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1.  Structure of MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) from Vibrio fischeri in complex with substrate UDP-N-acetylglucosamine and the drug fosfomycin 
The crystal structure of MurA from the marine Gram-negative bacterium Vibrio fischeri in complex with UNAG and the drug fosfomycin has been solved to a resolution of 1.93 Å.
The development of new antibiotics is necessitated by the rapid development of resistance to current therapies. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), which catalyzes the first committed step of bacterial peptidoglycan biosynthesis, is a prime candidate for therapeutic intervention. MurA is the target of the antibiotic fosfomycin, a natural product produced by Streptomyces. Despite possessing a high degree of sequence conservation with MurA enzymes from fosfomycin-susceptible organisms, recent microbiological studies suggest that MurA from Vibrio fischeri (VfiMurA) may confer fosfomycin resistance via a mechanism that is not yet understood. The crystal structure of VfiMurA in a ternary complex with the substrate UDP-N-acetylglucosamine (UNAG) and fosfomycin has been solved to a resolution of 1.93 Å. Fosfomycin is known to inhibit MurA by covalently binding to a highly conserved cysteine in the active site of the enzyme. A comparison of the title structure with the structure of fosfomycin-susceptible Haemophilus influenzae MurA (PDB entry 2rl2) revealed strikingly similar conformations of the mobile substrate-binding loop and clear electron density for a fosfomycin–cysteine adduct. Based on these results, there are no distinguishing sequence/structural features in VfiMurA that would translate to a diminished sensitivity to fosfomycin. However, VfiMurA is a robust crystallizer and shares high sequence identity with many clinically relevant bacterial pathogens. Thus, it would serve as an ideal system for use in the structure-guided optimization of new antibacterial agents.
doi:10.1107/S1744309112006720
PMCID: PMC3325803  PMID: 22505403
MurA; UDP-N-acetylglucosamine enolpyruvyl transferase; Vibrio fischeri; UDP-N-acetylglucosamine; fosfomycin
2.  Structures of the pleckstrin homology domain of Saccharomyces cerevisiae Avo1 and its human orthologue Sin1, an essential subunit of TOR complex 2 
The crystal structures of the C-terminal domain of S. cerevisiae Avo1 and of its human orthologue Sin1 have been determined. The structures show that the C-termini of the two proteins have the PH-domain fold with a putative binding site for phosphoinositides.
In eukaryotes, multiprotein complexes termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2) function as major regulators of cell growth, metabolism and ageing. The C-terminal domain of the Saccharomyces cerevisiae TORC2 component Avo1 is required for plasma-membrane localization of TORC2 and is essential for yeast viability. X-ray crystal structures of the C-­terminal domain of Avo1 and of its human orthologue Sin1 have been determined. The structures show that the C-termini of Avo1 and Sin1 both have the pleckstrin homology (PH) domain fold. Comparison with known PH-domain structures suggests a putative binding site for phosphoinositides.
doi:10.1107/S1744309112007178
PMCID: PMC3325804  PMID: 22505404
TOR signalling; TOR complex 2; Avo1; PH domains; Sin1
3.  Structure of the tetradecanucleotide d(CCCCGGTACCGGGG)2 as an A-DNA duplex 
The crystal structure of the tetradecanucleotide sequence d(CCCCGGTACCGGGG)2 as an A-DNA duplex and its interaction with a manganese ion are described.
The crystal structure of the tetradecanucleotide sequence d(CCCCGGTACC­GGGG)2 has been determined at 2.5 Å resolution in the tetragonal space group P41. This sequence was designed with the expectation of a four-way junction. However, the sequence crystallized as an A-DNA duplex and represents more than one full turn of the A-helix. The crystallographic asymmetric unit consists of one tetradecanucleotide duplex. The structural parameters of the A-type DNA duplex structure and the crystal-packing arrangement are described. One Mn2+ ion was identified with direct coordination to the N7 position of G13 and a water molecule at the major-groove side of the C2·G13 base pair.
doi:10.1107/S174430911200869X
PMCID: PMC3325805  PMID: 22505405
tetradecanucleotide; A-DNA duplex; crystal packing; ion interactions
4.  Structure of the His269Arg mutant of the rat aldose reductase-like protein AKR1B14 complexed with NADPH 
The 1.87 Å resolution crystal structure of the His269Arg mutant of rat aldose reductase-like protein (AKR1B14) complexed with NADPH is described.
