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1.  Purification, crystallization and structure determination of native GroEL from Escherichia coli lacking bound potassium ions 
Acta Crystallographica Section F  2007;63(Pt 6):457-461.
A 3.02 Å crystal structure of native GroEL from E. coli is presented.
GroEL is a member of the ATP-dependent chaperonin family that promotes the proper folding of many cytosolic bacterial proteins. The structures of GroEL in a variety of different states have been determined using X-ray crystallography and cryo-electron microscopy. In this study, a 3.02 Å crystal structure of the native GroEL complex from Escherichia coli is presented. The complex was purified and crystallized in the absence of potassium ions, which allowed evaluation of the structural changes that may occur in response to cognate potassium-ion binding by comparison to the previously determined wild-type GroEL structure (PDB code 1xck), in which potassium ions were observed in all 14 subunits. In general, the structure is similar to the previously determined wild-type GroEL crystal structure with some differences in regard to temperature-factor distribution.
doi:10.1107/S1744309107020295
PMCID: PMC2335072  PMID: 17554162
GroEL; chaperone proteins; ATP-binding proteins
2.  Crystallographic structure determination of B10 mutants of Vitreoscilla hemoglobin: role of Tyr29 (B10) in the structure of the ligand-binding site 
Acta Crystallographica Section F  2013;69(Pt 3):215-222.
The crystal structures of two mutants at position 29 of the dimeric hemoglobin from Vitreoscilla are reported, together with a discussion of the significance of these mutations.
Site-directed mutants of the gene encoding wild-type Vitreoscilla hemoglobin were made that changed Tyr29 (B10) of the wild-type Vitreoscilla hemoglobin (VHb) to either Phe or Ala. The wild-type and the two mutant hemoglobins were expressed in Escherichia coli and purified to homogeneity. The binding of the two mutants to CO was essentially identical to that of wild-type VHb as determined by CO-difference spectra. Circular-dichroism spectra also showed the two mutants to be essentially the same as wild-type VHb regarding overall helicity. All three VHbs were crystallized and their structures were determined at resolutions of 1.7–1.9 Å, which are similar to that of the original wild-type structure determination. The Tyr29Phe mutant has a structure that is essentially indistinguishable from that of the wild type. However, the structure of the Tyr29Ala mutant has significant differences from that of the wild type. In addition, for the Tyr29Ala mutant it was possible to determine the positions of most of the residues in the D region, which was disordered in the originally reported structure of wild-type VHb as well as in the wild-type VHb structure reported here. In the Tyr29Ala mutant, the five-membered ring of proline E8 (Pro54) occupies the space occupied by the aromatic ring of Tyr29 in the wild-type structure. These results are discussed in the context of the proposed role of Tyr29 in the structure of the oxygen-binding pocket.
doi:10.1107/S1744309112044818
PMCID: PMC3606562  PMID: 23519792
bacterial hemoglobin; Vitreoscilla; site-directed mutagenesis
3.  Structure of Vibrio cholerae ribosome hibernation promoting factor 
Acta Crystallographica Section F  2013;69(Pt 3):228-236.
The X-ray crystal structure of ribosome hibernation promoting factor from V. cholerae has been determined at 2.0 Å resolution. The crystal was phased by two-wavelength MAD using cocrystallized cobalt.
The X-ray crystal structure of ribosome hibernation promoting factor (HPF) from Vibrio cholerae is presented at 2.0 Å resolution. The crystal was phased by two-wavelength MAD using cocrystallized cobalt. The asymmetric unit contained two molecules of HPF linked by four Co atoms. The metal-binding sites observed in the crystal are probably not related to biological function. The structure of HPF has a typical β–α–β–β–β–α fold consistent with previous structures of YfiA and HPF from Escherichia coli. Comparison of the new structure with that of HPF from E. coli bound to the Thermus thermophilus ribosome [Polikanov et al. (2012 ▶), Science, 336, 915–918] shows that no significant structural changes are induced in HPF by binding.
doi:10.1107/S1744309113000961
PMCID: PMC3606564  PMID: 23519794
Vibrio cholerae; ribosome hibernation; hibernation promotion factor; ribosome-binding proteins; stationary phase; cold shock; MAD; cobalt; metal-binding site
4.  Arginine 116 stabilizes the entrance to the metal ion-binding site of the MntC protein 
Acta Crystallographica Section F  2013;69(Pt 3):237-242.
