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1.  Structure of Escherichia coli AdhP (ethanol-inducible dehydrogenase) with bound NAD 
The crystal structure of E. coli AdhP (ethanol-inducible dehydrogenase) in complex with NAD is reported.
The crystal structure of AdhP, a recombinantly expressed alcohol dehydrogenase from Escherichia coli K-12 (substrain MG1655), was determined to 2.01 Å resolution. The structure, which was solved using molecular replacement, also included the structural and catalytic zinc ions and the cofactor nicotinamide adenine dinucleotide (NAD). The crystals belonged to space group P21, with unit-cell parameters a = 68.18, b = 118.92, c = 97.87 Å, β = 106.41°. The final R factor and R free were 0.138 and 0.184, respectively. The structure of the active site of AdhP suggested a number of residues that may participate in a proton relay, and the overall structure of AdhP, including the coordination to structural and active-site zinc ions, is similar to those of other tetrameric alcohol dehydrogenase enzymes.
doi:10.1107/S1744309113015170
PMCID: PMC3702314  PMID: 23832197
alcohol dehydrogenases; AdhP; Escherichia coli
2.  Structures of MauG in complex with quinol and quinone MADH 
Crystal structures of MauG in complexes with MADH with its TTQ cofactor in the quinone and quinol forms are reported.
MauG has been cocrystallized with methylamine dehydrogenase (MADH) with its TTQ cofactor in the o-quinol (TTQOQ) and quinone (TTQOX) forms and the structures of the resulting complexes have been solved. The TTQOQ structure crystallized in either space group P21 or C2, while the TTQOX structure crystallized in space group P1. The previously solved structure of MauG in complex with MADH bearing an incompletely formed TTQ cofactor (preMADH) also crystallized in space group P1, although with different unit-cell parameters. Despite the changes in crystal form, the structures are virtually identical, with only very minor changes at the protein–protein interface. The relevance of these structures with respect to the measured changes in affinity between MauG and various forms of MADH is discussed.
doi:10.1107/S1744309113016539
PMCID: PMC3702316  PMID: 23832199
heme; tryptophan tryptophylquinone; long-range electron transfer; protein complex
3.  Conformational stability and crystal packing: polymorphism in Neurospora crassa CAT-3 
Comparisons between polymorphic structures in space groups P1, P212121 and P43212 highlight the conformational stability of tetrameric CAT-3 and reveal a distortion in the tetrameric structure that has not previously been described.
Polymorphism is frequently observed from different crystallization conditions. In proteins, the effect on conformational variability is poorly documented, with only a few reported examples. Here, three polymorphic crystal structures determined for a large-subunit catalase, CAT-3 from Neurospora crassa, are reported. Two of them belonged to new space groups, P1 and P43212, and a third structure belonged to the same space group, P212121, as the previously deposited 2.3 Å resolution structure (PDB entry 3ej6), but had a higher resolution (1.95 Å). Comparisons between these polymorphic structures highlight the conformational stability of tetrameric CAT-3 and reveal a distortion in the tetrameric structure that has not previously been described.
doi:10.1107/S1744309113013468
PMCID: PMC3702318  PMID: 23832201
4.  Crystallization and preliminary X-ray analysis of the C-terminal domain of δ-COP, a medium-sized subunit of the COPI complex involved in membrane trafficking 
The purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal domain of δ-COP, a medium-sized subunit of the COPI complex, are reported.
Coat protein I (COPI) is a protein complex composed of seven subunits that mediates retrograde transport of proteins and lipids from the cis-Golgi network to the endoplasmic reticulum and intra-Golgi membranes. The medium-sized δ subunit of COPI (δ-COP) is a 57 kDa protein with a C-terminal domain (CTD) and an N-­terminal longin domain. Here, the δ-COP CTD was successfully cloned, purified and crystallized. Diffraction data were collected from native and selenomethionyl crystals of δ-COP CTD to resolutions of 2.60 and 2.30 Å, respectively. Both crystals belonged to space group P21212, with similar unit-cell parameters. The native crystals had unit-cell parameters a = 100.23, b = 136.77, c = 44.39 Å.
doi:10.1107/S1744309112022798
PMCID: PMC3388933  PMID: 22750876
COPI complex; δ-COP C-terminal domain; membrane trafficking
5.  Purification and crystallization of yeast Ent1 ENTH domain 
The yeast Epsin-1 (ent1) gene has been cloned and expressed in Escherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method. Refined conditions containing polyethylene glycol 3350 and Tacsimate yielded thin rod-like crystals.
