Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form
Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical...
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: Baum, B.Lecker, L.S.M.Zoltner, M.Jaenicke, E.Schnell, R.Hunter, W.N.Brenk, R. Tags: fatty-acid biosynthesis FabF Gram-negative bacteria potassium binding Pseudomonas aeruginosa structure-based drug discovery research communications Source Type: research

Crystallographic analysis of FAD-dependent glucose dehydrogenase
An FAD-dependent glucose dehydrogenase (GDH) from Aspergillus terreus was purified and crystallized at 293 K using the sitting-drop vapour-diffusion method. A data set was collected to a resolution of 1.6 Å from a single crystal at 100 K using a rotating-anode X-ray source. The crystal belonged to space group P21, with unit-cell parameters a = 56.56, b = 135.74, c = 74.13 Å, β = 90.37°. The asymmetric unit contained two molecules of GDH. The Matthews coefficient was calculated to be 2.2 Å3 Da−1 and the solvent content was estimated to be 44%. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: Komori, H.Inaka, K.Furubayashi, N.Honda, M.Higuchi, Y. Tags: glucose dehydrogenase FAD glucose sensor research communications Source Type: research

Crystallographic studies of aspartate racemase from Lactobacillus sakei NBRC 15893
Aspartate racemase catalyzes the interconversion between l-aspartate and d-aspartate and belongs to the PLP-independent racemases. The enzyme from the lactic acid bacterium Lactobacillus sakei NBRC 15893, isolated from kimoto, is considered to be involved in d-aspartate synthesis during the brewing process of Japanese sake at low temperatures. The enzyme was crystallized at 293 K by the sitting-drop vapour-diffusion method using 25%(v/v) PEG MME 550, 5%(v/v) 2-propanol. The crystal belonged to space group P3121, with unit-cell parameters a = b = 104.68, c = 97.29 Å, and diffracted to 2.6 Å resolution. Str...
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: Fujii, T.Yamauchi, T.Ishiyama, M.Gogami, Y.Oikawa, T.Hata, Y. Tags: aspartate racemase Lactobacillus sakei d-amino acid crystallization research communications Source Type: research

Structure of α-carbonic anhydrase from the human pathogen Helicobacter pylori
The crystal structure of α-carbonic anhydrase, an enzyme present in the periplasm of Helicobacter pylori, a bacterium that affects humans and that is responsible for several gastric pathologies, is described. Two enzyme monomers are present in the asymmetric unit of the monoclinic space group P21, forming a dimer in the crystal. Despite the similarity of the enzyme structure to those of orthologues from other species, the H. pylori protein has adopted peculiar features in order to allow the bacterium to survive in the difficult environment of the human stomach. In particular, the crystal structure shows how the bacte...
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: Compostella, M.E.Berto, P.Vallese, F.Zanotti, G. Tags: Helicobacter pylori carbonic anhydrase metalloproteins periplasmic protein research communications Source Type: research

Phormidium phycoerythrin forms hexamers in crystals: a crystallographic study
The crystallographic analysis of a marine cyanobacterium (Phormidium sp. A09DM) phycoerythrin (PE) that shows distinct sequence features compared with known PE structures from cyanobacteria and red algae is reported. Phormidium PE was crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant. Diffraction data were collected on the protein crystallography beamline at the Indus-2 synchrotron. The crystals diffracted to about 2.1 Å resolution at 100 K. The crystals, with an apparent hexagonal morphology, belonged to space group P1, with unit-cell parameters a = 108.3, b = 108...
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: Sonani, R.R.Sharma, M.Gupta, G.D.Kumar, V.Madamwar, D. Tags: phycoerythrin crystal structure X-ray diffraction research communications Source Type: research

Cloning, expression, purification and crystallographic studies of galectin-11 from domestic sheep (Ovis aries)
Galectins are an evolutionarily conserved family of proteins that translate glycan recognition into cellular effects. Galectin-11 is a unique member of the galectin family that is only expressed in ruminants such as sheep, goat and cattle and that plays a critical role in several important biological processes, such as reproduction and parasite-mediated innate immune responses. Currently, these two areas are of major importance for the sustainability of ruminant livestock production. Despite the emerging biological significance of galectin-11, no structural information is available. It is expected that structural studies w...
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: Sakthivel, D.Littler, D.Shahine, A.Troy, S.Johnson, M.Rossjohn, J.Piedrafita, D.Beddoe, T. Tags: galectin parasite glycan livestock research communications Source Type: research

The soluble Y115E–Y117E variant of human glutaminyl cyclase is a valid target for X-ray and NMR screening of inhibitors against Alzheimer disease
Recent developments in molecular pathology and genetics have allowed the identification of human glutaminyl cyclase (hQC) among the abnormal proteins involved in many neurodegenerative disorders. Difficulties in obtaining large quantities of pure protein may limit the use of crystallographic screening for drug development on this target. Site-directed mutagenesis experiments have led to the identification of some solvent-exposed residues that are absolutely critical to achieve increased solubility and to avoid precipitation of the enzyme in inclusion bodies when expressed in Escherichia coli. The designed variant Y115E&nda...
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: DiPisa, F.Pozzi, C.Benvenuti, M.Andreini, M.Marconi, G.Mangani, S. Tags: glutaminyl cyclase Alzheimer disease inhibitor screening crystal structure inhibitors research communications Source Type: research