Rat aldose reductase-like protein (AKR1B14) is an orthologue of mouse vas deferens protein (AKR1B7) and plays roles in the detoxification of reactive aldehydes and synthesis of prostaglandin F2α. Here, the 1.87 Å resolution crystal structure of the His269Arg mutant of AKR1B14 complexed with NADPH is described and shows that the negatively charged 2′-phosphate group of the coenzyme forms an ionic interaction with the positively charged guanidinium group of Arg269 that is also observed in the human aldose reductase (AKR1B1) structure. Previous experiments on the site-directed mutagenesis of His269 to Arg, Phe and Met revealed fourfold, sevenfold and 127-fold increases in the K m for NADPH, respectively, which are in agreement with the present molecular-modelling and X-ray crystallographic studies. This is the first tertiary structure of a mutant form of this AKR1B7 orthologue to be reported in order to investigate the structure–function relationship of the nonconserved His269 and its role in coenzyme binding.
doi:10.1107/S1744309112008810
PMCID: PMC3325806  PMID: 22505406
AKR1B14; NADPH; aldose reductases
5.  High-resolution crystal structures of factor XIa coagulation factor in complex with nonbasic high-affinity synthetic inhibitors 
Two structures of factor XIa in complex with small synthetic inhibitors are reported. These are the first structures of factor XIa complexed to small non-basic inhibitors.
Factor XI (FXI) is a key enzyme in the coagulation pathway and an attractive target for the development of anticoagulant drugs. A small number of high-resolution crystal structures of FXIa in complex with small synthetic inhibitors have been published to date. All of these ligands have a basic P1 group and bind exclusively in the nonprime side of the active site of FXIa. Here, two structures of FXIa in complex with nonbasic inhibitors that occupy both the prime and nonprime sides of the active site are presented. These new structures could be valuable in the design and optimization of new FXIa synthethic inhibitors.
doi:10.1107/S1744309112009037
PMCID: PMC3325807  PMID: 22505407
factor XIa; inhibitors; coagulation factors
6.  Structure of AKR1C3 with 3-phenoxybenzoic acid bound 
The structure of the nonsteroidal anti-inflammatory drug analogue 3-phenoxybenzoic acid bound to aldo–keto reductase 1C3 (AKR1C3) is presented as a lead for the discovery and development of novel agents for inhibiting AKR1C3, an anticancer target.
Aldo–keto reductase 1C3 (AKR1C3) is a human enzyme that catalyzes the NADPH-dependent reduction of steroids and prostaglandins. AKR1C3 overexpression is associated with the proliferation of hormone-dependent cancers, most notably breast and prostate cancers. Nonsteroidal anti-inflammatory drugs (NSAIDs) and their analogues are well characterized inhibitors of AKR1C3. Here, the X-­ray crystal structure of 3-phenoxybenzoic acid in complex with AKR1C3 is presented. This structure provides useful information for the future development of new anticancer agents by structure-guided drug design.
doi:10.1107/S1744309112009049
PMCID: PMC3325808  PMID: 22505408
nonsteroidal anti-inflammatory drugs; NSAIDs; aldo–keto reductase 1C3; AKR1C3; drug discovery
7.  Structure of Escherichia coli aspartate α-decarboxylase Asn72Ala: probing the role of Asn72 in pyruvoyl cofactor formation 
The crystal structure of E. coli aspartate α-decarboxylase Asn72Ala reveals the structure of the previously unresolved C-terminus.
