringens, has been solved by SAD phasing using a mercury derivative. BgaR is an exquisite sensor of lactose, with a binding affinity in the low-micromolar range. This sensor and regulator has been captured bound to lactose and to lactulose as well as in a nominal apo form, and was compared with AraC, another saccharide-binding transcriptional regulator. It is shown that the saccharides bind in the N-terminal region of a jelly-roll fold, but that part of the saccharide is exposed to bulk solvent. This differs from the classical AraC s...
Source: Acta Crystallographica Section D - June 26, 2019 Category: Biochemistry Authors: Newman, J. Caron, K. Nebl, T. Peat, T.S. Tags: transcriptional regulators BgaR Clostridium perfringens lactose sensors SAD phasing research papers Source Type: research

Crystal structures of the naturally fused CS and cytochrome b5 reductase (b5R) domains of Ncb5or reveal an expanded CS fold, extensive CS – b5R interactions and productive binding of the NAD(P)+ nicotinamide ring
Ncb5or (NADH-cytochrome b5 oxidoreductase), a cytosolic ferric reductase implicated in diabetes and neurological diseases, comprises three distinct domains, cytochrome b5 (b5) and cytochrome b5 reductase (b5R) domains separated by a CHORD – Sgt1 (CS) domain, and a novel 50-residue N-terminal region. Understanding how interdomain interactions in Ncb5or facilitate the shuttling of electrons from NAD(P)H to heme, and how the process compares with the microsomal b5 (Cyb5A) and b5R (Cyb5R3) system, is of interest. A high-resolution structure of the b5 domain (PDB entry 3lf5) has previously been reported, which exhibits su...
Source: Acta Crystallographica Section D - June 26, 2019 Category: Biochemistry Authors: Benson, D.R. Lovell, S. Mehzabeen, N. Galeva, N. Cooper, A. Gao, P. Battaile, K.P. Zhu, H. Tags: crystal structure CHORD – Sgt1 domain CS domain FAD NAD NADP Ncb5or research papers Source Type: research

Structure of the N-terminal domain of Euprosthenops australis dragline silk suggests that conversion of spidroin dope to spider silk involves a conserved asymmetric dimer intermediate
Spider silk is a biomaterial with exceptional mechanical toughness, and there is great interest in developing biomimetic methods to produce engineered spider silk-based materials. However, the mechanisms that regulate the conversion of spider silk proteins (spidroins) from highly soluble dope into silk are not completely understood. The N-terminal domain (NT) of Euprosthenops australis dragline silk protein undergoes conformational and quaternary-structure changes from a monomer at a pH above 7 to a homodimer at lower pH values. Conversion from the monomer to the dimer requires the protonation of three conserved glutamic a...
Source: Acta Crystallographica Section D - June 26, 2019 Category: Biochemistry Authors: Jiang, W. Askarieh, G. Shkumatov, A. Hedhammar, M. Knight, S.D. Tags: spider silk assembly pH relay spidroin N-terminal domain Euprosthenops australis research papers Source Type: research

Crystal structure determination of Pseudomonas stutzeri A1501 endoglucanase Cel5A: the search for a molecular basis for glycosynthesis in GH5_5 enzymes
In this study, recombinant Ps_Cel5A from Pseudomonas stutzeri A1501, a novel member of the GH5_5 subfamily, was expressed, purified and crystallized. Preliminary experiments confirmed the ability of Ps_Cel5A to catalyze transglycosylation with cellotriose as a substrate. The crystal structure revealed several structural determinants in and around the positive subsites, providing a molecular basis for a better understanding of the mechanisms that promote and favour synthesis rather than hydrolysis. In the positive subsites, two nonconserved positively charged residues (Arg178 and Lys216) were found to interact with cellobio...
Source: Acta Crystallographica Section D - June 6, 2019 Category: Biochemistry Authors: Dutoit, R. Delsaute, M. Collet, L. Vander Wauven, C. Van Elder, D. Berlemont, R. Richel, A. Galleni, M. Bauvois, C. Tags: glycosyl hydrolase family 5 cellulases β -1,4-endoglucanases TIM barrel Ps_Cel5A Pst_2494 Pseudomonas stutzeri transglycosylation research papers Source Type: research

