An assessment of three human methylenetetrahydrofolate dehydrogenase/cyclohydrolase – ligand complexes following further refinement
The enzymes involved in folate metabolism are key drug targets for cell-growth modulation, and accurate crystallographic structures provide templates to be exploited for structure-based ligand design. In this context, three ternary complex structures of human methylenetetrahydrofolate dehydrogenase/cyclohydrolase have been published [Schmidt et al. (2000), Biochemistry, 39, 6325 – 6335] and potentially represent starting points for the development of new antifolate inhibitors. However, an inspection of the models and the deposited data revealed deficiencies and raised questions about the validity of the structures. A...
Source: Acta Crystallographica Section F - February 20, 2019 Category: Biochemistry Authors: Bueno, R. Dawson, A. Hunter, W.N. Tags: methylenetetrahydrofolate dehydrogenase methenyltetrahydrofolate cyclohydrolase antifolates structure-based ligand design structure validation re-refinement research communications Source Type: research

Crystal structure of the aromatic-amino-acid aminotransferase from Streptococcus mutans
In this study, the aromatic-amino-acid aminotransferase from Streptococcus mutans (SmAroAT) was recombinantly expressed in Escherichia coli. An effective purification protocol was established. The recombinant protein was crystallized using the hanging-drop vapor-diffusion method with PEG 3350 as the primary precipitant. The crystal structure of SmAroAT was solved at 2.2   Å resolution by the molecular-replacement method. Structural analysis indicated that the proteins of the aromatic-amino-acid aminotransferase family have conserved structural elements that might play a role in substrate binding. These results may ...
Source: Acta Crystallographica Section F - January 24, 2019 Category: Biochemistry Authors: Cong, X. Li, X. Li, S. Tags: Streptococcus mutans dental caries dental plaque infective endocarditis aromatic-amino-acid aminotransferase crystallization structure determination research communications Source Type: research

Penetration of dyes into protein crystals
Experiments were carried out on 15 different protein crystals with the objective of estimating the rates of penetration of dye molecules into the crystals. The dyes were in the molecular-weight range 250 – 1000   Da and the protein crystals were of dimensions of 0.7   mm or greater. Experiments were also conducted on protein crystals grown between glass cover slips (separation 200   µ m) that restricted the direction of diffusion. The rate of penetration of dyes into protein crystals depends very much on the degree of association between the dye and protein molecules. Dye penetration was not consistent wi...
Source: Acta Crystallographica Section F - January 24, 2019 Category: Biochemistry Authors: McPherson, A. Tags: penetration rates diffusion rates stains microscopy color time lapse research communications Source Type: research

Iterative screen optimization maximizes the efficiency of macromolecular crystallization
Advances in X-ray crystallography have streamlined the process of determining high-resolution three-dimensional macromolecular structures. However, a rate-limiting step in this process continues to be the generation of crystals that are of sufficient size and quality for subsequent diffraction experiments. Here, iterative screen optimization (ISO), a highly automated process in which the precipitant concentrations of each condition in a crystallization screen are modified based on the results of a prior crystallization experiment, is described. After designing a novel high-throughput crystallization screen to take full adv...
Source: Acta Crystallographica Section F - January 24, 2019 Category: Biochemistry Authors: Jones, H.G. Wrapp, D. Gilman, M.S.A. Battles, M.B. Wang, N. Sacerdote, S. Chuang, G.-Y. Kwong, P.D. McLellan, J.S. Tags: automated liquid handling crystallization screening macromolecular crystallography Sweet16 research communications Source Type: research

High-resolution structure of a Y27W mutant of the Dishevelled2 DIX domain
Dishevelled (Dvl) is a positive regulator of the canonical Wnt pathway that downregulates the phosphorylation of β -catenin and its subsequent degradation. Dvl contains an N-terminal DIX domain, which is involved in its homooligomerization and interactions with regulators of the Wnt pathway. The crystal structure of a Y27W mutant of the Dishevelled2 DIX domain (DIX-Y27W) has been determined at 1.64   Å resolution. DIX-Y27W has a compact ubiquitin-like fold and self-associates with neighbouring molecules through β -bridges, resulting in a head-to-tail helical molecular arrangement similar to previously rep...
Source: Acta Crystallographica Section F - January 23, 2019 Category: Biochemistry Authors: Yamanishi, K. Sin, Y. Terawaki, S. Higuchi, Y. Shibata, N. Tags: Wnt signalling pathway Dishevelled DIX domain X-ray structure analysis polymerizing domain research communications Source Type: research

Titration of ionizable groups in proteins using multiple neutron data sets from a single crystal: application to the small GTPase Ras
Neutron protein crystallography (NPC) reveals the three-dimensional structures of proteins, including the positions of H atoms. The technique is particularly suited to elucidate ambiguous catalytic steps in complex biochemical reactions. While NPC uniquely complements biochemical assays and X-ray structural analyses by revealing the protonation states of ionizable groups at and around the active site of enzymes, the technique suffers from a major drawback: large single crystals must be grown to compensate for the relatively low flux of neutron beams. However, in addition to revealing the positions of hydrogens involved in ...
Source: Acta Crystallographica Section F - January 23, 2019 Category: Biochemistry Authors: Knihtila, R. Volmar, A.Y. Meilleur, F. Mattos, C. Tags: in crystallo titration neutron crystallography pKa protonation radiation damage research communications Source Type: research

Crystallization and X-ray analysis of monodisperse human properdin
The 54   kDa protein properdin, also known as factor P (FP), plays a major role in the complement system through the stabilization of the alternative pathway convertases. FP circulates in the blood as cyclic dimers, trimers and tetramers, and this heterogeneity challenges detailed structural insight into the mechanism of convertase stabilization by FP. Here, the generation of an intact FP monomer and a variant monomer with the third thrombospondin repeat liberated is described. Both FP monomers were excised from recombinant full-length FP containing internal cleavage sites for TEV protease. These FP monomers could be cry...
Source: Acta Crystallographica Section F - January 23, 2019 Category: Biochemistry Authors: Pedersen, D.V. Revel, M. Gadeberg, T.A.F. Andersen, G.R. Tags: complement properdin modular proteins crystallization research communications Source Type: research