The structures of wild-type MntC and of an R116A mutant have been determined to 2.7 and 3.5 Å resolution, respectively. The role of Arg116 in the outer shell of the active site is described.
The cyanobacterium Synechocystis sp. PCC 6803 imports Mn2+ ions via MntCAB, an ABC transport system that is expressed at submicromolar Mn2+ concentrations. The structures of the wild type (WT) and a site-directed mutant of the MntC solute-binding protein have been determined at 2.7 and 3.5 Å resolution, respectively. The WT structure is significantly improved over the previously determined structure (PDB entry 1xvl), showing improved Mn2+ binding site parameters, disulfide bonds in all three monomers and ions bound to the protein surface, revealing the role of Zn2+ ions in the crystallization liquor. The structure of MntC reveals that the active site is surrounded by neutral-to-­positive electrostatic potential and is dominated by a network of polar interactions centred around Arg116. The mutation of this residue to alanine was shown to destabilize loops in the entrance to the metal-ion binding site and suggests a possible role in MntC function.
doi:10.1107/S174430911300153X
PMCID: PMC3606565  PMID: 23519795
ABC transporter; site-directed mutagenesis; cyanobacteria
5.  The structure of the FnI-EGF-like tandem domain of coagulation factor XII solved using SIRAS 
Acta Crystallographica Section F  2013;69(Pt 2):94-102.
The structure of the FnI-EGF-like tandem domain was solved to 1.6 Å using data sets of native and holmium chloride bound protein. Putative biological roles of this tandem domain are discussed based on the structure.
Coagulation factor XII (FXII) is a key protein in the intrinsic coagulation and kallikrein–kinin pathways. It has been found that negative surfaces and amyloids, such as Aβ fibrils, can activate FXII. Additionally, it has been suggested that FXII simulates cells and that it plays an important role in thrombosis. To date, no structural data on FXII have been deposited, which makes it difficult to support any hypothesis on the mechanism of FXII function. The crystal structure of the FnI-EGF-like tandem domain of FXII presented here was solved using experimental phases. To determine the phases, a SIRAS approach was used with a native and a holmium chloride-soaked data set. The holmium cluster was coordinated by the C-terminal tails of two symmetry-related molecules. Another observation was that the FnI domain was much more ordered than the EGF-like domain owing to crystal packing. Furthermore, the structure shows the same domain orientation as the homologous FnI-EGF-like tandem domain of tPA. The plausibility of several proposed inter­actions of these domains of FXII is discussed. Based on this FXII FnI-EGF-like structure, it could be possible that FXII binding to amyloid and negatively charged surfaces is mediated via this part of FXII.
doi:10.1107/S1744309113000286
PMCID: PMC3564606  PMID: 23385745
FXII; Hageman factor; experimental phasing; fibronectin type I domain; EGF-like domain
6.  Exploring the cross-reactivity of S25-2: complex with a 5,6-dehydro-Kdo disaccharide 
The structure of the near germline antibody S25-2 in complex with an unnatural Kdo disaccharide highlights the remarkable plasticity in antigen recognition of germline antibodies.