Structure of the human MLH1 N-terminus: implications for predisposition to Lynch syndrome
Mismatch repair prevents the accumulation of erroneous insertions/deletions and non-Watson–Crick base pairs in the genome. Pathogenic mutations in the MLH1 gene are associated with a predisposition to Lynch and Turcot's syndromes. Although genetic testing for these mutations is available, robust classification of variants requires strong clinical and functional support. Here, the first structure of the N-terminus of human MLH1, determined by X-ray crystallography, is described. The structure shares a high degree of similarity with previously determined prokaryotic MLH1 homologs; however, this structure affords a more...
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: Wu, H.Zeng, H.Lam, R.Tempel, W.Kerr, I.D.Min, J. Tags: ATPase GHKL Lynch syndrome mismatch repair research communications Source Type: research

Crystallographic analysis of the N-terminal domain of Middle East respiratory syndrome coronavirus nucleocapsid protein
In this study, the crystallization and crystallographic analysis of MERS-CoV NP-NTD (amino acids 39–165), with a molecular weight of 14.7 kDa, are reported. MERS-CoV NP-NTD was crystallized at 293 K using PEG 3350 as a precipitant and a 94.5% complete native data set was collected from a cooled crystal at 77 K to 2.63 Å resolution with an overall Rmerge of 9.6%. The crystals were monoclinic and belonged to space group P21, with unit-cell parameters a = 35.60, b = 109.64, c = 91.99 Å, β = 101.22°. The asymmetric unit contained four MERS-CoV NP-NTD molecules. (Source: Acta Cryst...
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: Wang, Y.-S.Chang, C.Hou, M.-H. Tags: Middle East respiratory syndrome coronavirus nucleocapsid protein N-terminal domain RNA binding research communications Source Type: research

Purification and crystallographic analysis of a FAD-dependent halogenase from Streptomyces sp. JCM9888
This study reports the overexpression and purification of HalY with an N-terminal hexahistidine tag, followed by crystallization experiments and X-ray crystallographic analysis. HalY was purified as a monomer in solution and crystallized to give X-ray diffraction to a resolution of 1.7 Å. The crystal belonged to the monoclinic space group P21, with unit-cell parameters a = 41.4, b = 113.4, c = 47.6 Å, α = γ = 90, β = 107.4°, and contained one monomer of HalY in the asymmetric unit, with a calculated Matthews coefficient of 2.3 Å3 Da−1 and a solvent content of 46%. The st...
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: Zhao, Y.Yan, B.Yang, T.Jiang, J.Wei, H.Zhu, X. Tags: FAD-dependent halogenase regioselective halogenation adenine research communications Source Type: research

Structure of the response regulator ChrA in the haem-sensing two-component system of Corynebacterium diphtheriae
ChrA is a response regulator (RR) in the two-component system involved in regulating the degradation and transport of haem (Fe–porphyrin) in the pathogen Corynebacterium diphtheriae. Here, the crystal structure of full-length ChrA is described at a resolution of 1.8 Å. ChrA consists of an N-terminal regulatory domain, a long linker region and a C-terminal DNA-binding domain. A structural comparison of ChrA with other RRs revealed substantial differences in the relative orientation of the two domains and the conformation of the linker region. The structural flexibility of the linker could be an important featu...
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: Doi, A.Nakamura, H.Shiro, Y.Sugimoto, H. Tags: transcription factor X-ray crystallography haem helix – turn phosphorylation research communications Source Type: research

Molecular cloning, overexpression, purification and crystallographic analysis of a GH43 β-xylosidase from Bacillus licheniformis
In this study, a GH43 β-xylosidase identified from the bacterium Bacillus licheniformis (BlXylA) was cloned into the the pET-28a bacterial expression vector, recombinantly overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity by metal-affinity and size-exclusion chromatography. The protein was crystallized in the presence of the organic solvent 2-methyl-2,4-pentanediol and a single crystal diffracted to 2.49 Å resolution. The X-ray diffraction data were indexed in the monoclinic space group C2, with unit-cell parameters a = 152.82, b = 41.9, c = 71.79 Å, β = 91.7°. Stru...
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: Diogo, J.A.Zanphorlin, L.M.Sato, H.H.Murakami, M.T.Ruller, R. Tags: β -xylosidase Bacillus licheniformis GH43 family research communications Source Type: research