A revisited version of the apo structure of the ligand-binding domain of the human nuclear receptor retinoic X receptor α
The retinoic X receptor (RXR) plays a crucial role in the superfamily of nuclear receptors (NRs) by acting as an obligatory partner of several nuclear receptors; its role as a transcription factor is thus critical in many signalling pathways, such as metabolism, cell development, differentiation and cellular death. The first published structure of the apo ligand-binding domain (LBD) of RXR α , which is still used as a reference today, contained inaccuracies. In the present work, these inaccuracies were corrected using modern crystallographic tools. The most important correction concerns the presence of a π -bulge ...
Source: Acta Crystallographica Section F - January 23, 2019 Category: Biochemistry Authors: Eberhardt, J. McEwen, A.G. Bourguet, W. Moras, D. Dejaegere, A. Tags: nuclear receptors retinoic acid ligand binding domains retinoic X receptor α research communications Source Type: research

Structure of an Influenza A virus N9 neuraminidase with a tetrabrachion-domain stalk
The influenza neuraminidase (NA) is a homotetramer with head, stalk, transmembrane and cytoplasmic regions. The structure of the NA head with a stalk has never been determined. The NA head from an N9 subtype influenza A virus, A/tern/Australia/G70C/1975 (H1N9), was expressed with an artificial stalk derived from the tetrabrachion (TB) tetramerization domain from Staphylothermus marinus. The NA was successfully crystallized both with and without the TB stalk, and the structures were determined to 2.6 and 2.3   Å resolution, respectively. Comparisons of the two NAs with the native N9 NA structure from egg-grown virus...
Source: Acta Crystallographica Section F - January 23, 2019 Category: Biochemistry Authors: Streltsov, V.A. Schmidt, P.M. McKimm-Breschkin, J.L. Tags: Influenza A virus neuraminidases neuraminidase stalk tetrabrachion tetramerization domain N9 neuraminidase structure research communications Source Type: research

Structures of the antibody 64M-5 Fab and its complex with dT(6 – 4)T indicate induced-fit and high-affinity mechanisms
DNA photoproducts with (6 – 4) pyrimidine – pyrimidone adducts produced by ultraviolet light are mutagenic and carcinogenic. The crystal structures of the anti-(6 – 4) photoproduct antibody 64M-5 Fab and of its complex with dT(6 – 4)T were determined at 2.5 and 2.0   Å resolution, respectively. A comparison between the dT(6 – 4)T-liganded and unliganded structures indicates that the side chain of His93L is greatly rotated and shifted on binding to dT(6 – 4)T, leading to the formation of an electrostatic interaction with the phosphate moiety of dT(6 – 4)T, which shows a rema...
Source: Acta Crystallographica Section F - January 23, 2019 Category: Biochemistry Authors: Yokoyama, H. Mizutani, R. Noguchi, S. Hayashida, N. Tags: antibodies Fabs crystal structure DNA (6 – 4) photoproduct ultraviolet light research communications Source Type: research

Crystal structure of the programmed cell death 5 protein from Sulfolobus solfataricus
Programmed cell death 5 (PDCD5) is a vital signaling protein in the apoptosis pathway in eukaryotes. It is known that there are two dissociated N-terminal regions and a triple-helix core in eukaryotic PDCD5. Structural and functional studies of PDCD5 from hyperthermophilic archaea have been limited to date. Here, the PDCD5 homolog Sso0352 (SsoPDCD5) was identified in Sulfolobus solfataricus, the SsoPDCD5 protein was expressed and crystallized, and the phase was identified by single-wavelength anomalous diffraction. The native SsoPDCD5 crystal belonged to space group C2 and diffracted to 1.49   Å resolution. This is...
Source: Acta Crystallographica Section F - January 23, 2019 Category: Biochemistry Authors: Lin, K.-F. Hsu, J.-Y. Hsieh, D.-L. Tsai, M.-J. Yeh, C.-H. Chen, C.-Y. Tags: Sulfolobus solfataricus DNA-binding protein programmed cell death X-ray crystallography PDCDS research communications Source Type: research

Visualization of biological macromolecules at near-atomic resolution: cryo-electron microscopy comes of age
Structural biology is going through a revolution as a result of transformational advances in the field of cryo-electron microscopy (cryo-EM) driven by the development of direct electron detectors and ultrastable electron microscopes. High-resolution cryo-EM images of isolated biomolecules (single particles) suspended in a thin layer of vitrified buffer are subjected to powerful image-processing algorithms, enabling near-atomic resolution structures to be determined in unprecedented numbers. Prior to these advances, electron crystallography of two-dimensional crystals and helical assemblies of proteins had established the f...
Source: Acta Crystallographica Section F - December 24, 2018 Category: Biochemistry Authors: Mitra, A.K. Tags: cryo-EM single-particle analysis three-dimensional reconstruction phase plates direct detectors topical reviews Source Type: research

Acta Crystallographica Section F – another home for cryo-electron microscopy contributions
(Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - December 24, 2018 Category: Biochemistry Authors: Mitra, A.K. van Raaij, M. Tags: cryo-EM introduction editorial Source Type: research