Crystal structures and snapshots along the reaction pathway of human phosphoserine phosphatase
The equilibrium between phosphorylation and dephosphorylation is one of the most important processes that takes place in living cells. Human phosphoserine phosphatase (hPSP) is a key enzyme in the production of serine by the dephosphorylation of phospho-l-serine. It is directly involved in the biosynthesis of other important metabolites such as glycine and d-serine (a neuromodulator). hPSP is involved in the survival mechanism of cancer cells and has recently been found to be an essential biomarker. Here, three new high-resolution crystal structures of hPSP (1.5 – 2.0   Å ) in complexes with phosphoserine and...
Source: Acta Crystallographica Section D - June 4, 2019 Category: Biochemistry Authors: Haufroid, M. Mirgaux, M. Leherte, L. Wouters, J. Tags: phosphoserine phosphatase mechanism cancer research papers Source Type: research

Crystal structures of the recombinant β -factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics
Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain ( β FXIIaHis) with yields of ∼ 1 – 2   mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP- β FXIIaHis). Crystal structures were determined of MBP- β FXIIaHis in complex with the inhibitor d-Phe-Pro-Arg chloromethyl ketone (PPACK) and of β FXIIaHis in iso...
Source: Acta Crystallographica Section D - June 4, 2019 Category: Biochemistry Authors: Pathak, M. Manna, R. Li, C. Kaira, B.G. Hamad, B.K. Belviso, B.D. Bonturi, C.R. Dreveny, I. Fischer, P.M. Dekker, L.V. Oliva, M.L.V. Emsley, J. Tags: recombinant β -factor XIIa protease coagulation factor XII serine protease crystal structure inhibitor complex substrate recognition research papers Source Type: research

Michael G. Rossmann (1930 – 2019), pioneer in macromolecular and virus crystallography: scientist, mentor and friend
Michael George Rossmann, who made monumental contributions to science, passed away peacefully in West Lafayette, Indiana on 14 May 2019 at the age of 88, following a courageous five-year battle with cancer. Michael was born in Frankfurt, Germany on 30 July 1930. As a young boy, he emigrated to England with his mother just as World War II ignited. Michael was a highly innovative and energetic person, well known for his intensity, persistence and focus in pursuing his research goals. Michael was a towering figure in crystallography as a highly distinguished faculty member at Purdue University for 55 years. Michael made many ...
Source: Acta Crystallographica Section D - June 1, 2019 Category: Biochemistry Authors: Arnold, E. Wu, H. Johnson, J.E. Tags: obituaries Michael Rossmann Source Type: research

Structural and functional characterization of CMP-N-acetylneuraminate synthetase from Vibrio cholerae
Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional...
Source: Acta Crystallographica Section D - May 31, 2019 Category: Biochemistry Authors: Bose, S. Purkait, D. Joseph, D. Nayak, V. Subramanian, R. Tags: CMP-N-acetylneuraminate synthetase Vibrio cholerae sialic acid structure function research papers Source Type: research

Structural insight into a matured humanized monoclonal antibody HuA21 against HER2-overexpressing cancer cells
HER2, a member of the epidermal growth factor receptor (EGFR) family, has been associated with human breast, ovarian and gastric cancers. Anti-HER2 monoclonal antibodies (mAbs) have demonstrated clinical efficacy for HER2-overexpressing breast cancer. A chimeric antibody chA21 that specifically inhibits the growth of HER2-overexpressing cancer cells both in vitro and in vivo has previously been developed. To reduce a potential human anti-mouse immune response, the humanized antibody HuA21 was developed and was further subjected to affinity maturation by phage display on the basis of chA21. Here, the crystal structure of Hu...
Source: Acta Crystallographica Section D - May 31, 2019 Category: Biochemistry Authors: Wang, Z. Cheng, L. Guo, G. Cheng, B. Hu, S. Zhang, H. Zhu, Z. Niu, L. Tags: HER2-overexpressing cancer humanized antibody EGFR family cell signaling crystal structure research papers Source Type: research

Phosphorus Chemistry. The Role of Phosphorus in Prebiotic Chemistry. By Yufen Zhao, Yan Liu, Xiang Gao and Pengxiang Xu. DeGruyter, 2018. Pp. XVI+166. EUR 119.95 USD 137.99 GBP 109.00, hardcover, ISBN 978-3-11-056237-8.
(Source: Acta Crystallographica Section D)
Source: Acta Crystallographica Section D - May 28, 2019 Category: Biochemistry Authors: Jacquot, J.-P. Tags: book reviews phosphorous chemistry prebiotic chemisitry Source Type: research