Purification and crystallographic studies of a putative carbohydrate-binding module from the Ruminococcus flavefaciens FD-1 endoglucanase Cel5A
Ruminant herbivores meet their carbon and energy requirements from a symbiotic relationship with cellulosome-producing anaerobic bacteria that efficiently degrade plant cell-wall polysaccharides. The assembly of carbohydrate-active enzymes (CAZymes) into cellulosomes enhances protein stability and enzyme synergistic interactions. Cellulosomes comprise diverse CAZymes displaying a modular architecture in which a catalytic domain is connected, via linker sequences, to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus facilitating catalysi...
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: Pires, A.J.Ribeiro, T.Thompson, A.Venditto, I.Fernandes, V.O.Bule, P.Santos, H.Alves, V.D.Pires, V.Ferreira, L.M.A.Fontes, C.M.G.A.Najmudin, S. Tags: Ruminococcus flavefaciens carbohydrate-binding module carbohydrate-active enzymes glycoside hydrolase cellulosomes research communications Source Type: research

Structure of recombinant prolidase from Thermococcus sibiricus in space group P21221
The crystal structure of recombinant prolidase from Thermococcus sibiricus was determined by X-ray diffraction at a resolution of 2.6 Å and was found to contain a tetramer in the asymmetric unit. A protein crystal grown in microgravity using the counter-diffusion method was used for X-ray studies. The crystal belonged to space group P21221, with unit-cell parameters a = 97.60, b = 123.72, c = 136.52 Å, α = β = γ = 90°. The structure was refined to an Rcryst of 22.1% and an Rfree of 29.6%. The structure revealed flexible folding of the N-terminal domain of the protein as well as high var...
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: Timofeev, V.Slutskaya, E.Gorbacheva, M.Boyko, K.Rakitina, T.Korzhenevskiy, D.Lipkin, A.Popov, V. Tags: archaeal proteins prolidase Thermococcus sibiricus crystallization crystallography research communications Source Type: research

Crystallographic studies of the extracytoplasmic function σ factor σJ from Mycobacterium tuberculosis
Mycobacterium tuberculosis has multiple σ factors which enable the bacterium to reprogram its transcriptional machinery under diverse environmental conditions. σJ, an extracytoplasmic function σ factor, is upregulated in late stationary phase cultures and during human macrophage infection. σJ governs the cellular response to hydrogen peroxide-mediated oxidative stress. σJ differs from other canonical σ factors owing to the presence of a SnoaL_2 domain at the C-terminus. σJ crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 133.85, c = 75.08 &A...
Source: Acta Crystallographica Section F - July 28, 2015 Category: Biochemistry Authors: Goutam, K.Gupta, A.K.Gopal, B. Tags: σ factor sigma factor SnoaL_2 domain potential drug target research communications Source Type: research

Structure of human collapsin response mediator protein 1: a possible role of its C-terminal tail
Collapsin response mediator protein 1 (CRMP-1) is the first identified member of the CRMP family and is crucial for both the mediation of neuronal differentiation and in suppressing the invasion of lung cancer. The crystal structure of full-length human CRMP-1 was determined at a resolution of 3 Å. Human CRMP-1 comprises a tetrameric assembly; its overall structure is similar to that of mouse CRMP-1, but the measured electron density of the C-terminal residues 488–496 show a randomly coiled link that connects the protomers to each other, within which residues 497–572 are proteolytically susceptible in v...
Source: Acta Crystallographica Section F - July 27, 2015 Category: Biochemistry Authors: Liu, S.-H.Huang, S.-F.Hsu, Y.-L.Pan, S.-H.Chen, Y.-J.Lin, Y.-H. Tags: collapsin response mediator protein 1 crystal structure non-small-cell lung cancer lung cancer suppressor circular dichoism research communications Source Type: research

Introduction to selected articles from the 15th ICCBM
(Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - July 1, 2015 Category: Biochemistry Authors: Einspahr, H.Kuta Smatanova, I.Betzel, C.Mesters, J. Tags: crystallization ICCBM15 research communications Source Type: research

Expression, purification, crystallization and crystallographic study of Lutzomyia longipalpis LJL143
Leishmaniasis is a neglected vector-borne disease with a global prevalence of over 12 million cases and 59 000 annual deaths. Transmission of the parasite requires salivary proteins, including LJL143 from the New World sandfly Lutzomyia longipalpis. LJL143 is a known marker of sandfly exposure in zoonotic hosts. LJL143 was crystallized from soluble protein expressed using Pichia pastoris. X-ray data were collected to 2.6 Å resolution from orthorhombic crystals belonging to space group P212121, with average unit-cell parameters a = 57.39, b = 70.24, c = 79.58 Å. The crystals are predicted to have a monomer...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Kelleher, A.Liu, Z.Seid, C.Zhan, B.Asojo, O.A. Tags: leishmaniasis Lutzomyia longipalpis neglected tropical diseases diagnosis sandfly salivary proteins transmission research communications Source Type: research