The crystal structure of the Asn72Ala site-directed mutant of Escherichia coli aspartate α-decarboxylase (ADC) has been determined at 1.7 Å resolution. The refined structure is consistent with the presence of a hydrolysis product serine in the active site in place of the pyruvoyl group required for catalysis, which suggests that the role of Asn72 is to protect the ester formed during ADC activation from hydrolysis. In previously determined structures of activated ADC, including the wild type and other site-directed mutants, the C-terminal region of the protein is disordered, but in the Asn72Ala mutant these residues are ordered owing to an interaction with the active site of the neighbouring symmetry-related multimer.
doi:10.1107/S1744309112009487
PMCID: PMC3325809  PMID: 22505409
pantothenate; pyruvoyl-dependent; autocatalytic activation; crystallographic artefacts
8.  Purification, crystallization and preliminary crystallographic studies of a PacL homologue from Listeria monocytogenes  
A PacL homologue from L. monocytogenes has been purified and crystallized. The crystals diffracted to 3.2 Å resolution.
Ca2+-ATPases are members of a large family of membrane proteins that maintain the selective movement of cations across biological membranes. A putative Listeria monocytogenes Ca2+-ATPase (Lmo0818) was crystallized in an unknown functional state. The crystal belonged to space group P212121 and a complete data set was collected to 3.2 Å resolution. The molecular-replacement solution obtained revealed that Lmo0818 is likely to adopt an E2-­like state mimicking the phosphorylated intermediate in the functional cycle of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and a stacked bilayer ‘type I’ packing in the crystal.
doi:10.1107/S1744309112004046
PMCID: PMC3325811  PMID: 22505411
PacL; Lmo0818; P-type ATPases; transporters; membrane proteins
9.  Crystallization and crystallographic analysis of the ligand-binding domain of the Pseudomonas putida chemoreceptor McpS in complex with malate and succinate 
The crystallization of the ligand-binding domain of the methyl-accepting chemotaxis protein chemoreceptor McpS (McpS-LBD) is reported.
Methyl-accepting chemotaxis proteins (MCPs) are transmembrane proteins that sense changes in environmental signals, generating a chemotactic response and regulating other cellular processes. MCPs are composed of two main domains: a ligand-binding domain (LBD) and a cytosolic signalling domain (CSD). Here, the crystallization of the LBD of the chemoreceptor McpS (McpS-LBD) is reported. McpS-LBD is responsible for sensing most of the TCA-cycle intermediates in the soil bacterium Pseudomonas putida KT2440. McpS-LBD was expressed, purified and crystallized in complex with two of its natural ligands (malate and succinate). Crystals were obtained by both the counter-diffusion and the hanging-drop vapour-diffusion techniques after pre-incubation of McpS-LBD with the ligands. The crystals were isomorphous and belonged to space group C2, with two molecules per asymmetric unit. Diffraction data were collected at the ESRF synchrotron X-ray source to resolutions of 1.8 and 1.9 Å for the malate and succinate complexes, respectively.
doi:10.1107/S1744309112004940
PMCID: PMC3325812  PMID: 22505412
methyl-accepting chemotaxis proteins; ligand-binding domains; Pseudomonas putida KT2440
10.  Preparation, crystallization and preliminary X-ray diffraction studies of the glycosylated form of human interleukin-23 
Interleukin-23 (IL-23), a member of the IL-12 family, is a heterodimeric cytokine composed of p19 and p40 subunits. Human p19 and p40 subunits were cloned and coexpressed in N-acetylglucosaminyltransferase I-negative 293S cells. The glycosylated human IL-23 was purified and crystallized by the hanging-drop vapour-diffusion method.
Interleukin-23 (IL-23), a member of the IL-12 family, is a heterodimeric cytokine composed of p19 and p40 subunits. IL-23 plays crucial roles in the activation, proliferation and survival of IL-17-producing helper T cells which induce various autoimmune diseases. Human p19 and p40 subunits were cloned and coexpressed in N-acetylglucosaminyltransferase I-negative 293S cells, which produce high-mannose-type glycosylated proteins in order to diminish the heterogeneity of modified N-linked glycans. The glycosylated human IL-23 was purified and crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were then collected to 2.6 Å resolution. The crystal belonged to space group P61 or P65, with unit-cell parameters a = b = 108.94, c = 83.79 Å, γ = 120°. Assuming that the crystal contains one molecule per asymmetric unit, the calculated Matthews coefficient was 2.69 Å3 Da−1, with a solvent content of 54.2%. The structure was determined by the molecular-replacement method, with an initial R factor of 52.6%. After subsequent rigid-body and positional refinement, the R work and R free values decreased to 31.4% and 38.7%, respectively.