Analysis of crystalline and solution states of ligand-free spermidine N-acetyltransferase (SpeG) from Escherichia coli
Spermidine N-acetyltransferase (SpeG) transfers an acetyl group from acetyl-coenzyme A to an N-terminal amino group of intracellular spermidine. This acetylation inactivates spermidine, reducing the polyamine toxicity that tends to occur under certain chemical and physical stresses. The structure of the SpeG protein from Vibrio cholerae has been characterized: while the monomer possesses a structural fold similar to those of other Gcn5-related N-acetyltransferase superfamily members, its dodecameric structure remains exceptional. In this paper, structural analyses of SpeG isolated from Escherichia coli are described. Like ...
Source: Acta Crystallographica Section D - May 28, 2019 Category: Biochemistry Authors: Filippova, E.V. Weigand, S. Kiryukhina, O. Wolfe, A.J. Anderson, W.F. Tags: Escherichia coli SpeG spermidine N-acetyltransferase GNAT family polyamine acetylation research papers Source Type: research

Structure of a hyperthermostable dimeric archaeal Rubisco from Hyperthermus butylicus
The crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the hyperthermophilic archaeon Hyperthermus butylicus is presented at 1.8   Å resolution. Previous structures of archaeal Rubisco have been found to assemble into decamers, and this oligomerization was thought to be required for a highly thermally stable enzyme. In the current study, H.   butylicus Rubisco is shown to exist as a dimer in solution, yet has a thermal denaturation midpoint of 114 ° C, suggesting that high thermal stability can be achieved without an increased oligomeric state. This increased thermal stabil...
Source: Acta Crystallographica Section D - May 28, 2019 Category: Biochemistry Authors: Bundela, R. Keown, J. Watkin, S. Pearce, F.G. Tags: ribulose-1,5-bisphosphate carboxylase oxygenase Rubisco Hyperthermus butylicus thermostability oligomerization research papers Source Type: research

CM01: a facility for cryo-electron microscopy at the European Synchrotron
Recent improvements in direct electron detectors, microscope technology and software provided the stimulus for a `quantum leap' in the application of cryo-electron microscopy in structural biology, and many national and international centres have since been created in order to exploit this. Here, a new facility for cryo-electron microscopy focused on single-particle reconstruction of biological macromolecules that has been commissioned at the European Synchrotron Radiation Facility (ESRF) is presented. The facility is operated by a consortium of institutes co-located on the European Photon and Neutron Campus and is managed...
Source: Acta Crystallographica Section D - May 28, 2019 Category: Biochemistry Authors: Kandiah, E. Giraud, T. de Maria Antolinos, A. Dobias, F. Effantin, G. Flot, D. Hons, M. Schoehn, G. Susini, J. Svensson, O. Leonard, G.A. Mueller-Dieckmann, C. Tags: cryo-TEM ESRF PSB cryo-EM platform CM01 feature articles Source Type: research

Analysis and validation of macromolecular B values
This paper describes a global analysis of macromolecular B values. It is shown that the distribution of B values generally follows the shifted inverse-gamma distribution (SIGD). The parameters of the SIGD are estimated using the Fisher scoring technique with the expected Fisher information matrix. It is demonstrated that a contour plot based on the parameters of the SIGD can play a role in the validation of macromolecular structures. The dependence of the peak-height distribution on resolution and atomic B values is also analysed. It is demonstrated that the B-value distribution can have a dramatically different effect on ...
Source: Acta Crystallographica Section D - April 30, 2019 Category: Biochemistry Authors: Masmaliyeva, R.C. Murshudov, G.N. Tags: macromolecular structures B values inverse-gamma distribution maximum-likelihood estimation research papers Source Type: research

Visualizing membrane trafficking through the electron microscope: cryo-tomography of coat complexes
Coat proteins mediate vesicular transport between intracellular compartments, which is essential for the distribution of molecules within the eukaryotic cell. The global arrangement of coat proteins on the membrane is key to their function, and cryo-electron tomography and subtomogram averaging have been used to study membrane-bound coat proteins, providing crucial structural insight. This review outlines a workflow for the structural elucidation of coat proteins, incorporating recent developments in the collection and processing of cryo-electron tomography data. Recent work on coat protein I, coat protein II and retromer ...
Source: Acta Crystallographica Section D - April 30, 2019 Category: Biochemistry Authors: Markova, E.A. Zanetti, G. Tags: cryo-electron tomography three-dimensional reconstruction coat proteins vesicular transport COPII subtomogram averaging research papers Source Type: research

Findable Accessible Interoperable Re-usable (FAIR) diffraction data are coming to protein crystallography
(Source: Acta Crystallographica Section D)
Source: Acta Crystallographica Section D - April 30, 2019 Category: Biochemistry Authors: Helliwell, J.R. Minor, W. Weiss, M.S. Garman, E.F. Read, R.J. Newman, J. van Raaij, M.J. Hajdu, J. Baker, E.N. Tags: FAIR diffraction data IUCr policy editorial Source Type: research