Members of the Epsin protein family regulate the ubiquitin/clathrin-dependent trafficking of transmembrane proteins. The yeast Epsin-1 (ent1) gene was cloned and expressed in Escherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method. Refined conditions containing polyethylene glycol 3350 and Tacsimate yielded thin rod-like crystals. X-ray analysis revealed that the crystallographic symmetry is primitive orthorhombic, space group P222, with unit-cell parameters a = 32.7, b = 35.5, c = 110.6 Å and a diffraction limit of 2.3 Å. Matthews coefficient calculations suggested that the crystal contained only the ENTH domain. This was corroborated by Coomassie Blue-stained SDS–PAGE analysis of dissolved crystals.
doi:10.1107/S1744309112022488
PMCID: PMC3388931  PMID: 22750874
Epsin; ENTH domain; ubiquitylation/clathrin-dependent endocytosis
6.  Purification, crystallization and preliminary X-ray diffraction analysis of a hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) from Coffea canephora involved in chlorogenic acid biosynthesis 
A hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase involved in chlorogenic acid biosynthesis in C. canephora was crystallized using the vapour-diffusion method. A diffraction data set was collected to 3.0 Å resolution on the microfocus beamline (ID23-2) at ESRF and a structure solution was obtained using molecular replacement.
Chlorogenic acids (CGAs) are a group of soluble phenolic compounds that are produced by a variety of plants, including Coffea canephora (robusta coffee). The last step in CGA biosynthesis is generally catalysed by a specific hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferase (HQT), but it can also be catalysed by the more widely distributed hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT). Here, the cloning and overexpression of HCT from C. canephora in Escherichia coli as well as its purification and crystallization are presented. Crystals were obtained by the sitting-drop technique at 293 K and X-ray diffraction data were collected on the microfocus beamline ID23-2 at the ESRF. The HCT crystals diffracted to better than 3.0 Å resolution, belonged to space group P42212 with unit-cell parameters a = b = 116.1, c = 158.9 Å and contained two molecules in the asymmetric unit. The structure was solved by molecular replacement and is currently under refinement. Such structural data are needed to decipher the molecular basis of the substrate specifities of this key enzyme, which belongs to the large plant acyl-CoA-dependent BAHD acyltransferase superfamily.
doi:10.1107/S1744309112019082
PMCID: PMC3388932  PMID: 22750875
Coffea canephora; phenylpropanoid-biosynthesis pathway; chlorogenic acids; plant acyl-CoA-dependent acyltransferase superfamily; hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase; molecular replacement
7.  Purification, crystallization and preliminary X-ray diffraction analysis of inner membrane complex (IMC) subcompartment protein 1 (ISP1) from Toxoplasma gondii  
To characterize the ISP family of proteins present in apicomplexan parasites, ISP1 from T. gondii was expressed, purified and crystallized. Two crystal forms (cubic and orthorhombic) were analyzed by X-ray diffraction and data were processed to 2.05 and 2.1 Å resolution, respectively.
The protozoan parasites of the Apicomplexa phylum are devastating global pathogens. Their success is largely due to phylum-specific proteins found in specialized organelles and cellular structures. The inner membrane complex (IMC) is a unique apicomplexan structure that is essential for motility, invasion and replication. The IMC subcompartment proteins (ISP) have recently been identified in Toxoplasma gondii and shown to be critical for replication, although their specific mechanisms are unknown. Structural characterization of TgISP1 was pursued in order to identify the fold adopted by the ISPs and to generate detailed insight into how this family of proteins functions during replication. An N-terminally truncated form of TgISP1 was purified from Escherichia coli, crystallized and subjected to X-ray diffraction analysis. Two crystal forms of TgISP1 belonging to space groups P4132 or P4332 and P212121 diffracted to 2.05 and 2.1 Å resolution, respectively.