The crystal and solution structure of YdiE from Escherichia coli
Iron-containing porphyrins are essential for all life as electron carriers. Since iron is poorly available in an oxidizing environment, bacterial growth may be restricted by iron limitation, and this has led to the evolution of a huge variety of iron-uptake systems. Among pathogens, iron scavenging from the haemoglobin of an animal host is a common means of acquiring sufficient iron for growth. The Isd system of Staphylococcus aureus is a well studied example; the bacterium devotes considerable resources to the construction of surface proteins that deftly remove haem from haemoglobin and pass it along a chain of related pr...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Nishimura, K.Addy, C.Shrestha, R.Voet, A.R.D.Zhang, K.Y.J.Ito, Y.Tame, J.R.H. Tags: YdiE Escherichia coli iron transport intrinsic disorder haem dimer small protein research communications Source Type: research

The structure of a GFP-based antibody (fluorobody) to TLH, a toxin from Vibrio parahaemolyticus
A fluorobody is a manmade hybrid molecule that is composed of green fluorescent protein (GFP) and a fragment of antibody, which combines the affinity and specificity of an antibody with the visibility of a GFP. It is able to provide a real-time indication of binding while avoiding the use of tags and secondary binding reagents. Here, the expression, purification and crystal structure of a recombinant fluorobody for TLH (thermolabile haemolysin), a toxin from the lethal food-borne disease bacterium Vibrio parahaemolyticus, are presented. This is the first structure of a fluorobody to be reported. Crystals belonging to space...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Chen, Y.Huang, X.Wang, R.Wang, S.Shi, N. Tags: fluorobody GFP scaffold CDR3 TLH research communications Source Type: research

The structure of nerve growth factor in complex with lysophosphatidylinositol
Nerve growth factor (NGF) is an important protein that is involved in a variety of physiological processes in cell survival, differentiation, proliferation and maintenance. The previously reported crystal structure of mouse NGF (mNGF) in complex with lysophosphatidylserine (LysoPS) showed that mNGF can bind LysoPS at its dimeric interface. To expand the understanding of the structural basis for specific lipid recognition by NGF, the crystal structure of mNGF complexed with lysophosphatidylinositol (13:0 LysoPI) was solved. Interestingly, in addition to Lys88, which interacts with the head glycerol group and the phosphate g...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Sun, H.-L.Jiang, T. Tags: nerve growth factor lysophosphatidylinositol LysoPI mNGF – LysoPI complex LysoPS research communications Source Type: research

Synthesis, purification and crystallographic studies of the C-terminal sterol carrier protein type 2 (SCP-2) domain of human hydroxysteroid dehydrogenase-like protein 2
In this study, the C-terminal SCP-2 domain of human HSDL2, including residues Lys318–Arg416, was produced in Escherichia coli, purified and crystallized. X-ray diffraction data were collected to 2.10 Å resolution. The crystal belonged to the trigonal space group P3121 (or P3221), with unit-cell parameters a = b = 70.4, c = 60.6 Å, α = β = 90, γ = 120°. Two protein molecules are present in the asymmetric unit, resulting in a Matthews coefficient of 2.16 Å3 Da−1 and an approximate solvent content of 43%. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Cheng, Z.Li, Y.Sui, C.Sun, X.Xie, Y. Tags: human HSDL2 SCP-2 domian crystallization X-ray diffraction research communications Source Type: research

Structure of human saposin A at lysosomal pH
The saposins are essential cofactors for the normal lysosomal degradation of complex glycosphingolipids by acid hydrolase enzymes; defects in either saposin or hydrolase function lead to severe metabolic diseases. Saposin A (SapA) activates the enzyme β-galactocerebrosidase (GALC), which catalyzes the breakdown of β-d-galactocerebroside, the principal lipid component of myelin. SapA is known to bind lipids and detergents in a pH-dependent manner; this is accompanied by a striking transition from a `closed' to an `open' conformation. However, previous structures were determined at non-lysosomal pH. This work descr...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Hill, C.H.Read, R.J.Deane, J.E. Tags: saposin A lipid-transfer protein sphingolipid activator protein GALC research communications Source Type: research

Expression, purification, crystallization and crystallographic study of the Aspergillus terreus aromatic prenyltransferase AtaPT
Prenylated aromatics are produced by aromatic prenyltransferases during the secondary metabolism of bacteria, fungi and plants. The prenylation of nonprenylated precursors can lead to great chemical diversity and extensive biological properties. Aspergillus terreus aromatic prenyltransferase (AtaPT), which has recently been discovered and characterized, is such an enzyme and is responsible for the prenylation of various aromatic compounds. Here, recombinant AtaPT was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to a resolution of 1.71 Å and the crystal belonged to sp...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Gao, B.Chen, R.Liu, X.Dai, J.Sun, F. Tags: aromatic prenyltransferase Aspergillus terreus prenylation crystallization research communications Source Type: research