AtNPR4 from Arabidopsis thaliana: expression, purification, crystallization and crystallographic analysis
In this study, Spodoptera frugiperda (Sf9) cells were used to express full-length AtNPR4 from Arabidopsis thaliana. To facilitate crystallization, T4 lysozyme (T4L) was added to the N-terminus of the AtNPR4 protein. The recombinant T4L-AtNPR4 protein was expressed, purified and crystallized using the sitting-drop and hanging-drop vapour-diffusion methods. The T4L-AtNPR4 crystals have symmetry consistent with space group C2, with unit-cell parameters a = 93.7, b = 85.8, c = 88.2   Å , β = 90 ° and one molecule per asymmetric unit. The best crystal diffracted to a resolution of 2.75   Å . Structure d...
Source: Acta Crystallographica Section F - December 22, 2018 Category: Biochemistry Authors: Yang, Q. Zhang, M. Zheng, J. Jia, Z. Tags: salicylic acid systemic acquired resistance protein purification regulatory mechanism crystal diffraction Arabidopsis thaliana research communications Source Type: research

The redefined DNA-binding domain of human xeroderma pigmentosum complementation group A: production, crystallization and structure solution
Human xeroderma pigmentosum complementation group A (XPA) is a scaffold protein that plays significant roles in DNA-damage verification and in recruiting downstream endonucleases to facilitate the repair of DNA lesions in nucleotide-excision repair. XPA98 – 219 (residues 98 – 219) has been identified as a DNA-binding domain and has been extensively studied in the last two decades. However, the most recent studies have redefined the DNA-binding domain as XPA98 – 239 (residues 98 – 239); it exerts a remarkably higher DNA-binding affinity than XPA98 – 219 and has a binding affinity that is quite ...
Source: Acta Crystallographica Section F - December 22, 2018 Category: Biochemistry Authors: Lian, F.-M. Yang, X. Yang, W. Jiang, Y.-L. Qian, C. Tags: XPA DNA-binding domain nucleotide-excision repair research communications Source Type: research

The structure and activity of the glutathione reductase from Streptococcus pneumoniae
The glutathione reductase (GR) from Streptococcus pneumoniae is a flavoenzyme that catalyzes the reduction of oxidized glutathione (GSSG) to its reduced form (GSH) in the cytoplasm of this bacterium. The maintenance of an intracellular pool of GSH is critical for the detoxification of reactive oxygen and nitrogen species and for intracellular metal tolerance to ions such as zinc. Here, S. pneumoniae GR (SpGR) was overexpressed and purified and its crystal structure determined at 2.56   Å resolution. SpGR shows overall structural similarity to other characterized GRs, with a dimeric structure that includes an antipa...
Source: Acta Crystallographica Section F - December 22, 2018 Category: Biochemistry Authors: Sikanyika, M. Arag ã o, D. McDevitt, C.A. Maher, M.J. Tags: glutathione reductase X-ray crystallography Streptococcus pneumoniae research communications Source Type: research

The X-ray crystal structure of human endothelin 1, a polypeptide hormone regulator of blood pressure
Human endothelin is a 21-amino-acid polypeptide, constrained by two intra-chain disulfide bridges, that is made by endothelial cells. It is the most potent vasoconstrictor in the body and is crucially important in the regulation of blood pressure. It plays a major role in a host of medical conditions, including hypertension, diabetes, stroke and cancer. Endothelin was crystallized 28 years ago in the putative space group P6122, but the structure was never successfully solved by X-ray diffraction. Using X-ray diffraction data from 1992, the structure has now been solved. Assuming a unit cell belonging to space group P61 and...
Source: Acta Crystallographica Section F - December 22, 2018 Category: Biochemistry Authors: McPherson, A. Larson, S.B. Tags: endothelin blood pressure vasoconstrictors polypeptide hormones twinned crystals crystallographic archeology research communications Source Type: research

Never too late for endothelin
(Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - December 22, 2018 Category: Biochemistry Authors: Stanfield, R.L. Tags: endothelin archiving raw data scientific commentaries Source Type: research

Single-particle reconstruction statistics: a diagnostic tool in solving biomolecular structures by cryo-EM
This study revisits the theory of 3D reconstruction and demonstrates how the associated statistics can provide a diagnostic tool to improve SPA. Small numbers of images already give sufficient information on micrograph quality and the amount of data required to reach high resolution. Such feedback allows the microscopist to improve sample-preparation and imaging parameters before committing to extensive data collection. Once a larger data set is available, a B factor can be determined describing the suppression of the signal owing to one or more causes, such as specimen movement, radiation damage, alignment inaccuracy and ...
Source: Acta Crystallographica Section F - December 22, 2018 Category: Biochemistry Authors: Heymann, J.B. Tags: cryo-EM cryo-electron microscopy spectral signal-to-noise ratio single-particle analysis Fourier shell correlation image processing research communications Source Type: research

Survey of the analysis of continuous conformational variability of biological macromolecules by electron microscopy
Single-particle analysis by electron microscopy is a well established technique for analyzing the three-dimensional structures of biological macromolecules. Besides its ability to produce high-resolution structures, it also provides insights into the dynamic behavior of the structures by elucidating their conformational variability. Here, the different image-processing methods currently available to study continuous conformational changes are reviewed. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - December 22, 2018 Category: Biochemistry Authors: Sorzano, C.O.S. Jim é nez, A. Mota, J. Vilas, J.L. Maluenda, D. Mart í nez, M. Ram í rez-Aportela, E. Majtner, T. Segura, J. S á nchez-Garc í a, R. Rancel, Y. del Ca ñ o, L. Conesa, P. Melero, R. Jonic, S. Vargas, J. Cazals, F. Freyberg, Z. Krieger, Tags: electron microscopy image processing continuous heterogeneity single-particle analysis normal-mode analysis research communications Source Type: research