The near-germline antibody S25-2 exhibits a remarkable cross-reactivity for oligosaccharides containing the bacterial lipopolysaccharide carbohydrate 3-­deoxy-d-manno-oct-2-ulosonic acid (Kdo). The recent synthesis of a variety of Kdo analogues permits a detailed structural analysis of the importance of specific interactions in antigen recognition by S25-2. The Kdo disaccharide analogue Kdo-(2→4)-5,6-dehydro-Kdo lacks a 5-OH group on the second Kdo residue and has been cocrystallized with S25-2. The structure reveals that the modification of the Kdo residue at position 5 results in a rearrangement of intramolecular hydrogen bonds in the antigen that allows it to assume a novel conformation in the antibody-combining site. The cross-reactive binding of S25-­2 to this synthetic ligand highlights the adaptability of this antibody to non-natural synthetic analogues.
doi:10.1107/S1744309112047422
PMCID: PMC3539693  PMID: 23295476
antibody–antigen interactions; protein–carbohydrate interactions; cross-reactive binding
7.  Structure of Pisum sativum Rubisco with bound ribulose 1,5-bisphosphate 
The crystallization and structure determination to 2.2 Å resolution is reported for a ribulose 1,5-bisphosphate-bound non-activated form of garden pea ribulose-1,5-bisphosphate carboxylase/oxygenase.
The first structure of a ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from a pulse crop is reported. Rubisco was purified from Pisum sativum (garden pea) and diffraction-quality crystals were obtained by hanging-drop vapour diffusion in the presence of the substrate ribulose 1,5-bisphosphate. X-ray diffraction data were recorded to 2.20 Å resolution from a single crystal at the Canadian Light Source. The overall quaternary structure of non-activated P. sativum Rubisco highlights the conservation of the form I Rubisco hexadecameric complex. The electron density places the substrate in the active site at the interface of the large-subunit dimers. Lys201 in the active site is not carbamylated as expected for this non-activated structure. Some heterogeneity in the small-subunit sequence is noted, as well as possible variations in the conformation and contacts of ribulose 1,5-bisphosphate in the large-subunit active sites. Overall, the active-site conformation most closely correlates with the ‘closed’ conformation observed in other substrate/inhibitor-bound Rubisco structures.
doi:10.1107/S1744309112047549
PMCID: PMC3539695  PMID: 23295478
Pisum sativum; Rubisco; molecular replacement; ribulose 1,5-bisphosphate
8.  Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus  
The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the ectodomain of BVDV E2 are described.
Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.
doi:10.1107/S1744309112049184
PMCID: PMC3539699  PMID: 23295482
Pestivirus; BVDV; envelope glycoprotein E2
9.  Purification, crystallization and preliminary X-ray crystallographic analysis of diaminopimelate epimerase from Acinetobacter baumannii  
The crystallization and preliminary X-ray crystallographic analysis of diaminopimelate epimerase from A. baumannii are reported.
The meso isomer of diaminopimelate (meso-DAP) is a biosynthetic precursor of l-lysine in bacteria and plants, and is a key component of the peptidoglycan layer in the cell walls of Gram-negative and some Gram-positive bacteria. Diaminopimelate epimerase (DapF) is a pyridoxal-5′-phosphate-independent racemase which catalyses the interconversion of (6S,2S)-2,6-diaminopimelic acid (ll-DAP) and meso-DAP. In this study, DapF from Acinetobacter baumannii was overexpressed in Escherichia coli strain SoluBL21, purified and crystallized using a vapour-diffusion method. A native crystal diffracted to a resolution of 1.9 Å and belonged to space group P31 or P32, with unit-cell parameters a = b = 74.91, c = 113.35 Å, α = β = 90, γ = 120°. There were two molecules in the asymmetric unit.
doi:10.1107/S1744309112048506
PMCID: PMC3539701  PMID: 23295484
meso-diaminopimelate; diaminopimelate epimerase; Acinetobacter baumannii
10.  Crystallization and preliminary X-ray analysis of human MTH1 with a homogeneous N-terminus 
A mutant human MTH1 protein [hMTH1(G2K)] with a homogeneous N-terminus produced high-quality crystals which diffracted to near 1.1 Å resolution using synchrotron radiation.