doi:10.1107/S174430911202297X
PMCID: PMC3388934  PMID: 22750877
Toxoplasma gondii; inner membrane complex; ISP1
8.  Expression, purification, crystallization and preliminary X-ray diffraction analysis of Deg5 from Arabidopsis thaliana  
A protease involved in turnover of a photosynthesis reaction centre protein has been crystallized and preliminary diffraction analysis was performed.
Arabidopsis thaliana Deg5 is an ATP-independent serine protease which resides on the luminal side of the thylakoid in chloroplasts. Deg5 and another Deg/HtrA-family protease, Deg8, have a synergistic function in the turnover of the D1 protein of photosystem II (PSII), which is prone to damage arising from high light exposure. An inactive mutant of the protein, Deg5S266A, was overexpressed in Escherichia coli. After purification and crystallization, crystals that diffracted to 2.6 Å resolution were obtained. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 109.1, b = 126.0, c = 83.3 Å, β = 102.9°, and contained three molecules in the asymmetric unit. The calculated Matthews coefficient and solvent content were 3.0 Å3 Da−1 and 59.0%, respectively.
doi:10.1107/S1744309112023603
PMCID: PMC3388936  PMID: 22750879
Deg5; photosystem II
9.  Purification, crystallization and preliminary X-ray diffraction analysis of the effector protein PevD1 from Verticillium dahliae  
The overexpression, purification, crystallization and preliminary X-ray diffraction analysis of protein elicitor PevD1 from Verticillium dahliae are reported.
The effector protein PevD1 from the pathogenic fungus Verticillium dahliae was purified and crystallized using the hanging-drop vapour-diffusion method. Native crystals appeared in a solution consisting of 4.0 M sodium formate. A native data set was collected at 1.9 Å resolution at 100 K using an in-house X-ray source. Because of the absence of useful methinione in the protein sequence, derivative crystals that contained iodine were obtained by soaking in 1.25 M potassium iodide, and a data set that contained anomalous signal was collected using the same X-ray facility at a wavelength of 1.54 Å. The single-wavelength anomalous dispersion method was used to successfully solve the structure based on the anomalous signal generated from iodine.
doi:10.1107/S1744309112020556
PMCID: PMC3388926  PMID: 22750869
PevD1; effector proteins; Verticillium dahliae
10.  Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp1 
The extracellular region of mouse Enpp1 was expressed, purified and crystallized. An X-ray diffraction data set was collected to 3.0 Å resolution by employing a helical data-collection strategy involving a micro-focus synchrotron beam.
Enpp1 is an extracellular membrane-bound glycoprotein that regulates bone mineralization by hydrolyzing ATP to generate pyrophosphate. The extracellular region of mouse Enpp1 was expressed in HEK293S GnT1− cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-­ray diffraction data set was collected to 3.0 Å resolution. The crystal belonged to space group P31, with unit-cell parameters a = b = 105.3, c = 173.7 Å. A single-wavelength anomalous dispersion (SAD) data set was also collected to 2.7 Å resolution using a selenomethionine-labelled crystal. The experimental phases determined by the SAD method produced an interpretable electron-density map.
doi:10.1107/S1744309112019306
PMCID: PMC3388920  PMID: 22750863
Enpp1
11.  Crystallization and preliminary X-ray diffraction analysis of the high molecular weight ketoacyl reductase FabG4 complexed with NADH 
The cloning, overexpression, purification, crystallization in a triclinic space group and preliminary X-ray diffraction analysis of the high-molecular-weight ketoacyl reductase FabG4 complexed with NADH are reported.