On cross-correlations, averages and noise in electron microscopy
Biological samples are radiation-sensitive and require imaging under low-dose conditions to minimize damage. As a result, images contain a high level of noise and exhibit signal-to-noise ratios that are typically significantly smaller than 1. Averaging techniques, either implicit or explicit, are used to overcome the limitations imposed by the high level of noise. Averaging of 2D images showing the same molecule in the same orientation results in highly significant projections. A high-resolution structure can be obtained by combining the information from many single-particle images to determine a 3D structure. Similarly, a...
Source: Acta Crystallographica Section F - December 21, 2018 Category: Biochemistry Authors: Radermacher, M. Ruiz, T. Tags: image processing signal-to-noise ratio cross-correlation multireference alignment 3D reference-based projection alignment research communications Source Type: research

The structure of SDS22 provides insights into the mechanism of heterodimer formation with PP1
Protein phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets by associating with nearly 200 regulatory proteins to form highly specific holoenzymes. The vast majority of regulators are intrinsically disordered proteins (IDPs) and bind PP1 via short linear motifs within their intrinsically disordered regions. One of the most ancient PP1 regulators is SDS22, a protein that is conserved from yeast to mammals. Sequence analysis of SDS22 revealed that it is a leucine-rich repeat (LRR) protein, suggesting that SDS22, unlike nearly every other known PP1 regulator, is not an IDP but instead is fully structured. ...
Source: Acta Crystallographica Section F - November 30, 2018 Category: Biochemistry Authors: Choy, M.S. Bolik-Coulon, N. Archuleta, T.L. Peti, W. Page, R. Tags: SDS22 protein phosphatase 1 LRR protein PP1 regulator research communications Source Type: research

Crystal structure of the Agrobacterium tumefaciens type VI effector – immunity complex
The type VI secretion system (T6SS) comprises needle-shaped multisubunit complexes that play a role in the microbial defense systems of Gram-negative bacteria. Some Gram-negative bacteria harboring a T6SS deliver toxic effector proteins into the cytoplasm or periplasm of competing bacteria in order to lyse and kill them. To avoid self-cell disruption, these bacteria have cognate immunity proteins that inhibit their toxic effector proteins. T6SS amidase effector protein 4 (Tae4) and T6SS amidase immunity protein 4 (Tai4) are a representative of the toxic effector – immunity pairs of the T6SS. Here, the three-dimension...
Source: Acta Crystallographica Section F - November 30, 2018 Category: Biochemistry Authors: Fukuhara, S. Nakane, T. Yamashita, K. Ishii, R. Ishitani, R. Nureki, O. Tags: Agrobacterium tumefaciens type VI effector – immunity complex crystal structure Tae4 Tai4 research communications Source Type: research

The crystal structure of Mycobacterium tuberculosis high-temperature requirement A protein reveals an autoregulatory mechanism
The crystal structure of Mycobacterium tuberculosis high-temperature requirement A (HtrA) protein was determined at 1.83   Å resolution. This membrane-associated protease is essential for the survival of M. tuberculosis. The crystal structure reveals that interactions between the PDZ domain and the catalytic domain in HtrA lead to an inactive conformation. This finding is consistent with its proposed role as a regulatory protease that is conditionally activated upon appropriate environmental triggers. The structure provides a basis for directed studies to evaluate the role of this essential protein and the regulato...
Source: Acta Crystallographica Section F - November 29, 2018 Category: Biochemistry Authors: Gupta, A.K. Behera, D. Gopal, B. Tags: regulated proteolysis high-temperature requirement A protein PDZ domain Mycobacterium tuberculosis research communications Source Type: research

An inexpensive system for imaging the contents of multi-well plates
An inexpensive system for automated imaging of the contents of 12-, 24- and 96-well plates has been built. The xyz stage is constructed from parts from a light-duty computer numerical control wood-carving/engraving machine, and the Arduino-based board was wired so that it can trigger still images or movies though a microscope-mounted digital camera. The translation stage provides reproducible three-dimensional movement of the sample over a volume of 160   mm in x, 100   mm in y and 40   mm in z. A Python script generates the G-code command file that scans the plate and collects a series of z-stacked images of each sa...
Source: Acta Crystallographica Section F - November 29, 2018 Category: Biochemistry Authors: Bohm, A. Tags: automated microscope imaging system multi-well plates research communications Source Type: research

Structural studies of a glycoside hydrolase family 3 β -glucosidase from the model fungus Neurospora crassa
The glycoside hydrolase family 3 (GH3) β -glucosidases are a structurally diverse family of enzymes. Cel3A from Neurospora crassa (NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. β -Glucosidases hydrolyse the β -1,4 bond at the nonreducing end of cellodextrins. The hydrolysis of cellobiose is of special importance as its accumulation inhibits other cellulases acting on crystalline cellulose. Here, the crystal structure of the biologically relevant dimeric form of NcCel3A is reported. The structure has been refined to 2.25   Å resolution, with an Rcr...
Source: Acta Crystallographica Section F - November 26, 2018 Category: Biochemistry Authors: Karkehabadi, S. Hansson, H. Mikkelsen, N.E. Kim, S. Kaper, T. Sandgren, M. Gudmundsson, M. Tags: glycoside hydrolase family 3 β -glucosidase biodegradation crystal structure Neurospora crassa NcCel3A research communications Source Type: research

Crystal structure of the dimethylsulfide monooxygenase DmoA from Hyphomicrobium sulfonivorans
DmoA is a monooxygenase which uses dioxygen (O2) and reduced flavin mononucleotide (FMNH2) to catalyze the oxidation of dimethylsulfide (DMS). Although it has been characterized, the structure of DmoA remains unknown. Here, the crystal structure of DmoA was determined to a resolution of 2.28   Å and was compared with those of its homologues LadA and BdsA. The results showed that their overall structures are similar: they all share a conserved TIM-barrel fold which is composed of eight α -helices and eight β -strands. In addition, they all have five additional insertions. Detailed comparison showed that t...
Source: Acta Crystallographica Section F - November 26, 2018 Category: Biochemistry Authors: Cao, H.-Y. Wang, P. Peng, M. Shao, X. Chen, X.-L. Li, C.-Y. Tags: dimethylsulfide monooxygenase sulfur cycle TIM-barrel fold substrate-binding pocket Hyphomicrobium sulfonivorans research communications Source Type: research