Structural characterization of the principal mRNA-export factor Mex67–Mtr2 from Chaetomium thermophilum
Members of the Mex67–Mtr2/NXF–NXT1 family are the principal mediators of the nuclear export of mRNA. Mex67/NXF1 has a modular structure based on four domains (RRM, LRR, NTF2-like and UBA) that are thought to be present across species, although the level of sequence conservation between organisms, especially in lower eukaryotes, is low. Here, the crystal structures of these domains from the thermophilic fungus Chaetomium thermophilum are presented together with small-angle X-ray scattering (SAXS) and in vitro RNA-binding data that indicate that, not withstanding the limited sequence conservation between differen...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Aibara, S.Valkov, E.Lamers, M.H.Dimitrova, L.Hurt, E.Stewart, M. Tags: nuclear transport Mex67 Chaetomium thermophilum RNA binding research communications Source Type: research

Crystallographic analysis of the Arabidopsis thaliana BAG5–calmodulin protein complex
Arabidopsis thaliana BAG5 (AtBAG5) belongs to the plant BAG (Bcl-2-associated athanogene) family that performs diverse functions ranging from growth and development to abiotic stress and senescence. BAG family members can act as nucleotide-exchange factors for heat-shock protein 70 (Hsp70) through binding of their evolutionarily conserved BAG domains to the Hsp70 ATPase domain, and thus may be involved in the regulation of chaperone-mediated protein folding in plants. AtBAG5 is distinguished from other family members by the presence of a unique IQ motif adjacent to the BAG domain; this motif is specific for calmodulin (CaM...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Cui, B.Fang, S.Xing, Y.Shen, Y.Yang, X. Tags: BAG5 calmodulin Arabidopsis thaliana research communications Source Type: research

Fusion-protein-assisted protein crystallization
Fusion proteins can be used directly in protein crystallization to assist crystallization in at least two different ways. In one approach, the `heterologous fusion-protein approach', the fusion partner can provide additional surface area to promote crystal contact formation. In another approach, the `fusion of interacting proteins approach', protein assemblies can be stabilized by covalently linking the interacting partners. The linker connecting the proteins plays different roles in the two applications: in the first approach a rigid linker is required to reduce conformational heterogeneity; in the second, conversely, a f...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Kobe, B.Ve, T.Williams, S.J. Tags: linker heterologous fusion-protein approach fusion of interacting proteins approach membrane-protein crystallization protein interactions recombinant fusion protein research communications Source Type: research

Batch crystallization of rhodopsin for structural dynamics using an X-ray free-electron laser
Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Wu, W.Nogly, P.Rheinberger, J.Kick, L.M.Gati, C.Nelson, G.Deupi, X.Standfuss, J.Schertler, G.Panneels, V. Tags: batch crystallization GPCR serial crystallography FEL dynamics research communications Source Type: research

Crystallographic analysis of a novel aldo-keto reductase from Thermotoga maritima in complex with NADP+
In this study, Tm1743 was overexpressed in Escherichia coli BL21(DE3) cells with an N-terminal His6 tag and was purified by Ni2+-chelating affinity and size-exclusion chromatography. Purified recombinant enzyme was incubated with its cofactor NADP+ and its substrate ethyl 2-oxo-4-phenylbutyrate (EOPB) for crystallization. Two X-ray diffraction data sets were collected at 2.0 and 1.7 Å resolution from dodecahedral crystals grown from samples containing Tm1743–NADP+–EOPB and Tm1743–NADP+, respectively. Both crystals belonged to space group P3121, with similar unit-cell parameters. However, in the re...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Hou, H.Li, R.Wang, X.Yuan, Z.Liu, X.Chen, Z.Xu, X. Tags: aldo-keto reductases thermostable Thermotoga maritima research communications Source Type: research

Use of dynamic light scattering and small-angle X-ray scattering to characterize new surfactants in solution conditions for membrane-protein crystallization
The structural and interactive properties of two novel hemifluorinated surfactants, F2H9-β-M and F4H5-β-M, the syntheses of which were based on the structure and hydrophobicity of the well known dodecyl-β-maltoside (DD-β-M), are described. The shape of their micellar assemblies was characterized by small-angle X-ray scattering and their intermicellar interactions in crystallizing conditions were measured by dynamic light scattering. Such information is essential for surfactant phase-diagram determination and membrane-protein crystallization. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Dahani, M.Barret, L.-A.Raynal, S.Jungas, C.Pernot, P.Polidori, A.Bonneté, F. Tags: membrane protein surfactant micelle scattering techniques phase diagram crystallization research communications Source Type: research

The MORPHEUS II protein crystallization screen
High-quality macromolecular crystals are a prerequisite for the process of protein structure determination by X-ray diffraction. Unfortunately, the relative yield of diffraction-quality crystals from crystallization experiments is often very low. In this context, innovative crystallization screen formulations are continuously being developed. In the past, MORPHEUS, a screen in which each condition integrates a mix of additives selected from the Protein Data Bank, a cryoprotectant and a buffer system, was developed. Here, MORPHEUS II, a follow-up to the original 96-condition initial screen, is described. Reagents were selec...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Gorrec, F. Tags: macromolecular crystallography protein crystallization crystallization screening crystallization additives heavy-atom derivatization MORPHEUS II research communications Source Type: research