FabG4 from Mycobacterium tuberculosis belongs to the high molecular weight ketoacyl reductases (HMwFabGs). The enzyme requires NADH for β-ketoacyl reductase activity. The protein was overexpressed, purified to homogeneity and crystallized as a FabG4–NADH complex. A mountable FabG4:NADH complex crystal diffracted to 2.59 Å resolution and belonged to space group P1, with unit-cell parameters a = 63.07, b = 71.03, c = 92.92 Å, α = 105.02, β = 97.06, γ = 93.66°. The Matthews coefficient suggested the presence of four monomers in the unit cell. In addition, a self-rotation function revealed the presence of two twofold NCS axes and one fourfold NCS axis. At χ = 180° the highest peak corresponds to the twofold NCS between two monomers, whereas the second peak corresponds to the twofold NCS between two dimers.
doi:10.1107/S1744309112020301
PMCID: PMC3388922  PMID: 22750865
FabG4; high-molecular-weight ketoacyl reductases; Mycobacterium tuberculosis
12.  Expression, purification, crystallization and preliminary crystallographic analysis of the phosphoglycerate kinase from Acinetobacter baumannii  
Approximately five decades have passed with only one or two new antibiotics making it into clinical use. Phosphoglycerate kinase from A. baumanii has been selected as a potential target for antibiotic development; this paper presents the initial structural biological results from this research.
Acinetobacter baumannii is a common multidrug-resistant clinical pathogen that is often found in hospitals. The A. baumannii phosphoglycerate kinase (AbPGK) is involved in the key energy-producing pathway of glycolysis and presents a potential target for antibiotic development. AbPGK has been expressed and purified; it was crystallized using lithium sulfate as the precipitant. The AbPGK crystals belonged to space group P2221. They diffracted to a resolution of 2.5 Å using synchrotron radiation at the Canadian Light Source.
doi:10.1107/S1744309112020222
PMCID: PMC3388923  PMID: 22750866
phosphoglycerate kinase; Acinetobacter baumannii
13.  Purification, crystallization and preliminary crystallographic analysis of the adhesion domain of Epf from Streptococcus pyogenes  
The putative adhesion domain of the multidomain protein Epf from S. pyogenes has been crystallized in space groups P21 and P212121. The crystals diffracted to 2.0 and 1.6 Å resolution, respectively, at the Australian Synchrotron.
The extracellular protein Epf from Streptococcus pyogenes is important for streptococcal adhesion to human epithelial cells. However, Epf has no sequence identity to any protein of known structure or function. Thus, several predicted domains of the 205 kDa protein Epf were cloned separately and expressed in Escherichia coli. The N-terminal domain of Epf was crystallized in space groups P21 and P212121 in the presence of the protease chymotrypsin. Mass spectrometry showed that the species crystallized corresponded to a fragment comprising residues 52–357 of Epf. Complete data sets were collected to 2.0 and 1.6 Å resolution, respectively, at the Australian Synchrotron.
doi:10.1107/S1744309112020313
PMCID: PMC3388924  PMID: 22750867
Epf; Streptococcus pyogenes; adhesion domain
14.  Crystallization and preliminary X-ray diffraction studies of the GhKCH2 motor domain: alteration of pH significantly improved the quality of the crystals 
The GhKCH2 motor domain was crystallized and the pH of the crystallization buffer was shown to have a significant effect on the crystal morphology and diffraction quality.
GhKCH2, a member of the kinesin superfamily, is a plant-specific microtubule-dependent motor protein from cotton with the ability to bind to both microtubules and microfilaments. Here, the motor domain of GhKCH2 (GhKCH2MD; amino acids 371–748) was overexpressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method. The pH of the crystallization buffer was shown to have a significant effect on the crystal morphology and diffraction quality. The crystals belonged to space group P212121, with unit-cell parameters a = 60.7, b = 78.6, c = 162.8 Å, α = β = γ = 90°. The Matthews coefficient and solvent content were calculated as 2.27 Å3 Da−1 and 45.87%, respectively. X-ray diffraction data for GhKCH2MD were collected on beamline BL17U1 at Shanghai Synchrotron Radiation Facility and processed to 2.8 Å resolution.
doi:10.1107/S1744309112016351
PMCID: PMC3388925  PMID: 22750868
kinesins; pH; crystal morphology; diffraction quality
15.  Crystallization and preliminary X-ray crystallographic analysis of an ice-binding protein (FfIBP) from Flavobacterium frigoris PS1 
The ice-binding protein FfIBP from F. frigoris PS1 was overexpressed, purified, characterized and crystallized, and preliminary X-ray crystallographic analysis was performed.