Structures of endo-1,5- α -l-arabinanase mutants from Bacillus thermodenitrificans TS-3 in complex with arabino-oligosaccharides
In this study, the crystal structures of inactive ABN-TS mutants, D27A and D147N, were determined in complex with arabino-oligosaccharides. The crystal structures revealed that ABN-TS has at least six subsites in the deep V-shaped cleft formed across one face of the propeller structure. The structural features indicate that substrate recognition is profoundly influenced by the remote subsites as well as by the subsites surrounding the active center. The `open' structure of the substrate-binding cleft of the endo-acting ABN-TS is suitable for the random binding of several sugar units in polymeric substrates. (Source: Acta C...
Source: Acta Crystallographica Section F - November 26, 2018 Category: Biochemistry Authors: Yamaguchi, A. Sogabe, Y. Fukuoka, S. Sakai, T. Tada, T. Tags: arabinanases endo-acting enzymes biofuels Bacillus thermodenitrificans endo-1,5- α -l-arabinanase mutants crystal structure – substrate complex glycoside hydrolase family 43 research communications Source Type: research

Neutron and X-ray crystal structures of Lactobacillus brevis alcohol dehydrogenase reveal new insights into hydrogen-bonding pathways
Lactobacillus brevis alcohol dehydrogenase (LbADH) is a well studied homotetrameric enzyme which catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. LbADH is stable and enzymatically active at elevated temperatures and accepts a broad range of substrates, making it a valuable tool in industrial biocatalysis. Here, the expression, purification and crystallization of LbADH to generate large, single crystals with a volume of up to 1   mm3 suitable for neutron diffraction studies are described. Neutron diffraction data were collected from an H/D-exchanged LbADH crystal using...
Source: Acta Crystallographica Section F - November 26, 2018 Category: Biochemistry Authors: Hermann, J. Nowotny, P. Schrader, T.E. Biggel, P. Hekmat, D. Weuster-Botz, D. Tags: short-chain dehydrogenases/reductases protein crystallization neutron diffraction hydrogen-bonding network Lactobacillus brevis alcohol dehydrogenase research communications Source Type: research

X-ray crystallographic analysis of the catalytic domain of α -1,3-glucanase FH1 from Paenibacillus glycanilyticus overexpressed in Brevibacillus choshinensis
In this study, the recombinant catalytic domain of α -1,3-glucanase FH1 from Paenibacillus glycanilyticus FH11, which is classified into glycoside hydrolase family 87, was prepared using a Brevibacillus choshinensis expression system and purified in a soluble form. Crystals of the purified protein were produced by the sitting-drop vapor-diffusion method. Diffraction data were collected to a resolution of 1.6   Å using synchrotron radiation. The crystals obtained belonged to the tetragonal space group P41212 or P43212, with unit-cell parameters a   =   b   =   132.6, c = 76.1   Å . ...
Source: Acta Crystallographica Section F - November 16, 2018 Category: Biochemistry Authors: Intuy, R. Itoh, T. Suyotha, W. Hayashi, J. Yano, S. Makabe, K. Wakayama, M. Hibi, T. Tags: α -1,3-glucanase catalytic domains Paenibacillus glycanilyticus research communications Source Type: research

Structure of undecaprenyl pyrophosphate synthase from Acinetobacter baumannii
Undecaprenyl pyrophosphate (UPP) is an important carrier of the oligosaccharide component in peptidoglycan synthesis. Inhibition of UPP synthase (UPPS) may be an effective strategy in combating the pathogen Acinetobacter baumannii, which has evolved to be multidrug-resistant. Here, A. baumannii UPPS (AbUPPS) was cloned, expressed, purified and crystallized, and its structure was determined by X-ray diffraction. Each chain of the dimeric protein folds into a central β -sheet with several surrounding α -helices, including one at the C-terminus. In the active site, two molecules of citrate interact with the side ch...
Source: Acta Crystallographica Section F - November 16, 2018 Category: Biochemistry Authors: Ko, T.-P. Huang, C.-H. Lai, S.-J. Chen, Y. Tags: undecaprenyl pyrophosphate synthase Acinetobacter baumannii peptidoglycan research communications Source Type: research

Combining crystallogenesis methods to produce diffraction-quality crystals of a psychrophilic tRNA-maturation enzyme
The determination of conditions for the reproducible growth of well diffracting crystals is a critical step in every biocrystallographic study. On the occasion of a new structural biology project, several advanced crystallogenesis approaches were tested in order to increase the success rate of crystallization. These methods included screening by microseed matrix screening, optimization by counter-diffusion and crystal detection by trace fluorescent labeling, and are easily accessible to any laboratory. Their combination proved to be particularly efficient in the case of the target, a 48   kDa CCA-adding enzyme from the p...
Source: Acta Crystallographica Section F - October 31, 2018 Category: Biochemistry Authors: de Wijn, R. Hennig, O. Ernst, F.G.M. Lorber, B. Betat, H. M ö rl, M. Sauter, C. Tags: microseeding counter-diffusion trace fluorescent labeling optimization crystallogenesis tRNA maturation CCA-adding enzyme Planococcus halocryophilus research communications Source Type: research