Human MTH1 (hMTH1) is an enzyme that hydrolyses several oxidized purine nucleoside triphosphates to their corresponding nucleoside monophosphates. Crystallographic studies have shown that the accurate mode of interaction between 8-oxoguanine and hMTH1 cannot be understood without determining the positions of the H atoms, as can be observed in neutron and/or ultrahigh-resolution X-ray diffraction studies. The hMTH1 protein prepared in the original expression system from Escherichia coli did not appear to be suitable for obtaining high-quality crystals because the hMTH1 protein had heterogeneous N-termini of Met1 and Gly2 that resulted from N-terminal Met excision by methionine aminopeptidase from the E. coli host. To obtain homogeneous hMTH1, the Gly at the second position was replaced by Lys. As a result, mutant hMTH1 protein [hMTH1(G2K)] with a homogeneous N-terminus could be prepared and high-quality crystals which diffracted to near 1.1 Å resolution using synchrotron radiation were produced. The new crystals belonged to space group P212121, with unit-cell parameters a = 46.36, b = 47.58, c = 123.89 Å.
doi:10.1107/S1744309112048002
PMCID: PMC3539702  PMID: 23295485
human MTH1
11.  Crystallization and preliminary X-ray diffraction analysis of the Nif3-family protein MJ0927 from Methanocaldococcus jannaschii  
The hypothetical protein MJ0927 of the Nif3 family has been crystallized and X-ray diffraction data have been collected to a resolution of 2.47 Å.
MJ0927 is a member of the Nif3 family and is widely distributed across living organisms. Although several crystal structures of Nif3 proteins have been reported, structural information on archaeal Nif3 is still limited. To understand the structural differences between bacterial and archaeal Nif3 proteins, MJ0927 from Methanocaldococcus jannaschii was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.47 Å and belonged to the orthorhombic space group C222, with unit-cell parameters a = 81.21, b = 172.94, c = 147.42 Å. Determination of this structure may provide insights into the function of MJ0927.
doi:10.1107/S1744309112049408
PMCID: PMC3539711  PMID: 23295494
Nif3; Methanocaldococcus jannaschii; metal-binding proteins
12.  Preliminary crystallographic analysis of a polyadenylate synthase from Megavirus  
The preliminary crystallographic analysis of Megavirus chilensis Mg561, which has a predicted polyadenylate synthase function, is reported. The crystals belonged to space group P212121, with two monomers per asymmetric unit.
Megavirus chilensis, a close relative of the Mimivirus giant virus, is also the most complex virus sequenced to date, with a 1.26 Mb double-stranded DNA genome encoding 1120 genes. The two viruses share common regulatory elements such as a peculiar palindrome governing the termination/polyadenylation of viral transcripts. They also share a predicted polyadenylate synthase that presents a higher than average percentage of residue conservation. The Megavirus enzyme Mg561 was overexpressed in Escherichia coli, purified and crystallized. A 2.24 Å resolution MAD data set was recorded from a single crystal on the ID29 beamline at the ESRF.
doi:10.1107/S1744309112048257
PMCID: PMC3539704  PMID: 23295487
Megavirus chilensis; Megaviridae; giant viruses; transcription; poly(A) polymerase; polyadenylate synthase
13.  Overexpression, crystallization and preliminary X-ray crystallographic analysis of the phosphotriesterase from Mycobacterium tuberculosis  
Crystals of the phosphotriesterase from M. tuberculosis were obtained and diffraction data were collected and processed to 2.27 Å resolution. An analytical ultracentrifugation experiment suggested that mPHP exists as dimers in solution.
Organophosphates (OPs) are extremely toxic compounds that are used as insecticides or even as chemical warfare agents. Phosphotriesterases (PHPs) are responsible for the detoxification of OPs by catalysing their degradation. Almost 100 PHP structures have been solved to date, yet the crystal structure of the phosphotriesterase from Mycobacterium tuberculosis (mPHP) remains unavailable. This study reports the first crystallization of mPHP. The crystal belonged to space group C2221, with unit-cell parameters a = 68.03, b = 149.60, c = 74.23 Å, α = β = γ = 90°. An analytical ultracentrifugation experiment suggested that mPHP exists as a dimer in solution, even though one molecule is calculated to be present in the asymmetric unit according to the structural data.
doi:10.1107/S1744309112049032
PMCID: PMC3539705  PMID: 23295488
Mycobacterium tuberculosis; phosphotriesterases
14.  Crystallization and preliminary X-ray diffraction studies of Drosophila melanogaster Gαo-subunit of heterotrimeric G protein in complex with the RGS domain of CG5036 
D. melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization and preliminary X-ray crystallographic analysis of the complex of the two proteins are reported.