Ice growth in a cold environment is fatal for polar organisms, not only because of the physical destruction of inner cell organelles but also because of the resulting chemical damage owing to processes such as osmotic shock. The properties of ice-binding proteins (IBPs), which include antifreeze proteins (AFPs), have been characterized and IBPs exhibit the ability to inhibit ice growth by binding to specific ice planes and lowering the freezing point. An ice-binding protein (FfIBP) from the Gram-negative bacterium Flavobacterium frigoris PS1, which was isolated from the Antarctic, has recently been overexpressed. Interestingly, the thermal hysteresis activity of FfIBP was approximately 2.5 K at 50 µM, which is ten times higher than that of the moderately active IBP from Arctic yeast (LeIBP). Although FfIBP closely resembles LeIBP in its amino-acid sequence, the antifreeze activity of FfIBP appears to be much greater than that of LeIBP. In an effort to understand the reason for this difference, an attempt was made to solve the crystal structure of FfIBP. Here, the crystallization and X-­ray diffraction data of FfIBP are reported. FfIBP was crystallized using the hanging-drop vapour-diffusion method with 0.1 M sodium acetate pH 4.4 and 3 M sodium chloride as precipitant. A complete diffraction data set was collected to a resolution of 2.9 Å. The crystal belonged to space group P4122, with unit-cell parameters a = b = 69.4, c = 178.2 Å. The asymmetric unit contained one monomer.
doi:10.1107/S1744309112020465
PMCID: PMC3388927  PMID: 22750870
antifreeze proteins; ice-binding proteins; Flavobacterium frigoris PS1; psychrophilic bacteria
16.  Expression, purification, crystallization and preliminary X-ray analysis of the d-alanyl carrier protein DltC from Staphylococcus epidermidis  
The S. epidermidis carrier protein DltC has been crystallized in order to elucidate the functional role of DltC in the alanylation of lipoteichoic acids in bacteria.
The d-alanyl lipoteichoic acids (d-alanyl LTAs) present in the cell walls of Gram-positive bacteria play crucial roles in autolysis, cation homeostasis and biofilm formation. The alanylation of LTAs requires the d-alanyl carrier protein DltC to transfer d-Ala onto a membrane-associated LTA. Here, DltC from Staphylococcus epidermidis (SeDltC) was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.83 Å and belonged to space group P2, with unit-cell parameters a = 66.26, b = 53.28, c = 88.05 Å, β = 98.22°. The results give a preliminary crystallographic analysis of SeDltC and shed light on the functional role of DltC in the alanylation of LTAs.
doi:10.1107/S1744309112021720
PMCID: PMC3388928  PMID: 22750871
d-alanyl lipoteichoic acids; lipoteichoic acids; Staphylococcus epidermidis
17.  Expression, purification, crystallization and preliminary X-ray analysis of the human NORE1 SARAH domain 
The crystallization of the human NORE1 SARAH domain is reported.
NORE1 is an important tumour suppressor in human cancers that interacts with the pro-apoptotic protein kinase MST1/2 through SARAH domains. The SARAH domain (residues 366–413) of human NORE1 was expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal diffracted to 2.7 Å resolution and belonged to space group P6122, with unit-cell parameters a = b = 73.041, c = 66.092 Å, α = β = 90, γ = 120°.
doi:10.1107/S1744309112021744
PMCID: PMC3388929  PMID: 22750872
NORE1; apoptosis; tumour suppressors; Ras
18.  Crystallization and preliminary crystallographic studies of CCM3 in complex with the C-terminal domain of MST4 
The complex of CCM3 and the C-terminal domain of MST4 has been successfully constructed, purified and crystallized. The crystal diffracted to a resolution of 2.4 Å.