doi:10.1107/S1744309112005295
PMCID: PMC3325813  PMID: 22505413
interleukin-23; cytokines; glycosylation
11.  Crystallization and preliminary crystallographic characterization of the N-terminal Kunitz domain of boophilin 
The N-terminal Kunitz domain of boophilin, a specific thrombin inhibitor, was crystallized. The orthorhombic crystals had an unusually low solvent content and diffracted to beyond 0.87 Å resolution at a synchrotron source.
Boophilin is a tight-binding thrombin inhibitor composed of two canonical Kunitz-type domains in a tandem arrangement. Thrombin-bound boophilin can inhibit a second trypsin-like serine proteinase, most likely through the reactive loop of its N-terminal Kunitz domain. Here, the crystallization and preliminary crystallographic analysis of the isolated N-terminal domain of boophilin is reported. The crystals belonged to the orthorhombic space group P212121 and diffracted to beyond 1.8 Å resolution using a sealed-tube home source and to 0.87 Å resolution at a synchrotron source.
doi:10.1107/S1744309112005532
PMCID: PMC3325814  PMID: 22505414
thrombin inhibitors; noncovalent; blood coagulation; atomic resolution
12.  Overexpression, crystallization and preliminary X-­ray crystallographic analysis of pyridoxal biosynthesis lyase PdxS from Pyrococcus horikoshii  
Pyridoxal biosynthesis lyase PdxS from P. horikoshii has been overexpressed and crystallized. X-ray diffraction data have been collected to 2.61 Å resolution.
Pyridoxal biosynthesis lyase (PdxS) is an important player in the biosynthesis of pyridoxal 5′-phosphate (PLP), the biologically active form of vitamin B6. PLP is an important cofactor involved in the metabolic pathway of amine-containing natural products such as amino acids and amino sugars. PdxS catalyzes the condensation of ribulose 5-phosphate (Ru5P), glyceraldehyde 3-phosphate (G3P) and ammonia, while glutamine amidotransferase (PdxT) catalyzes the production of ammonia from glutamine. PdxS and PdxT form a complex, PLP synthase, and widely exist in eubacteria, archaea, fungi and plants. To facilitate further structural comparisons among PdxS proteins, the structural analysis of PdxS from Pyrococcus horikoshii encoded by the Ph1355 gene was initiated. PdxS from P. horikoshii was overexpressed in Escherichia coli and crystallized at 296 K using 2-methyl-2,4-pentanediol as a precipitant. Crystals of P. horikoshii PdxS diffracted to 2.61 Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 59.30, b = 178.56, c = 109.23 Å, β = 102.97°. The asymmetric unit contained six monomers, with a corresponding V M of 2.54 Å3 Da−1 and a solvent content of 51.5% by volume.
doi:10.1107/S1744309112005829
PMCID: PMC3325815  PMID: 22505415
Pyrococcus horikoshii; pdxS; pyridoxal biosynthesis lyase; pyridoxal 5′-phosphate
13.  Crystallization and preliminary X-ray diffraction analysis of a novel wild-type blue fluorescent protein from Vibrio vulnificus CKM-1 
The wild type BFPvv has been crystallized for the first time in order to obtain its tertiary structure and to further understand how chromophore formation occurs via a different oxygen-independent mechanism.