Towards time-resolved serial crystallography in a microfluidic device
Serial methods for crystallography have the potential to enable dynamic structural studies of protein targets that have been resistant to single-crystal strategies. The use of serial data-collection strategies can circumvent challenges associated with radiation damage and repeated reaction initiation. This work utilizes a microfluidic crystallization platform for the serial time-resolved Laue diffraction analysis of macroscopic crystals of photoactive yellow protein (PYP). Reaction initiation was achieved via pulsed laser illumination, and the resultant electron-density difference maps clearly depict the expected pR1/pRE46...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Pawate, A.S.Šrajer, V.Schieferstein, J.Guha, S.Henning, R.Kosheleva, I.Schmidt, M.Ren, Z.Kenis, P.J.A.Perry, S.L. Tags: serial crystallography Laue diffraction time-resolved protein crystallography protein crystallization microfluidics photoactive yellow protein research communications Source Type: research

Do protein crystals nucleate within dense liquid clusters?
Protein-dense liquid clusters are regions of high protein concentration that have been observed in solutions of several proteins. The typical cluster size varies from several tens to several hundreds of nanometres and their volume fraction remains below 10−3 of the solution. According to the two-step mechanism of nucleation, the protein-rich clusters serve as locations for and precursors to the nucleation of protein crystals. While the two-step mechanism explained several unusual features of protein crystal nucleation kinetics, a direct observation of its validity for protein crystals has been lacking. Here, two inde...
Source: Acta Crystallographica Section F - June 27, 2015 Category: Biochemistry Authors: Maes, D.Vorontsova, M.A.Potenza, M.A.C.Sanvito, T.Sleutel, M.Giglio, M.Vekilov, P.G. Tags: nucleation two-step mechanism protein-rich clusters crystallization research communications Source Type: research

Trace fluorescent labeling for protein crystallization
Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace-labeling approach, the protein is covalently derivatized with a high-quantum-yield visible-wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate-screening experiment in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A stu...
Source: Acta Crystallographica Section F - June 26, 2015 Category: Biochemistry Authors: Pusey, M.Barcena, J.Morris, M.Singhal, A.Yuan, Q.Ng, J. Tags: trace fluorescence labeling crystallization screening intensity research communications Source Type: research

Practical macromolecular cryocrystallography
Cryocrystallography is an indispensable technique that is routinely used for single-crystal X-ray diffraction data collection at temperatures near 100 K, where radiation damage is mitigated. Modern procedures and tools to cryoprotect and rapidly cool macromolecular crystals with a significant solvent fraction to below the glass-transition phase of water are reviewed. Reagents and methods to help prevent the stresses that damage crystals when flash-cooling are described. A method of using isopentane to assess whether cryogenic temperatures have been preserved when dismounting screened crystals is also presented. (Source: ...
Source: Acta Crystallographica Section F - June 1, 2015 Category: Biochemistry Authors: Pflugrath, J.W. Tags: cryocrystallography cryoprotectant flash-cooling crystal mounting annealing automounter high pressure research communications Source Type: research

Mode of binding of the antithyroid drug propylthiouracil to mammalian haem peroxidases. Erratum
The results in the article by Singh et al. [(2015), Acta Cryst. F71, 304–310] have been brought into question and the article is retracted. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 27, 2015 Category: Biochemistry Authors: Singh, R.P.Singh, A.Kushwaha, G.S.Singh, A.K.Kaur, P.Sharma, S.Singh, T.P. Tags: lactoperoxidase propylthiouracil distal haem side antithyroid drug retraction addenda and errata Source Type: research

Structure–activity correlations for three pyrido[2,3-d]pyrimidine antifolates binding to human and Pneumocystis carinii dihydrofolate reductase
To further define the interactions that enhance the selectivity of binding and to directly compare the binding of the most potent analogue {N6-methyl-N6-(3,4,5-trifluorophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine; compound 26} in the series of bicyclic pyrido[2,3-d]pyrimidine analogues of piritrexim (PTX) with native human (h), Pneumocystis carinii (pc) and Pneumocystis jirovecii (pj) dihydrofolate reductase (DHFR) enzymes, the crystal structures of hDHFR complexed with N6-methyl-N6-(4-isopropylphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (compound 22), of hDHFR complexed with compound 26 and of pcDHFR complexed with N6...
Source: Acta Crystallographica Section F - May 27, 2015 Category: Biochemistry Authors: Cody, V.Pace, J.Namjoshi, O.A.Gangjee, A. Tags: dihydrofolate reductase Pneumocystis carinii pathogens antifolate inhibitors conformational analysis research communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of the two distinct types of zebrafish β2-microglobulin
β2-Microglobulin (β2m) noncovalently associates with the heavy chain of major histocompatibility complex class I (MHC I) molecules, which bind foreign antigen peptides to control the cytotoxic T lymphocyte (CTL) immune response. In contrast to mammals, there are distinct types of β2ms derived from two loci in a number of teleost species. In order to clarify the structures of the β2ms, the zebrafish (Danio rerio) β2ms Dare-β2m-I and Dare-β2m-II were expressed in Escherichia coli, purified and crystallized, and diffraction data were collected to 1.6 and 1.9 Å resolution, respectivel...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Chen, Z.Zhang, N.Lu, S.Tariq, M.Wang, J.Xia, C. Tags: zebrafish β 2-microglobulin MHC class I research communications Source Type: research