Regulator of G-protein signalling (RGS) proteins negatively regulate heterotrimeric G-protein signalling through their conserved RGS domains. RGS domains act as GTPase-activating proteins, accelerating the GTP hydrolysis rate of the activated form of Gα-subunits. Although omnipresent in eukaryotes, RGS proteins have not been adequately analysed in non-mammalian organisms. The Drosophila melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization of the complex of the two proteins using PEG 4000 as a crystallizing agent and preliminary X-ray crystallographic analysis are reported. Diffraction data were collected to 2.0 Å resolution using a synchrotron-radiation source.
doi:10.1107/S174430911204804X
PMCID: PMC3539706  PMID: 23295489
Drosophila melanogaster; Gαo-subunit; G-protein signalling
15.  Crystallization and preliminary X-ray crystallographic analysis of UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase (VvGT5) from the grapevine Vitis vinifera  
The UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase (VvGT5) from the grapevine V. vinifera was purified and crystallized. The best crystal diffracted X-rays to 2.2 Å resolution and belonged to space group P6122.
Grapevine (Vitis vinifera) glycosyltransferase 5 (VvGT5) is a UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase that catalyses the 3-O-specific glucuronosylation of flavonols using UDP-glucuronic acid as a sugar donor to produce flavonol 3-O-glucosides, which are important bioactive phytochemicals. Recombinant VvGT5 expressed in Escherichia coli cells was purified and crystallized by the sitting-drop vapour-diffusion method. A full set of X-ray diffraction data was collected to 2.2 Å Bragg spacing from a single crystal using a synchrotron-radiation source. The crystal was hexagonal, belonging to space group P6122, with unit-cell parameters a = b = 102.70, c = 535.92 Å. The initial phases were determined by the molecular-replacement method.
doi:10.1107/S1744309112045095
PMCID: PMC3539707  PMID: 23295490
VvGT5; UGT; glycosyltransferase; flavonol; flavonoid
16.  Expression, purification, crystallization and preliminary X-ray diffraction analysis of Arabidopsis thaliana Deg8 
Crystals of Deg8, an ATP-independent serine endopeptidase from A. thaliana, were monoclinic, belonging to space group C2 with unit-cell parameters a = 129.5, b = 124.2, c = 93.3 Å, β = 132.4°, and diffracted to 2.0 Å resolution.
Arabidopsis thaliana Deg8, an ATP-independent serine endopeptidase, is involved in the repair of photosystem II (PSII), specifically the degradation of the photo-damaged PSII reaction centre D1 protein. To understand the molecular mechanism underlying the participation of Deg8 in the degradation of the photo-damaged D1 protein, the structure of Deg8 is needed. Until recently, however, no structure of Deg8 had been solved. In this study, Deg8 from A. thaliana was cloned, overexpressed and purified in Escherichia coli. Crystallization was performed at 277 K using tribasic sodium citrate as the precipitant and the crystals diffracted to 2.0 Å resolution, belonging to space group C2 with unit-cell parameters a = 129.5, b = 124.2, c = 93.3 Å, α = γ = 90, β = 132.4°. Assuming one trimer in the asymmetric unit, the Matthews coefficient and the solvent content were calculated to be 2.35 Å3 Da−1 and 47.6%, respectively.
doi:10.1107/S1744309112048774
PMCID: PMC3539708  PMID: 23295491
Deg8; photosystem II; Arabidopsis thaliana
17.  Crystallization and preliminary X-ray diffraction analysis of the organophosphorus hydrolase OPHC2 from Pseudomonas pseudoalcaligenes  
An organophosphorus hydrolase from P. pseudoalcaligenes named OPHC2 has been crystallized. Combined with biochemical characterization, it is expected that the structure of this protein will provide insight into the catalytic mechanism of organophosphorus hydrolysis and will highlight the role of key residues involved in substrate specificity.