The use of green fluorescent protein (GFP) for non-invasive in vivo imaging is limited to aerobic systems, as chromophore formation requires oxygen. However, a novel NADPH-dependent blue fluorescent protein from Vibrio vulnificus CKM-1 (BFPvv) that emits blue fluorescence in both aerobic and anaerobic systems has recently been discovered. Wild-type BFPvv was overexpressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method. The resulting BFPvv crystals diffracted to a resolution of 1.9 Å and belonged to space group P3, with unit-cell parameters a = b = 96.62, c = 214.511 Å. Assuming the presence of eight molecules in the unit cell, the solvent content was estimated to be ∼56.16%.
doi:10.1107/S1744309112006355
PMCID: PMC3325817  PMID: 22505417
blue fluorescent protein; Vibrio vulnificus CKM-1
14.  Crystallization of a novel metal-containing cupin from Acidobacterium sp. and preliminary diffraction data analysis 
Recombinant AciX9_0562 from Acidobacterium sp. MP5ACTX9 containing sequence motifs characteristic of the RmlC-type cupins superfamily and containing Pfam motif PF07883 has been successfully cloned, expressed and purified, and crystallized in a number of conditions from the Morpheus protein crystallization screen.
Recombinant AciX9_0562 from Acidobacterium sp. MP5ACTX9 (UniProt ID E8WYN5) containing sequence motifs characteristic of the RmlC-type cupins superfamily and containing Pfam motif PF07883 has been successfully cloned, expressed and purified. AciX9_0562 crystallized in a number of conditions from the Morpheus protein crystallization screen. The best crystal diffracted to 2.7 Å resolution (space group C2221; unit-cell parameters a = 125.29, b = 254.63, c = 82.99 Å). Structure solution was facilitated by the automated molecular-replacement pipeline BALBES. The initial solution was automatically rebuilt using the PHENIX AutoBuild wizard, with final R and R free values of 0.23 and 0.26, respectively. The structure is currently undergoing manual refinement.
doi:10.1107/S1744309112006550
PMCID: PMC3325818  PMID: 22505418
cupins; AciX9_0562; Acidobacterium sp. MP5ACTX9
15.  Towards the crystal structure elucidation of eukaryotic UDP-galactopyranose mutase 
UDP-galactopyranose mutase from A. fumigatus was crystallized and structure solution is in progress.
UDP-galactopyranose mutase (UGM) catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose. Eukaryotic UGMs from Aspergillus fumigatus and Leishmania major have been purified to homogeneity by means of Ni2+-affinity chromatography and crystallized. Eukaryotic UGM structure elucidation was not straightforward owing to high pseudo-symmetry, twinning and very low anomalous signal. Phasing to 2.8 Å resolution using SAD was successful for L. major UGM. However, the maps could only be improved by iterative density modification and manual model building. High pseudo-symmetry and twinning prevented correct space-group assignment and the completion of structure refinement. The structure of A. fumigatus UGM to 2.52 Å resolution was determined by molecular replacement using the incomplete 2.8 Å resolution L. major UGM model.
doi:10.1107/S1744309112006914
PMCID: PMC3325819  PMID: 22505419
UDP-galactopyranose mutase; cell-wall biosynthesis; Aspergillus fumigatus
16.  Expression, purification, crystallization and preliminary X-ray diffraction analysis of nurse shark β2-microglobulin 
A crystal of nurse shark β2-microglobulin diffracted to 2.3 Å resolution and belonged to space group P3221, with unit-cell parameters a = b = 88.230, c = 67.146 Å and two molecules per asymmetric unit. The Matthews coefficient V M was calculated to be about 3.28 Å3 Da−1, corresponding to 62.5% solvent content.
β2-Microglobulin (β2m) is an essential subunit of the major histocompatibility complex (MHC) class I molecule that helps to stabilize the structure of peptide–MHC I (pMHC I). It is also one of the typical immunoglobulin superfamily (IgSF) molecules in the adaptive immune system (AIS). Sharks belong to the cartilaginous fish, which are the oldest jawed vertebrate ancestors with an AIS to exist in the world. Thus, the study of cartilaginous fish β2m would help in understanding the evolution of IgSF molecules. In order to demonstrate this, β2m from a cartilaginous fish, nurse shark (Ginglymostoma cirratum), was expressed, refolded, purified and crystallized. Diffraction data were collected to a resolution of 2.3 Å. The crystal belonged to space group P3221, with unit-cell parameters a = b = 88.230, c = 67.146 Å. The crystal structure contained two molecules in the asymmetric unit. The results will provide structural information for study of the evolution of β2m and IgSF in the AIS.
doi:10.1107/S1744309112006811
PMCID: PMC3325820  PMID: 22505420
nurse shark; β2-microglobulin
17.  Crystallization and preliminary X-ray analysis of stationary phase survival protein E (SurE) from Xylella fastidiosa in two crystal forms 
The crystallization and preliminary analysis of two crystal forms of survival protein E from X. fastidiosa are reported.