Crystal structure of glycosyltrehalose synthase from Sulfolobus shibatae DSM5389
Glycosyltrehalose synthase (GTSase) converts the glucosidic bond between the last two glucose residues of amylose from an α -1,4 bond to an α -1,1 bond, generating a nonreducing glycosyl trehaloside, in the first step of the biosynthesis of trehalose. To better understand the structural basis of the catalytic mechanism, the crystal structure of GTSase from the hyperthermophilic archaeon Sulfolobus shibatae DSM5389 (5389-GTSase) has been determined to 2.4   Å resolution by X-ray crystallography. The structure of 5389-GTSase can be divided into five domains. The central domain contains the ( β / &alp...
Source: Acta Crystallographica Section F - October 31, 2018 Category: Biochemistry Authors: Okazaki, N. Blaber, M. Kuroki, R. Tamada, T. Tags: trehalose glycosyltransferase GH13 family glycosyltrehalose synthase Sulfolobus shibatae research communications Source Type: research

Crystal structure and kinetic analyses of a hexameric form of (S)-3-hydroxybutyryl-CoA dehydrogenase from Clostridium acetobutylicum
(S)-3-Hydroxybutyryl-CoA dehydrogenase (HBD) has been gaining increased attention recently as it is a key enzyme in the enantiomeric formation of (S)-3-hydroxybutyryl-CoA [(S)-3HB-CoA]. It converts acetoacetyl-CoA to (S)-3HB-CoA in the synthetic metabolic pathway. (S)-3HB-CoA is further modified to form (S)-3-hydroxybutyrate, which is a source of biodegradable polymers. During the course of a study to develop biodegradable polymers, attempts were made to determine the crystal structure of HBD from Clostridium acetobutylicum (CacHBD), and the crystal structures of both apo and NAD+-bound forms of CacHBD were determined. The...
Source: Acta Crystallographica Section F - October 31, 2018 Category: Biochemistry Authors: Takenoya, M. Taguchi, S. Yajima, S. Tags: bio-based plastics enzyme kinetics fatty-acid metabolism SDR superfamily X-ray protein crystallography research communications Source Type: research

Quo vadis, Acta Crystallographica F?
(Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - October 31, 2018 Category: Biochemistry Authors: van Raaij, M.J. Tags: editorial Acta Crystallographica F publishing scientific society journals Source Type: research

Crystal structures and kinetics of N-acetylneuraminate lyase from Fusobacterium nucleatum
In this study, atomic resolution structures of NanA from Fusobacterium nucleatum (FnNanA) in ligand-free and ligand-bound forms are reported at 2.32 and 1.76   Å resolution, respectively. F. nucleatum is a Gram-negative pathogen that causes gingival and periodontal diseases in human hosts. Like other bacterial N-acetylneuraminate lyases, FnNanA also shares the triosephosphate isomerase (TIM)-barrel fold. As observed in other homologous enzymes, FnNanA forms a tetramer. In order to characterize the structure – function relationship, the steady-state kinetic parameters of the enzyme are also reported. (Source: ...
Source: Acta Crystallographica Section F - October 17, 2018 Category: Biochemistry Authors: Kumar, J.P. Rao, H. Nayak, V. Ramaswamy, S. Tags: N-acetylneuraminate lyase sialic acid catabolism enzyme kinetics Fusobacterium nucleatum research communications Source Type: research

High-resolution structures of inhibitor complexes of human indoleamine 2,3-dioxygenase 1 in a new crystal form
Human indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-dependent enzyme with important roles in many cellular processes and is a potential target for drug discovery against cancer and other diseases. Crystal structures of IDO1 in complex with various inhibitors have been reported. Many of these crystals belong to the same crystal form and most of the reported structures have resolutions in the range 3.2 – 2.3   Å . Here, three new crystal forms of human IDO1 obtained by introducing a surface mutation, K116A/K117A, distant from the active site are reported. One of these crystal forms diffracted to 1.5   Å...
Source: Acta Crystallographica Section F - October 17, 2018 Category: Biochemistry Authors: Luo, S. Xu, K. Xiang, S. Chen, J. Chen, C. Guo, C. Tong, Y. Tong, L. Tags: indoleamine 2,3-dioxygenase 1 cancer immunotherapy heme proteins tryptophan metabolism crystal engineering research communications Source Type: research

BpeB, a major resistance-nodulation-cell division transporter from Burkholderia cenocepacia: construct design, crystallization and preliminary structural analysis
Burkholderia cenocepacia is an opportunistic pathogen that infects cystic fibrosis patients, causing pneumonia and septicemia. B. cenocepacia has intrinsic antibiotic resistance against monobactams, aminoglycosides, chloramphenicol and fluoroquinolones that is contributed by a homologue of BpeB, which is a member of the resistance-nodulation-cell division (RND)-type multidrug-efflux transporters. Here, the cloning, overexpression, purification, construct design for crystallization and preliminary X-ray diffraction analysis of this BpeB homologue from B. cenocepacia are reported. Two truncation variants were designed to rem...
Source: Acta Crystallographica Section F - October 16, 2018 Category: Biochemistry Authors: Horikawa, T. Hung, L.-W. Kim, H.-B. Shaya, D. Kim, C.-Y. Terwilliger, T.C. Yamashita, E. Aoki, M. Okada, U. Murakami, S. Tags: membrane transporter multidrug resistance drug exporter Burkholderia research communications Source Type: research

Crystal structures of the N-terminal domain of the Staphylococcus aureus DEAD-box RNA helicase CshA and its complex with AMP
In this study, the crystal structures of the N-terminal RecA-like domain 1 of S. aureus CshA (SaCshAR1) and of its complex with AMP (SaCshAR1 – AMP) are reported at resolutions of 1.5 and 1.8   Å , respectively. SaCshAR1 adopts a conserved α / β RecA-like structure with seven parallel strands surrounded by nine α -helices. The Q motif and motif I are responsible for the binding of the adenine group and phosphate group of AMP, respectively. Structure comparison of SaCshAR1 – AMP and SaCshAR1 reveals that motif I undergoes a conformational change upon AMP binding. Isothermal titration cal...
Source: Acta Crystallographica Section F - October 16, 2018 Category: Biochemistry Authors: Chen, X. Wang, C. Zhang, X. Tian, T. Zang, J. Tags: CshA DEAD-box RNA helicase Staphylococcus aureus AMP crystallography research communications Source Type: research