MST4 is a member of the GCKIII kinases. The interaction between cerebral cavernous malformation 3 (CCM3) and GCKIII kinases plays a critical role in cardiovascular development and in cerebral cavernous malformations. The complex of CCM3 and the C-terminal domain of MST4 has been constructed, purified and crystallized, and a diffraction data set has been collected to 2.4 Å resolution. The crystal of the CCM3–MST4 C-terminal domain complex belonged to space group P41212 or P43212, with unit-cell parameters a = 69.10, b = 69.10, c = 117.57 Å.
doi:10.1107/S1744309112016843
PMCID: PMC3388915  PMID: 22750858
cerebral cavernous malformations; GCKIII kinases; CCM3–MST4 C-terminal domain complex
19.  Structure of the signal transduction protein TRAP (target of RNAIII-activating protein) 
The 1.85 Å resolution structure of the signal transduction protein TRAP is presented. The overall fold of TRAP is an unsymmetrical eight-stranded β-barrel with five helices.
The crystal structure of the signal transduction protein TRAP is reported at 1.85 Å resolution. The structure of TRAP consists of a central eight-stranded β-­barrel flanked asymmetrically by helices and is monomeric both in solution and in the crystal structure. A formate ion was found bound to TRAP identically in all four molecules in the asymmetric unit.
doi:10.1107/S1744309112020167
PMCID: PMC3388912  PMID: 22750855
TRAP; Staphylococcus aureus; signal transduction
20.  Structure of the RBD-PRDI fragment of the antiterminator protein GlcT 
The crystal structure of the RBD-PRDI fragment of the antiterminator protein GlcT from Bacillus subtilis has been solved at 2 Å resolution. The structure represents an inactive state of the protein.
GlcT is a transcriptional antiterminator protein that is involved in regulation of glucose metabolism in Bacillus subtilis. Antiterminator proteins bind specific RNA sequences, thus preventing the formation of overlapping terminator stem-loops. The structure of a fragment (residues 3–170) comprising the RNA-binding domain (RBD) and the first regulatory domain (PRDI) of GlcT was solved at 2.0 Å resolution with one molecule in the asymmetric unit. The two domains are connected by a helical linker. Their interface is mostly constituted by hydrophobic interactions.
doi:10.1107/S1744309112020635
PMCID: PMC3388913  PMID: 22750856
antiterminator proteins; GlcT; Bacillus subtilis; RBD-PRDI fragment
21.  Crystallization and preliminary X-ray crystallographic analysis of Pz peptidase B from Geobacillus collagenovorans MO-1 
In this work, Pz peptidase B, an intracellular M3 metallopeptidase that is found in the thermophile Geobacillus collagenovorans MO-1, was crystallized using the counter-diffusion method.
Pz peptidase B is an intracellular M3 metallopeptidase that is found together with Pz peptidase A in the thermophile Geobacillus collagenovorans MO-1 and recognizes collagen-specific tripeptide units (-Gly-Pro-X-). These peptidases have low homology in their primary structures; however, their cleavage patterns towards peptide substrates are similar. In this work, Pz peptidase B was crystallized using the counter-diffusion method. Data were collected to a resolution of 1.6 Å at 100 K from a crystal obtained in the Japanese Experiment Module (JEM; also known as ‘Kibo’) at the International Space Station (ISS). The crystal belonged to the trigonal space group P3121, with unit-cell parameters a = b = 87.64, c = 210.5 Å. A complete data set was also obtained from crystals of selenomethionine-substituted protein.
doi:10.1107/S1744309112018969
PMCID: PMC3388914  PMID: 22750857
Pz peptidase B; Geobacillus collagenovorans MO-1; microgravity
22.  Purification, crystallization and preliminary crystallographic analysis of histone lysine demethylase NO66 from Homo sapiens  
The JmjC domain-containing histone demethylase NO66 from H. sapiens was overproduced in E. coli, purified and crystallized. Diffraction data were collected to 2.29 Å resolution.