The bacterium Xylella fastidiosa is a phytopathogenic organism that causes citrus variegated chlorosis, a disease which attacks economically important crops, mainly oranges. In this communication, the crystallization and preliminary X-ray crystallographic analysis of XfSurE, a survival protein E from X. fastidiosa, are reported. Data were collected for two crystal forms, I and II, to 1.93 and 2.9 Å resolution, respectively. Crystal form I belonged to space group C2, with unit-cell parameters a = 172.36, b = 84.18, c = 87.24 Å, α = γ = 90, β = 96.59°, whereas crystal form II belonged to space group C2, with unit-cell parameters a = 88.05, b = 81.26, c = 72.84 Å, α = γ = 90, β = 94.76°.
doi:10.1107/S1744309112007129
PMCID: PMC3325821  PMID: 22505421
SurE; Xylella fastidiosa; citrus variegated chlorosis
18.  Crystallization and preliminary X-ray diffraction analysis of human endoplasmic reticulum aminopeptidase 2 
The luminal domain of human endoplasmic reticulum aminopeptidase 2 has been expressed, purified and crystallized. The crystals belonged to the orthorhombic space group P21212 and diffracted anisotropically to 3.3 Å resolution in the best direction on an in-house source.
Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a critical enzyme involved in the final processing of MHC class I antigens. Peptide trimming by ERAP2 and the other members of the oxytocinase subfamily is essential to customize longer precursor peptides in order to fit them to the correct length required for presentation on major histocompatibility complex class I molecules. While recent structures of ERAP1 have provided an understanding of the ‘molecular-ruler’ mechanism of substrate selection, little is known about the complementary activities of its homologue ERAP2 despite their sharing 49% sequence identity. In order to gain insights into the structure–function relationship of the oxytocinase subfamily, and in particular ERAP2, the luminal region of human ERAP2 has been crystallized in the presence of the inhibitor bestatin. The crystals belonged to an orthorhombic space group and diffracted anisotropically to 3.3 Å resolution in the best direction on an in-house X-ray source. A molecular-replacement solution suggested that the enzyme has adopted the closed state as has been observed in other inhibitor-bound aminopeptidase structures.
doi:10.1107/S1744309112006963
PMCID: PMC3325822  PMID: 22505422
aminopeptidases; antigen processing; endoplasmic reticulum aminopeptidase 2; ERAP2; MHC presentation
19.  Crystallization and preliminary crystallographic analysis of the complex between triiodothyronine and the bb′ fragment of rat protein disulfide isomerase 
Protein disulfide isomerase (PDI) is known to bind to the thyroid hormone triiodothyronine (T3). Here, the expression, crystallization and preliminary X-ray studies of the b and b′ domains of rat PDI in ligand-free and T3-complexed states are reported.
Protein disulfide isomerase (PDI) is a multifunctional protein that catalyzes the formation of a disulfide bond in nascent and misfolded proteins and is also known to bind to the thyroid hormone triiodothyronine (T3). When T3 is bound to PDI its catalytic activity is inhibited, but the biological function of this binding is not well understood. In previous studies, it was found that T3 binds to the bb′ fragment of PDI. Therefore, to clarify the structure of the complex consisting of PDI bound to T3, a crystallographic analysis of the three-dimensional structure of the T3–rat PDI bb′ complex was performed. Native bb′ crystals and T3–bb′ complex crystals were both obtained using the hanging-drop vapour-diffusion technique with 1.6 M trisodium citrate pH 6.2 as a precipitant. The space group of the native bb′ crystals was found to be C222, with unit-cell parameters a = 94.8, b = 114.9, c = 182.9 Å, while the space group of the T3–bb′ complex crystals was P212121, with unit-cell parameters a = 99.9, b = 184.5, c = 232.2 Å. Diffraction data for the native and complex crystals were collected to resolutions of 3.06 and 3.00 Å, respectively.