Sir Aaron Klug OM FRS (1926 – 2018)
(Source: Acta Crystallographica Section D)
Source: Acta Crystallographica Section D - April 29, 2019 Category: Biochemistry Authors: Crowther, R.A. Tags: Sir Aaron Klug structural molecular biology obituaries Source Type: research

Structural analysis of a replication protein encoded by a plasmid isolated from a multiple sclerosis patient
In this study, a Rep WH1 domain encoded on a BMMF (MSBI1.176) isolated from a multiple sclerosis human brain sample was determined to 1.53   Å resolution using X-ray crystallography. The overall structure of the MSBI1.176 WH1 domain was remarkably similar to other Rep structures, despite having a low (28%) amino-acid sequence identity. The MSBI1.176 WH1 domain contained elements common to other Reps, including five α -helices, five β -strands and a hydrophobic pocket. These new findings suggest that the MSBI1.176 Rep might have comparable roles and functions to other known Reps of different origins. (Sou...
Source: Acta Crystallographica Section D - April 29, 2019 Category: Biochemistry Authors: Kilic, T. Popov, A.N. Burk-K ö rner, A. Koromyslova, A. zur Hausen, H. Bund, T. Hansman, G.S. Tags: bovine meat and milk factors episomal circular DNA trans-acting replication initiator protein multiple sclerosis X-ray crystal structure research papers Source Type: research

Crystal structure of the leucine-rich repeat ectodomain of the plant immune receptor kinase SOBIR1
Plant-unique membrane receptor kinases with leucine-rich repeat (LRR) extracellular domains are key regulators of development and immune responses. Here, the 1.55   Å resolution crystal structure of the immune receptor kinase SOBIR1 from Arabidopsis is presented. The ectodomain structure reveals the presence of five LRRs sandwiched between noncanonical capping domains. The disulfide-bond-stabilized N-terminal cap harbours an unusual β -hairpin structure. The C-terminal cap features a highly positively charged linear motif which was found to be largely disordered in this structure. Size-exclusion chromatography...
Source: Acta Crystallographica Section D - April 29, 2019 Category: Biochemistry Authors: Hohmann, U. Hothorn, M. Tags: leucine-rich repeat ectodomain receptor kinase plant immune signalling Arabidopsis cell signalling membrane receptor research papers Source Type: research

Structural and functional insights into phosphomannose isomerase: the role of zinc and catalytic residues
Phosphomannose isomerase (PMI) is a housekeeping enzyme that is found in organisms ranging from bacteria to fungi to mammals and is important for cell-wall synthesis, viability and signalling. PMI is a zinc-dependent enzyme that catalyses the reversible isomerization between mannose 6-phosphate (M6P) and fructose 6-phosphate (F6P), presumably via the formation of a cis-enediol intermediate. The reaction is hypothesized to involve ring opening of M6P, the transfer of a proton from the C2 atom to the C1 atom and between the O1 and O2 atoms of the substrate, followed by ring closure resulting in the product F6P. Several attem...
Source: Acta Crystallographica Section D - April 29, 2019 Category: Biochemistry Authors: Bangera, M. Gowda K, G. Sagurthi, S.R. Murthy, M.R.N. Tags: phosphomannose isomerase zinc binding mechanism of isomerization catalytic base research papers Source Type: research

Announcing mandatory submission of PDBx/mmCIF format files for crystallographic depositions to the Protein Data Bank (PDB)
(Source: Acta Crystallographica Section D)
Source: Acta Crystallographica Section D - April 8, 2019 Category: Biochemistry Authors: Adams, P.D. Afonine, P.V. Baskaran, K. Berman, H.M. Berrisford, J. Bricogne, G. Brown, D.G. Burley, S.K. Chen, M. Feng, Z. Flensburg, C. Gutmanas, A. Hoch, J.C. Ikegawa, Y. Kengaku, Y. Krissinel, E. Kurisu, G. Liang, Y. Liebschner, D. Mak, L. Markley, J.L Tags: PDB mmCIF OneDep wwPDB data dictionary data archiving biocuration validation macromolecular crystallography data standards PDBx/mmCIF format Protein Data Bank Worldwide Protein Data Bank letters to the editor Source Type: research

Protein Tyrosine Phosphatases. Structure, Signaling and Drug Discovery. Edited by L. G. Ahuja. De Gruyter, 2018. Pp. 282. Price (hardback) 139.95 EUR, 160.99 USD, 127 GBP. ISBN 978-3-11-042643-4.
(Source: Acta Crystallographica Section D)
Source: Acta Crystallographica Section D - April 8, 2019 Category: Biochemistry Authors: Jacquot, J.-P. Tags: book reviews protein tyrosine phosphatases Source Type: research