A preliminary X-ray study of 3-deoxy-d-manno-oct-2-ulosonic acid 8-phosphate phosphatase (YrbI) from Burkholderia pseudomallei
In this study, YrbI from Burkholderia pseudomallei, the causative agent of melioidosis, has been cloned, expressed, purified and crystallized. Synchrotron X-ray data were also collected to 2.25 Å resolution. The crystal belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 63.7, b = 97.5, c = 98.0 Å. A full structural determination is in progress to elucidate the structure–function relationship of this protein. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Park, J.Lee, D.Kim, M.-S.Kim, D.Y.Shin, D.H. Tags: 3-deoxy-d-manno-oct-2-ulosonic acid 8-phosphate phosphatase YrbI Burkholderia pseudomallei research communications Source Type: research

Expression, purification, crystallization and preliminary X-ray analysis of CttA, a putative cellulose-binding protein from Ruminococcus flavefaciens
A number of anaerobic microorganisms produce multi-modular, multi-enzyme complexes termed cellulosomes. These extracellular macromolecular nanomachines are designed for the efficient degradation of plant cell-wall carbohydrates to smaller sugars that are subsequently used as a source of carbon and energy. Cellulolytic strains from the rumens of mammals, such as Ruminococcus flavefaciens, have been shown to have one of the most complex cellulosomal systems known. Cellulosome assembly requires the binding of dockerin modules located in cellulosomal enzymes to cohesin modules located in a macromolecular scaffolding protein. O...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Venditto, I.Bule, P.Thompson, A.Sanchez-Weatherby, J.Sandy, J.Ferreira, L.M.A.Fontes, C.M.G.A.Najmudin, S. Tags: X-dockerin cellulosome CttA Ruminococcus flavefaciens cell surface attachment cellulose-binding protein research communications Source Type: research

Crystallization and preliminary crystallographic analysis of the major acid phosphatase from Legionella pneumophila
The major acid phosphatase from Legionella pneumophila (LpMAP) belongs to the histidine acid phosphatase superfamily. It contains the characteristic histidine acid phosphatase (HAP) sequence motif RHGXRXP responsible for the hydrolysis of a phosphoryl group from phosphate monoesters under acidic conditions. Here, the crystallization and preliminary X-ray analysis of crystals of LpMAP in the apo form and in complex with l-(+)-tartrate are described. By using the hanging-drop vapour-diffusion method, apo LpMAP and LpMAP–tartrate were crystallized in space group P21, with unit-cell parameters a = 91.50, b = 56.48, ...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Zhou, D.Pan, Y.Chen, X.Zhang, N.Ge, H. Tags: Legionella pneumophila major acid phosphatase histidine acid phosphatase research communications Source Type: research

Protein production, crystallization and preliminary crystallographic analysis of the four N-terminal immunoglobulin domains of Down syndrome cell adhesion molecule 1
In this study, eight different isforms of Dscam1 Ig1–4 have been cloned, overexpressed, purified to homogeneity and crystallized. X-ray data were collected to 1.9–4.0 Å resolution. These structures will provide the opportunity to perform extensive structural comparisons of different Dscam1 isoforms and provide insight into its specificity. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Cheng, L.Li, S.-A.Yu, Y.Chen, Q. Tags: Dscam1 RNA splicing homophilic dimer horseshoe configuration crystallization research communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of the Csu pili CsuC–CsuA/B chaperone–major subunit pre-assembly complex from Acinetobacter baumannii
The attachment of many Gram-negative pathogens to biotic and abiotic surfaces is mediated by fimbrial adhesins, which are assembled via the classical, alternative and archaic chaperone–usher (CU) pathways. The archaic CU fimbrial adhesins have the widest phylogenetic distribution, yet very little is known about their structure and mechanism of assembly. To elucidate the biogenesis of archaic CU systems, structural analysis of the Csu fimbriae, which are used by Acinetobacter baumannii to form stable biofilms and cause nosocomial infection, was focused on. The major fimbriae subunit CsuA/B complexed with the CsuC chap...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Pakharukova, N.Tuittila, M.Paavilainen, S.Zavialov, A. Tags: chaperone – usher pathway archaic fimbriae biofilm Acinetobacter baumannii assembly adhesion research communications Source Type: research

Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of NAD synthetase from methicillin-resistant Staphylococcus aureus
Staphylococcus aureus is an important human and animal pathogen that causes a wide range of infections. The prevalence of multidrug-resistant S. aureus strains in both hospital and community settings makes it imperative to characterize new drug targets to combat S. aureus infections. In this context, enzymes involved in NAD metabolism and synthesis are significant drug targets as NAD is a central player in several cellular processes. NAD synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide, making it a crucial intermediate enzyme linked to the biosynthesis of several amino acids, puri...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Arbade, G.K.Srivastava, S.K. Tags: NAD synthetase Staphylococcus aureus research communications Source Type: research