Enzymes that are capable of degrading neurotoxic organophosphorus compounds are of increasing interest because of the lack of efficient and clean methods for their removal. Recently, a novel organophosphorus hydrolase belonging to the metallo-β-lactamase superfamily was identified and isolated from the mesophilic bacterium Pseudomonas pseudoalcaligenes. This enzyme, named OPHC2, is endowed with significant thermal and pH stability, making it an appealing candidate for engineering studies to develop an efficient organophosphorus biodecontaminant. Combined with biochemical studies, structural information will help decipher the catalytic mechanism of organo­phosphorus hydrolysis by OPHC2 and identify the residues involved in its substrate specificity. Here, the expression, purification, crystallization and X-ray data collection at 2.1 Å resolution of OPHC2 are presented.
doi:10.1107/S174430911205049X
PMCID: PMC3539709  PMID: 23295492
organophosphorus; quorum sensing; phosphotriesterases; lactonases; nerve agents; pesticides; quorum quenching; Pseudomonas pseudoalcaligenes
18.  Crystallization and preliminary X-ray diffraction analysis of YisP protein from Bacillus subtilis subsp. subtilis strain 168 
A bacteria biofilm formation involved enzyme, BsYisP, from Bacillus subtilis subsp. subtilis strain 168, was crystallized and diffracted to 1.92 Å.
YisP is an enzyme involved in the pathway of biofilm formation in bacteria and is predicted to possess squalene synthase activity. A BlastP search using the YisP protein sequence from Bacillus subtilis subsp. subtilis strain 168 shows that it shares 23% identity with the dehydrosqualene synthase from Staphylococcus aureus. The YisP from B. subtilis 168 was expressed in Escherichia coli and the recombinant protein was purified and crystallized. The crystals, which belong to the orthorhombic space group P212121, with unit-cell parameters a = 43.966, b = 77.576, c = 91.378 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 1.92 Å resolution. Structure determination using MAD and MIR methods is in progress.
doi:10.1107/S1744309112049330
PMCID: PMC3539710  PMID: 23295493
YisP; squalene synthase; dehydrosqualene synthase; Bacillus subtilis subsp. subtilis; drug design; synchrotron radiation
19.  Use of europium ions for SAD phasing of lysozyme at the Cu Kα wavelength 
Acta Crystallographica Section F  2012;69(Pt 1):20-24.
Europium(III) ions bound to the surface of hen egg-white lysozyme were found to exhibit good anomalous signal facilitating SAD phasing using laboratory-source data and automated model building. The europium ion-binding sites were observed up to the 15σ level.
Europium is shown to be a good anomalous scatterer in SAD phasing for solving the structure of biological macromolecules. The large value of the anomalous contribution of europium, f′′ = 11.17 e−, at the Cu Kα wavelength is an advantage in de novo phasing and automated model building. Tetragonal crystals of hen egg-white lysozyme (HEWL) incorporating europium(III) chloride (50 mM) were obtained which diffracted to a resolution of 2.3 Å at a wavelength of 1.54 Å (Cu Kα). The master data set (360° frames) was split and analyzed for anomalous signal-to-noise ratio, multiplicity, completeness, SAD phasing and automated building. The structure solution and model building of the split data sets were carried out using phenix.autosol and phenix.autobuild. The contributions of the Eu ions to SAD phasing using in-house data collection are discussed. This study revealed successful lysozyme phasing by SAD using laboratory-source data involving Eu ions, which are mainly coordinated by the side chains of Asn46, Asp52 and Asp101 together with some water molecules.
doi:10.1107/S1744309112047562
PMCID: PMC3539697  PMID: 23295480
europium; SAD; lysozyme
20.  Interactions of Mn2+ with a non-self-complementary Z-type DNA duplex 
Crystal structures of the hexanucleotide d(CACGCG)·d(CGCGTG) were determined in the presence of Mn2+ ions in two crystal lattices and provide insights into ion interactions.