doi:10.1107/S1744309112007439
PMCID: PMC3325824  PMID: 22505424
protein disulfide isomerase; triiodothyronine; bisphenol A; bb′ fragment
20.  Crystallization and preliminary X-ray diffraction studies of the abscisic acid receptor PYL3 and its complex with pyrabactin 
Crystals of the abscisic acid receptor PYL3 and of the PYL3–pyrabactin complex were obtained and optimized in order to obtain high-quality diffraction data. Diffraction data sets were collected and processed to 2.5 and 1.83 Å resolution, respectively.
Abscisic acid (ABA) modulates many developmental processes and responses to environmental stress. Recently, a family of pyrabactin resistance-like proteins (PYLs) in Arabidopsis thaliana were identified to be abscisic acid receptors. Although the 14 PYLs members share a similar sequence identity, they exhibit different responses toward pyrabactin. Apo-PYL3 is a dimer; however, its oligomeric state changes greatly on the addition of pyrabactin. Moreover, pyrabactin binds dimeric PYL3 in a nonproductive mode which prevents receptor activation and inhibition of PP2Cs. Here, the expression, purification and crystallization of apo-PYL3 and of PYL3 complexed with pyrabactin are reported. Diffraction data were optimized to 2.5 Å resolution for apo-PYL3 and to 1.83 Å resolution for PYL3–pyrabactin. The crystals of apo-PYL3 and PYL3–pyrabactin belonged to space groups P41212 and P212121, respectively.
doi:10.1107/S1744309112007506
PMCID: PMC3325825  PMID: 22505425
PYL3; abscisic acid receptors; pyrabactin
21.  Cocrystallization and preliminary crystallographic analysis of an inactive MaoC-like hydratase mutant with the substrate crotonyl-CoA 
An inactive MaoC-like hydratase mutant from P. capsici was obtained and purified, and the purified mutant was cocrystallized in the presence of the substrate crotonyl-CoA.
MaoC-like hydratase (MaoC) is a recently identified enzyme involved in the biosynthetic pathway of polyhydroxyalkanoates (PHAs), which are completely biodegradable polymers used to produce green plastics. The inactive mutant D194N-MaoC was crystallized in the presence of the substrate crotonyl-CoA. Crystals were grown in a number of conditions, but only those produced using 20%(v/v) ethylene glycol were suitable for structural studies. Data were collected to 2.10 Å resolution using X-radiation and the crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 81.40, b = 82.58, c = 123.99 Å.
doi:10.1107/S1744309112007671
PMCID: PMC3325826  PMID: 22505426
MaoC-like hydratase; crotonyl-CoA; Phytophthora capsici
22.  In praise of impurity: 30S ribosomal S15 protein-assisted crystallization of turnip yellow mosaic virus proteinase 
Diffraction-quality crystals of the turnip yellow mosaic virus proteinase/ubiquitin hydrolase could only be obtained from a protein preparation that was heavily contaminated with E. coli 30S ribosomal S15 protein. Crystal packing reveals the basis of this observation.
Turnip yellow mosaic virus is an excellent model for eukaryotic positive-stranded RNA virus replication. Correct processing of the replication polyprotein is dependent on the virally encoded cysteine proteinase (PRO) domain. Crystalline needles obtained from highly pure preparations of the recombinant 17.6 kDa PRO did not diffract. In contrast, small hexagonal prisms that were obtained together with the needles under the same conditions but from a poorly purified preparation diffracted to 2 Å resolution and allowed structure determination by MIRAS. It turned out that the hexagonal crystals contained stoichiometric amounts of PRO and Escherichia coli 30S ribosomal S15, a 10.1 kDa protein commonly co-purified by immobilized metal-affinity chromatography. The solvent content is nearly 70%, with S15 bridging parallel infinite helices of PRO across large solvent channels. With hindsight, this spurious interaction not only yielded diffraction-quality crystals but would also have allowed structure determination by molecular replacement using S15 as a search model and subsequent automatic rebuilding of the asymmetric unit.
doi:10.1107/S1744309112008445
PMCID: PMC3325827  PMID: 22505427
Tymoviridae; proteinases; 30S ribosomal S15 protein
23.  Purification, crystallization and preliminary X-ray analysis of OsAREB8 from rice, a member of the AREB/ABF family of bZIP transcription factors, in complex with its cognate DNA 
OsAREB8 from rice (O. sativa), a member of the AREB/ABF family of bZIP transcription factors, was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. A crystal of OsAREB8 in complex with its cognate DNA diffracted X-rays to 3.65 Å resolution.