NO66 is a JmjC domain-containing histone demethylase with specificity towards histone H3 methylated on both Lys4 and Lys36 in vitro and in vivo. A fragment of NO66 lacking the N-terminal 167 amino-acid residues was overexpressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to a resolution of 2.29 Å. NO66 crystallized in space group P31 or P32, with unit-cell parameters a = 89.35, b = 89.35, c = 304.86 Å, α = β = 90, γ = 120°, and the crystal is likely to contain four molecules in the asymmetric unit.
doi:10.1107/S174430911201740X
PMCID: PMC3388916  PMID: 22750859
histone demethylases; NO66; JmjC domain
23.  Crystallization and preliminary X-ray analysis of a novel halotolerant feruloyl esterase identified from a soil metagenomic library 
A novel feruloyl esterase (EstF27) identified from a soil metagenomic library has been crystallized and a complete data set was collected from a single cooled crystal using an in-house X-ray source.
Feruloyl esterase cleaves the ester linkage formed between ferulic acid and polysaccharides in plant cell walls and thus has wide potential industrial applications. A novel feruloyl esterase (EstF27) identified from a soil metagenomic library was crystallized and a complete data set was collected from a single cooled crystal using an in-house X-ray source. The crystal diffracted to 2.9 Å resolution and belonged to space group P212121, with unit-cell parameters a = 94.35, b = 106.19, c = 188.51 Å, α = β = γ = 90.00°. A Matthews coefficient of 2.55 Å3 Da−1, with a corresponding solvent content of 51.84%, suggested the presence of ten protein subunits in the asymmetric unit.
doi:10.1107/S1744309112017812
PMCID: PMC3388917  PMID: 22750860
feruloyl esterase
24.  Crystallization of an atypical short-chain dehydrogenase from Vibrio vulnificus lacking the conserved catalytic tetrad 
An atypical short-chain dehydrogenase/reductase from Vibrio vulnificus was expressed, purified and cocrystallized with NADPH by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.80 Å.
Short-chain dehydrogenases/reductases (SDRs) are a rapidly expanding superfamily of enzymes that are found in all kingdoms of life. Hallmarked by a highly conserved Asn-Ser-Tyr-Lys catalytic tetrad, SDRs have a broad substrate spectrum and play diverse roles in key metabolic processes. Locus tag VVA1599 in Vibrio vulnificus encodes a short-chain dehydrogenase (hereafter referred to as SDRvv) which lacks the signature catalytic tetrad of SDR members. Structure-based protein sequence alignments have suggested that SDRvv may harbour a unique binding site for its nicotinamide cofactor. To date, structural studies of SDRs with altered catalytic centres are underrepresented in the scientific literature, thus limiting understanding of their spectrum of substrate and cofactor preferences. Here, the expression, purification and crystallization of recombinant SDRvv are presented. Two well diffracting crystal forms could be obtained by cocrystallization in the presence of the reduced form of the phosphorylated nicotinamide cofactor NADPH. The collected data were of sufficient quality for successful structure determination by molecular replacement and subsequent refinement. This work sets the stage for deriving the identity of the natural substrate of SDRvv and the structure–function landscape of typical and atypical SDRs.
doi:10.1107/S1744309112018672
PMCID: PMC3388918  PMID: 22750861
short-chain dehydrogenases/reductases; Vibrio vulnificus
25.  Cloning, purification, crystallization and preliminary X-ray diffraction crystallographic study of acyl-protein thioesterase 1 from Saccharomyces cerevisiae  
This paper reports the cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of acyl-protein thioesterase 1 from S. cerevisiae.
Palmitoylation/depalmitoylation plays an important role in protein modification. yApt1 is the only enzyme in Saccharomyces cerevisiae that catalyses depalmitoylation. In the present study, recombinant full-length yApt1 was cloned, expressed, purified and crystallized. The crystals diffracted to 2.40 Å resolution and belonged to space group P42212, with unit-cell parameters a = b = 146.43, c = 93.29 Å. A preliminary model of the three-dimensional structure has been built and further refinement is ongoing.
doi:10.1107/S1744309112019276
PMCID: PMC3388919  PMID: 22750862
palmitoylation; acyl-protein thioesterase 1; Saccharomyces cerevisiae

Results 1-25 (2579)