Crystallization of ectonucleotide phosphodiesterase/pyrophosphatase-3 and orientation of the SMB domains in the full-length ectodomain
Ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3, ENPP3) is an ATP-hydrolyzing glycoprotein that is located in the extracellular space. The full-length ectodomain of rat NPP3 was expressed in HEK293S GntI − cells, purified using two chromatographic steps and crystallized. Its structure at 2.77   Å resolution reveals that the active-site zinc ions are missing and a large part of the active site and the surrounding residues are flexible. The SMB-like domains have the same orientation in all four molecules in the asymmetric unit. The SMB2 domain is oriented as in NPP2, but the SMB1 domain does not intera...
Source: Acta Crystallographica Section F - October 16, 2018 Category: Biochemistry Authors: D ö hler, C. Zebisch, M. Krinke, D. Robitzki, A. Str ä ter, N. Tags: ectonucleotide phosphodiesterase/pyrophosphatase NPP3 ectonucleotidase ATP hydrolysis allergic response research communications Source Type: research

Characterization and structure determination of a llama-derived nanobody targeting the J-base binding protein 1
J-base binding protein 1 (JBP1) contributes to the biosynthesis and maintenance of base J ( β -d-glucosylhydroxymethyluracil), a modification of thymidine confined to some protozoa. Camelid (llama) single-domain antibody fragments (nanobodies) targeting JBP1 were produced for use as crystallization chaperones. Surface plasmon resonance screening identified Nb6 as a strong binder, recognizing JBP1 with a 1:1 stoichiometry and high affinity (Kd = 30   nM). Crystallization trials of JBP1 in complex with Nb6 yielded crystals that diffracted to 1.47   Å resolution. However, the dimensions of the asymmetric unit a...
Source: Acta Crystallographica Section F - October 16, 2018 Category: Biochemistry Authors: van Beusekom, B. Heidebrecht, T. Adamopoulos, A. Fish, A. Pardon, E. Steyaert, J. Joosten, R.P. Perrakis, A. Tags: nanobodies J-base binding protein 1 llamas immune system research communications Source Type: research

Cryo-neutron crystallographic data collection and preliminary refinement of left-handed Z-DNA d(CGCGCG)
Crystals of left-handed Z-DNA [d(CGCGCG)]2 diffract X-rays to beyond 1   Å resolution, feature a small unit cell ( ∼ 18 × 31 × 44   Å ) and are well hydrated, with around 90 water molecules surrounding the duplex in the asymmetric unit. The duplex shows regular hydration patterns in the narrow minor groove, on the convex surface and around sugar – phosphate backbones. Therefore, Z-DNA offers an ideal case to test the benefits of low-temperature neutron diffraction data collection to potentially determine the donor – acceptor patterns of first- and second-shell water molecules. Nu...
Source: Acta Crystallographica Section F - September 21, 2018 Category: Biochemistry Authors: Harp, J.M. Coates, L. Sullivan, B. Egli, M. Tags: neutron diffraction cryogenic data collection oligonucleotide hydration Z-DNA research communications Source Type: research

Redox manipulation of the manganese metal in human manganese superoxide dismutase for neutron diffraction
Human manganese superoxide dismutase (MnSOD) is one of the most significant enzymes in preventing mitochondrial dysfunction and related diseases by combating reactive oxygen species (ROS) in the mitochondrial matrix. Mitochondria are the source of up to 90% of cellular ROS generation, and MnSOD performs its necessary bioprotective role by converting superoxide into oxygen and hydrogen peroxide. This vital catalytic function is conducted via cyclic redox reactions between the substrate and the active-site manganese using proton-coupled electron transfers. Owing to protons being difficult to detect experimentally, the series...
Source: Acta Crystallographica Section F - September 21, 2018 Category: Biochemistry Authors: Azadmanesh, J. Lutz, W.E. Weiss, K.L. Coates, L. Borgstahl, G.E.O. Tags: manganese superoxide dismutase neutron diffraction perdeuteration human oxidation reduction large unit cell research communications Source Type: research

X-ray structure of Arthrobacter globiformis M30 ketose 3-epimerase for the production of d-allulose from d-fructose
The X-ray structure of ketose 3-epimerase from Arthrobacter globiformis M30, which was previously reported to be a d-allulose 3-epimerase (AgD-AE), was determined at 1.96   Å resolution. The crystal belonged to the hexagonal space group P6522, with unit-cell parameters a = b = 103.98, c = 256.53   Å . The structure was solved by molecular replacement using the structure of Mesorhizobium loti l-ribulose 3-epimerase (MlL-RE), which has 41% sequence identity, as a search model. A hexagonal crystal contained two molecules in the asymmetric unit, and AgD-AE formed a homotetramer with twofold symmetry. The overal...
Source: Acta Crystallographica Section F - September 21, 2018 Category: Biochemistry Authors: Yoshida, H. Yoshihara, A. Gullapalli, P.K. Ohtani, K. Akimitsu, K. Izumori, K. Kamitori, S. Tags: Arthrobacter globiformis ketose 3-epimerase β / α -barrel d-allulose X-ray structure research communications Source Type: research