The AREB/ABF family of bZIP transcription factors play a key role in drought stress response and tolerance during the vegetative stage in plants. To reveal the DNA-recognition mechanism of the AREB/ABF family of proteins, the bZIP domain of OsAREB8, an AREB/ABF-family protein from Oryza sativa, was expressed in Escherichia coli, purified and crystallized with its cognate DNA. Crystals of the OsAREB8–DNA complex were obtained by the sitting-drop vapour-diffusion method at 277 K with a reservoir solution consisting of 50 mM MES pH 6.4, 29% MPD, 2 mM spermidine, 20 mM magnesium acetate and 100 mM sodium chloride. A crystal diffracted X-rays to 3.65 Å resolution and belonged to space group C222, with unit-cell parameters a = 155.1, b = 206.7, c = 38.5 Å. The crystal contained one OsAREB8–DNA complex in the asymmetric unit.
doi:10.1107/S1744309112009384
PMCID: PMC3325828  PMID: 22505428
AREB/ABF family; stress response; Oryza sativa
24.  Fast high-pressure freezing of protein crystals in their mother liquor 
Protein crystals were vitrified using high-pressure freezing in their mother liquor at 210 MPa and 77 K without cryoprotectants or oil coating. The method was successfully applied to photosystem II, which is representative of a membrane protein with a large unit cell and weak crystal contacts.
High-pressure freezing (HPF) is a method which allows sample vitrification without cryoprotectants. In the present work, protein crystals were cooled to cryogenic temperatures at a pressure of 210 MPa. In contrast to other HPF methods published to date in the field of cryocrystallography, this protocol involves rapid sample cooling using a standard HPF device. The fast cooling rates allow HPF of protein crystals directly in their mother liquor without the need for cryoprotectants or external reagents. HPF was first attempted with hen egg-white lysozyme and cubic insulin crystals, yielding good to excellent diffraction quality. Non-cryoprotected crystals of the membrane protein photosystem II have been successfully cryocooled for the first time. This indicates that the presented HPF method is well suited to the vitrification of challenging systems with large unit cells and weak crystal contacts.
doi:10.1107/S1744309112009670
PMCID: PMC3325829  PMID: 22505429
high-pressure freezing; cryocooling
25.  Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix 
The deer mouse, Peromyscus maniculatus, exhibits altitude-associated variation in hemoglobin oxygen affinity. To examine the structural basis of this functional variation, the structure of the hemoglobin has been solved.
The deer mouse, Peromyscus maniculatus, exhibits altitude-associated variation in hemoglobin oxygen affinity. To examine the structural basis of this functional variation, the structure of the hemoglobin was solved. Recombinant hemoglobin was expressed in Escherichia coli and was purified by ion-exchange chromatography. Recombinant hemoglobin was crystallized by the hanging-drop vapor-diffusion method using polyethylene glycol as a precipitant. The obtained orthorhombic crystal contained two subunits in the asymmetric unit. The refined structure was interpreted as the aquo-met form. Structural comparisons were performed among hemoglobins from deer mouse, house mouse and human. In contrast to human hemoglobin, deer mouse hemoglobin lacks the hydrogen bond between α1Trp14 in the A helix and α1Thr67 in the E helix owing to the Thr67Ala substitution. In addition, deer mouse hemoglobin has a unique hydrogen bond at the α1β1 interface between residues α1Cys34 and β1Ser128.
doi:10.1107/S1744309113005708
PMCID: PMC3614163  PMID: 23545644
hemoglobin; deer mouse; oxygen affinity; Peromyscus maniculatus

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