Crystal structures of the hexanucleotide d(CACGCG)·d(CGCGTG) were determined in two crystal lattices when different concentrations of the counterion Mn2+ were used in crystallization. The availability of Mn2+ during the crystallization process appears to play an important role in inducing different crystal packings that lead to crystals belonging to the two space groups P21 and P65. Analysis of the molecular interactions of Mn2+ with the Z-form duplexes shows direct coordination to the purine residues G and A.
doi:10.1107/S1744309112041759
PMCID: PMC3509959  PMID: 23192018
Z-DNA; crystal packing; DNA–ion interactions; manganese ions
21.  ATP-dependent DNA ligase from Thermococcus sp. 1519 displays a new arrangement of the OB-fold domain 
The structure of ATP-dependent DNA ligase from Thermococcus sp. 1519 was determined at a resolution of 3.02 Å, showing a new relative arrangement of the OB-fold domain.
DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5′-phosphate and 3′-­hydroxyl termini. Their function is essential for maintaining genome integrity in the replication, recombination and repair of DNA. High flexibility is important for the function of DNA ligase molecules. Two types of overall conformations of archaeal DNA ligase that depend on the relative position of the OB-fold domain have previously been revealed: closed and open extended conformations. The structure of ATP-dependent DNA ligase from Thermococcus sp. 1519 (LigTh1519) in the crystalline state determined at a resolution of 3.02 Å shows a new relative arrangement of the OB-fold domain which is intermediate between the positions of this domain in the closed and the open extended conformations of previously determined archaeal DNA ligases. However, small-angle X-ray scattering (SAXS) measurements indicate that in solution the LigTh1519 molecule adopts either an open extended conformation or both an intermediate and an open extended conformation with the open extended conformation being dominant.
doi:10.1107/S1744309112043394
PMCID: PMC3509962  PMID: 23192021
ATP-dependent DNA ligase; OB fold; Thermococcus sp. 1519; DNA ligases
22.  Structure of the α-1,6/α-1,4-specific glucansucrase GTFA from Lactobacillus reuteri 121 
A 118 kDa fragment, comprising the catalytic domain and four other domains, of the glucansucrase GTFA from L. reuteri 121, which synthesizes α-glucans with both α-1,6- and α-1,4-glycosidic linkages, was crystallized. The weakly diffracting crystals, which contained 85% solvent, were used to determine the structure at 3.6 Å resolution.
The reuteransucrase GTFA from Lactobacillus reuteri 121, which belongs to glycosyl hydrolase family GH70, synthesizes branched α-glucans with both α-­1,6- and α-1,4-glycosidic linkages (reuteran) from sucrose. The crystal structure of GTFA-ΔN, a 118 kDa fragment of GTFA comprising residues 745–1763 and including the catalytic domain, was determined at 3.6 Å resolution by molecular replacement. The crystals have large solvent channels and an unusually high solvent content of 85%. GTFA-ΔN has the same domain arrangement and domain topologies as observed in previously determined GH70 glucansucrase structures. The architecture of the GTFA-ΔN active site and binding pocket confirms that glucansucrases have a conserved substrate specificity for sucrose. However, this first crystal structure of an α-1,6/α-1,4-specific glucansucrase shows that residues from conserved sequence motif IV (1128–1136 in GTFA-ΔN) contribute to the acceptor-binding subsites and that they display differences compared with other structurally characterized glucansucrases. In particular, the structure clarifies the importance of residues following the transition-state stabilizer for product specificity, and especially residue Asn1134, which is in a position to interact with sugar units in acceptor subsite +2.
doi:10.1107/S1744309112044168
PMCID: PMC3509963  PMID: 23192022
lactic acid bacteria; glucansucrase; reuteransucrase
23.  Structure of the Rho-specific guanine nucleotide-exchange factor Xpln 
The Xpln crystal structure provides structural insights into Rho GTPase binding.