Purification, crystallization and preliminary X-ray diffraction analysis of 3-ketoacyl-CoA thiolase A1887 from Ralstonia eutropha H16
The gene product of A1887 from Ralstonia eutropha (ReH16_A1887) has been annotated as a 3-ketoacyl-CoA thiolase, an enzyme that catalyzes the fourth step of β-oxidation degradative pathways by converting 3-ketoacyl-CoA to acyl-CoA. ReH16_A1887 was overexpressed and purified to homogeneity by affinity and size-exclusion chromatography. The degradative thiolase activity of the purified ReH16_A1887 was measured and enzyme-kinetic parameters for the protein were obtained, with Km, Vmax and kcat values of 158 µM, 32 mM min−1 and 5 × 106 s−1, respectively. The ReH16_A1887 protein was crystall...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Kim, J.Kim, K.-J. Tags: 3-ketoacyl-CoA thiolase Ralstonia eutropha β -oxidation degradative pathway research communications Source Type: research

Structure of the catalytic domain of Mre11 from Chaetomium thermophilum
Together with the Rad50 ATPase, the Mre11 nuclease forms an evolutionarily conserved protein complex that plays a central role in the repair of DNA double-strand breaks (DSBs). Mre11–Rad50 detects and processes DNA ends, and has functions in the tethering as well as the signalling of DSBs. The Mre11 dimer can bind one or two DNA ends or hairpins, and processes DNA endonucleolytically as well as exonucleolytically in the 3′-to-5′ direction. Here, the crystal structure of the Mre11 catalytic domain dimer from Chaetomium thermophilum (CtMre11CD) is reported. CtMre11CD crystals diffracted to 2.8 Å res...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Seifert, F.U.Lammens, K.Hopfner, K.-P. Tags: Mre11 nuclease MRN complex research communications Source Type: research

Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora
Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P212121 and P1211 and diffracted to ∼1.65 Å resolution. Co...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Molitor, C.Mauracher, S.G.Rompel, A. Tags: aurone synthase latent proenzyme polyphenol oxidase liquid – liquid phase separation polyoxometalate research communications Source Type: research

Structure of the MarR family protein Rv0880 from Mycobacterium tuberculosis
Rv0880 from the pathogen Mycobacterium tuberculosis is classified as a MarR family protein in the Pfam database. It consists of 143 amino acids and has an isoelectric point of 10.9. Crystals of Rv0880 belonged to space group P1, with unit-cell parameters a = 54.97, b = 69.60, c = 70.32 Å, α = 103.71, β = 111.06, γ = 105.83°. The structure of the MarR family transcription regulator Rv0880 was solved at a resolution of 2.0 Å with an Rcryst and Rfree of 21.2 and 24.9%, respectively. The dimeric structure resembles that of other MarR proteins, with each subunit comprising a winged helix&nda...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Gao, Y.-R.Feng, N.Chen, T.Li, D.-F.Bi, L.-J. Tags: MarR Mycobacterium tuberculosis transcription factor MarR family protein research communications Source Type: research

Crystallization and preliminary X-ray diffraction analysis of the CRISPR–Cas RNA-silencing Cmr complex
Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNA (crRNA) and CRISPR-associated (Cas) proteins constitute a prokaryotic adaptive immune system (CRISPR–Cas system) that targets and degrades invading genetic elements. The type III-B CRISPR–Cas Cmr complex, composed of the six Cas proteins (Cmr1–Cmr6) and a crRNA, captures and cleaves RNA complementary to the crRNA guide sequence. Here, a Cmr1-deficient functional Cmr (CmrΔ1) complex composed of Pyrococcus furiosus Cmr2–Cmr3, Archaeoglobus fulgidus Cmr4–Cmr5–Cmr6 and the 39-mer P. furiosus 7.01-crRNA was pr...
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Osawa, T.Inanaga, H.Numata, T. Tags: Cmr complex CRISPR-Cas crRNA research communications Source Type: research

Purification, crystallization and X-ray crystallographic studies of a Bacillus cereus MepR-like transcription factor, BC0657
In this study, a Bacillus cereus MepR-like transcription regulator, BC0657, was crystallized. The BC0657 crystals diffracted to 2.05 Å resolution and belonged to either space group P6222 or P6422, with unit-cell parameters a = 110.57, b = 110.57, c = 67.29 Å. There was one molecule per asymmetric unit. Future comparative structural studies on BC0657 would extend knowledge of ligand-induced transcriptional regulatory mechanisms in the MarR family and would make a significant contribution to the design of antibiotic drugs against multidrug-resistant bacteria. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - May 22, 2015 Category: Biochemistry Authors: Cho, M.U.Kim, M.I.Hong, M. Tags: MepR transcription factor Bacillus cereus research communications Source Type: research