Re-refinement of Plasmodium falciparum orotidine 5 ′ -monophosphate decarboxylase provides a clearer picture of an important malarial drug target
The development of antimalarial drugs remains a public health priority, and the orotidine 5 ′ -monophosphate decarboxylase from Plasmodium falciparum (PfOMPDC) has great potential as a drug target. The crystallization of PfOMPDC with substrate bound represents an important advance for structure-based drug-design efforts [Tokuoka et al. (2008), J. Biochem. 143, 69 – 78]. The complex of the enzyme bound to the substrate OMP (PDB entry 2za1) would be of particular utility in this regard. However, re-refinement of this structure of the Michaelis complex shows that the bound ligand is the product rather than the sub...
Source: Acta Crystallographica Section F - September 21, 2018 Category: Biochemistry Authors: Novak, W.R.P. West, K.H.J. Kirkman, L.M.D. Brandt, G.S. Tags: orotidine 5 ′ -monophosphate decarboxylase Plasmodium falciparum Michaelis complex re-refinement research communications Source Type: research

Comparative structure analysis of the ETSi domain of ERG3 and its complex with the E74 promoter DNA sequence
ERG3 (ETS-related gene) is a member of the ETS (erythroblast transformation-specific) family of transcription factors, which contain a highly conserved DNA-binding domain. The ETS family of transcription factors differ in their binding to promoter DNA sequences, and the mechanism of their DNA-sequence discrimination is little known. In the current study, crystals of the ETSi domain (the ETS domain of ERG3 containing a CID motif) in space group P41212 and of its complex with the E74 DNA sequence (DNA9) in space group C2221 were obtained and their structures were determined. Comparative structure analysis of the ETSi domain ...
Source: Acta Crystallographica Section F - September 21, 2018 Category: Biochemistry Authors: Sharma, R. Gangwar, S.P. Saxena, A.K. Tags: prostate cancer ETS transcription factor ERG3 E74 promoter DNA X-ray structure comparative structure analysis research communications Source Type: research

Crystal structures of green fluorescent protein with the unnatural amino acid 4-nitro-l-phenylalanine
The X-ray crystal structures of two superfolder green fluorescent protein (sfGFP) constructs containing a genetically incorporated spectroscopic reporter unnatural amino acid, 4-nitro-l-phenylalanine (pNO2F), at two unique sites in the protein have been determined. Amber codon-suppression methodology was used to site-specifically incorporate pNO2F at a solvent-accessible (Asp133) and a partially buried (Asn149) site in sfGFP. The Asp133pNO2F sfGFP construct crystallized with two molecules per asymmetric unit in space group P3221 and the crystal structure was refined to 2.05   Å resolution. Crystals of Asn149pNO2F s...
Source: Acta Crystallographica Section F - September 19, 2018 Category: Biochemistry Authors: Maurici, N. Savidge, N. Lee, B.U. Brewer, S.H. Phillips-Piro, C.M. Tags: 4-nitro-l-phenylalanine unnatural amino acids noncanonical amino acids green fluorescent protein research communications Source Type: research

The novel metallo- β -lactamase PNGM-1 from a deep-sea sediment metagenome: crystallization and X-ray crystallographic analysis
In this study, PNGM-1 was overexpressed, purified and crystallized. Crystals of native and selenomethionine-substituted PNGM-1 diffracted to 2.10 and 2.30   Å resolution, respectively. Both the native and the selenomethionine-labelled PNGM-1 crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 122, b = 83, c = 163   Å , β = 110 ° . Matthews coefficient (VM) calculations suggested the presence of 6 – 10 molecules in the asymmetric unit, corresponding to a solvent content of ∼ 31 – 58%. Structure determination is currently in progress. (Source: Acta Cryst...
Source: Acta Crystallographica Section F - September 19, 2018 Category: Biochemistry Authors: Park, K.S. Hong, M.-K. Jeon, J.W. Kim, J.H. Jeon, J.H. Lee, J.H. Kim, T.Y. Karim, A.M. Malik, S.K. Kang, L.-W. Lee, S.H. Tags: deep-sea sediment Edison Seamount metagenome antibiotic resistance metallo- β -lactamase research communications Source Type: research

Crystal structure of mutant carboxypeptidase T from Thermoactinomyces vulgaris with an implanted S1 ′ subsite from pancreatic carboxypeptidase B
A site-directed mutagenesis method has been used to obtain the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT), in which the amino-acid residues of the S1 ′ subsite are substituted by the corresponding residues from pancreatic carboxypeptidase B (CPB). It was shown that the mutant enzyme retained the broad, mainly hydrophobic selectivity of wild-type CPT. The mutant containing the implanted CPB S1 ′ subsite was crystallized and its three-dimensional structure was determined at 1.29   Å resolution by X-ray crystallography. A comparison of the three-dimensi...
Source: Acta Crystallographica Section F - September 19, 2018 Category: Biochemistry Authors: Akparov, V.K. Timofeev, V.I. Kuranova, I.P. Rakitina, T.V. Tags: metallocarboxypeptidase T Thermoactinomyces vulgaris metallocarboxypeptidase B S1 ′ subsite substrate selectivity X-ray crystallography research communications Source Type: research

Crystal structure of the ribonuclease-P-protein subunit from Staphylococcus aureus
Staphylococcus aureus ribonuclease-P-protein subunit (RnpA) is a promising antimicrobial target that is a key protein component for two essential cellular processes, RNA degradation and transfer-RNA (tRNA) maturation. The first crystal structure of RnpA from the pathogenic bacterial species, S. aureus, is reported at 2.0   Å resolution. The structure presented maintains key similarities with previously reported RnpA structures from bacteria and archaea, including the highly conserved RNR-box region and aromatic residues in the precursor-tRNA 5 ′ -leader-binding domain. This structure will be instrumental in t...
Source: Acta Crystallographica Section F - September 19, 2018 Category: Biochemistry Authors: Ha, L. Colquhoun, J. Noinaj, N. Das, C. Dunman, P.M. Flaherty, D.P. Tags: ribonucleoprotein structure RnpA RNase P protein Staphylococcus aureus tRNA research communications Source Type: research