Xpln is a guanine nucleotide-exchange factor (GEF) for Rho GTPases. A Dbl homology (DH) domain followed by a pleckstrin homology (PH) domain is a widely adopted GEF-domain architecture. The Xpln structure solely comprises these two domains. Xpln activates RhoA and RhoB, but not RhoC, although their GTPase sequences are highly conserved. The molecular mechanism of the selectivity of Xpln for Rho GTPases is still unclear. In this study, the crystal structure of the tandemly arranged DH-PH domains of mouse Xpln, with a single molecule in the asymmetric unit, was determined at 1.79 Å resolution by the multiwavelength anomalous dispersion method. The DH-PH domains of Xpln share high structural similarity with those from neuroepithelial cell-transforming gene 1 protein, PDZ-RhoGEF, leukaemia-associated RhoGEF and intersectins 1 and 2. The crystal structure indicated that the α4–α5 loop in the DH domain is flexible and that the DH and PH domains interact with each other intramolecularly, thus suggesting that PH-domain rearrangement occurs upon RhoA binding.
doi:10.1107/S1744309112045265
PMCID: PMC3509964  PMID: 23192023
GEF proteins; DH-PH module structure
24.  Inorganic pyrophosphatase crystals from Thermococcus thioreducens for X-ray and neutron diffraction 
Inorganic pyrophosphatase from T. thioreducans has been crystallized and the crystals were deemed to be suitable for both X-ray and neutron diffraction at room temperature.
Inorganic pyrophosphatase (IPPase) from the archaeon Thermococcus thioreducens was cloned, overexpressed in Escherichia coli, purified and crystallized in restricted geometry, resulting in large crystal volumes exceeding 5 mm3. IPPase is thermally stable and is able to resist denaturation at temperatures above 348 K. Owing to the high temperature tolerance of the enzyme, the protein was amenable to room-temperature manipulation at the level of protein preparation, crystallization and X-ray and neutron diffraction analyses. A complete synchrotron X-ray diffraction data set to 1.85 Å resolution was collected at room temperature from a single crystal of IPPase (monoclinic space group C2, unit-cell parameters a = 106.11, b = 95.46, c = 113.68 Å, α = γ = 90.0, β = 98.12°). As large-volume crystals of IPPase can be obtained, preliminary neutron diffraction tests were undertaken. Consequently, Laue diffraction images were obtained, with reflections observed to 2.1 Å resolution with I/σ(I) greater than 2.5. The preliminary crystallographic results reported here set in place future structure–function and mechanism studies of IPPase.
doi:10.1107/S1744309112032447
PMCID: PMC3509969  PMID: 23192028
inorganic pyrophosphatase; Thermococcus thioreducens; neutron diffraction
25.  Crystallization and preliminary X-ray crystallographic analysis of quinolinate phosphoribosyltransferase from porcine kidney in complex with nicotinate mononucleotide 
Crystals of S. scrofa quinolinate phosphoribosyltransferase purified from porcine kidney in complex with nicotinate mononucleotidewere obtained and diffraction data were collected and processed to 2.1 Å resolution.
Quinolinate phosphoribosyltransferase (QAPRTase) is a key enzyme in NAD biosynthesis; it catalyzes the formation of nicotinate mononucleotide (NAMN) from quinolinate and 5-phosphoribosyl-1-pyrophosphate. In order to elucidate the mechanism of NAMN biosynthesis, crystals of Sus scrofa QAPRTase (Ss-­QAPRTase) purified from porcine kidney in complex with NAMN were obtained and diffraction data were collected and processed to 2.1 Å resolution. The Ss-QAPRTase–NAMN cocrystals belonged to space group P321, with unit-cell parameters a = 119.1, b = 119.1, c = 93.7 Å, γ = 120.0°. The Matthews coefficient and the solvent content were estimated as 3.10 Å3 Da–1 and 60.3%, respectively, assuming the presence of two molecules in the asymmetric unit.
doi:10.1107/S1744309112040638
PMCID: PMC3509970  PMID: 23192029
quinolinate phosphoribosyltransferase; NAD biosynthesis; nicotinate mononucleotide

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