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1.  Structure of WbdD: a bifunctional kinase and methyltransferase that regulates the chain length of the O antigen in Escherichia coli O9a 
Molecular microbiology  2012;86(3):730-742.
Summary
The Escherichia coli serotype O9a O-antigen polysaccharide (O-PS) is a model for glycan biosynthesis and export by the ATP-binding cassette transporter-dependent pathway. The polymannose O9a O-PS is synthesized as a polyprenol-linked glycan by mannosyltransferase enzymes located at the cytoplasmic membrane. The chain length of the O9a O-PS is tightly regulated by the WbdD enzyme. WbdD first phosphorylates the terminal non-reducing mannose of the O-PS and then methylates the phosphate, stopping polymerization. The 2.2 Å resolution structure of WbdD reveals a bacterial methyltransferase domain joined to a eukaryotic kinase domain. The kinase domain is again fused to an extended C-terminal coiled-coil domain reminiscent of eukaryotic DMPK (Myotonic Dystrophy Protein Kinase) family kinases such as Rho-associated protein kinase (ROCK). WbdD phosphorylates 2-α-d-mannosyl-d-mannose (2α-MB), a short mimic of the O9a polymer. Mutagenesis identifies those residues important in catalysis and substrate recognition and the in vivo phenotypes of these mutants are used to dissect the termination reaction. We have determined the structures of co-complexes of WbdD with two known eukaryotic protein kinase inhibitors. Although these are potent inhibitors in vitro, they do not show any in vivo activity. The structures reveal new insight into O-PS chain-length regulation in this important model system.
doi:10.1111/mmi.12014
PMCID: PMC3482155  PMID: 22970759
2.  Structure of WbdD: a bifunctional kinase and methyltransferase that regulates the chain length of the O antigen in Escherichia coli O9a 
Molecular Microbiology  2012;86(3):730-742.
Summary
The Escherichia coli serotype O9a O-antigen polysaccharide (O-PS) is a model for glycan biosynthesis and export by the ATP-binding cassette transporter-dependent pathway. The polymannose O9a O-PS is synthesized as a polyprenol-linked glycan by mannosyltransferase enzymes located at the cytoplasmic membrane. The chain length of the O9a O-PS is tightly regulated by the WbdD enzyme. WbdD first phosphorylates the terminal non-reducing mannose of the O-PS and then methylates the phosphate, stopping polymerization. The 2.2 Å resolution structure of WbdD reveals a bacterial methyltransferase domain joined to a eukaryotic kinase domain. The kinase domain is again fused to an extended C-terminal coiled-coil domain reminiscent of eukaryotic DMPK (Myotonic Dystrophy Protein Kinase) family kinases such as Rho-associated protein kinase (ROCK). WbdD phosphorylates 2-α-d-mannosyl-d-mannose (2α-MB), a short mimic of the O9a polymer. Mutagenesis identifies those residues important in catalysis and substrate recognition and the in vivo phenotypes of these mutants are used to dissect the termination reaction. We have determined the structures of co-complexes of WbdD with two known eukaryotic protein kinase inhibitors. Although these are potent inhibitors in vitro, they do not show any in vivo activity. The structures reveal new insight into O-PS chain-length regulation in this important model system.
doi:10.1111/mmi.12014
PMCID: PMC3482155  PMID: 22970759
3.  Crystallization and preliminary X-ray crystallographic analysis of the cysteine protease inhibitor clitocypin 
Clitocypin is a cysteine protease inhibitor from the mushroom Clitocybe nebularis. The protein has been purified from natural sources and crystallized in a variety of non-isomorphous forms belonging to monoclinic and triclinic space groups.
Clitocypin is a cysteine protease inhibitor from the mushroom Clitocybe nebularis. The protein has been purified from natural sources and crystallized in a variety of non-isomorphous forms belonging to monoclinic and triclinic space groups. A diffraction data set to 1.55 Å resolution was obtained from a crystal belonging to space group P2, with unit-cell parameters a = 38.326, b = 33.597, c = 55.568 Å, β = 104°. An inability to achieve isomorphism forced the use of MAD and SAD phasing methods. Phasing is in progress.
doi:10.1107/S1744309105038534
PMCID: PMC2150921  PMID: 16511249
cysteine protease inhibitors; clitocypin
4.  Localization and orientation of heavy-atom cluster compounds in protein crystals using molecular replacement 
A new approach is presented that allows the efficient localization and orientation of heavy-atom cluster compounds used in experimental phasing by a molecular replacement procedure. This permits the calculation of meaningful phases up to the highest resolution of the diffraction data.
Heavy-atom clusters (HA clusters) containing a large number of specifically arranged electron-dense scatterers are especially useful for experimental phase determination of large complex structures, weakly diffracting crystals or structures with large unit cells. Often, the determination of the exact orientation of the HA cluster and hence of the individual heavy-atom positions proves to be the critical step in successful phasing and subsequent structure solution. Here, it is demonstrated that molecular replacement (MR) with either anomalous or isomorphous differences is a useful strategy for the correct placement of HA cluster compounds. The polyoxometallate cluster hexasodium α-metatungstate (HMT) was applied in phasing the structure of death receptor 6. Even though the HA cluster is bound in alternate partially occupied orientations and is located at a special position, its correct localization and orientation could be determined at resolutions as low as 4.9 Å. The broad applicability of this approach was demonstrated for five different derivative crystals that included the compounds tantalum tetradeca­bromide and trisodium phosphotungstate in addition to HMT. The correct placement of the HA cluster depends on the length of the intramolecular vectors chosen for MR, such that both a larger cluster size and the optimal choice of the wavelength used for anomalous data collection strongly affect the outcome.
doi:10.1107/S0907444912046008
PMCID: PMC3565441  PMID: 23385464
experimental phasing; heavy-metal cluster; hexasodium α-metatungstate; molecular replacement; death receptor 6
5.  Evaluating Patterns of a White-Band Disease (WBD) Outbreak in Acropora palmata Using Spatial Analysis: A Comparison of Transect and Colony Clustering 
PLoS ONE  2011;6(7):e21830.
Background
Despite being one of the first documented, there is little known of the causative agent or environmental stressors that promote white-band disease (WBD), a major disease of Caribbean Acropora palmata. Likewise, there is little known about the spatiality of outbreaks. We examined the spatial patterns of WBD during a 2004 outbreak at Buck Island Reef National Monument in the US Virgin Islands.
Methodology/Principal Findings
Ripley's K statistic was used to measure spatial dependence of WBD across scales. Localized clusters of WBD were identified using the DMAP spatial filtering technique. Statistics were calculated for colony- (number of A. palmata colonies with and without WBD within each transect) and transect-level (presence/absence of WBD within transects) data to evaluate differences in spatial patterns at each resolution of coral sampling. The Ripley's K plots suggest WBD does cluster within the study area, and approached statistical significance (p = 0.1) at spatial scales of 1100 m or less. Comparisons of DMAP results suggest the transect-level overestimated the prevalence and spatial extent of the outbreak. In contrast, more realistic prevalence estimates and spatial patterns were found by weighting each transect by the number of individual A. palmata colonies with and without WBD.
Conclusions
As the search for causation continues, surveillance and proper documentation of the spatial patterns may inform etiology, and at the same time assist reef managers in allocating resources to tracking the disease. Our results indicate that the spatial scale of data collected can drastically affect the calculation of prevalence and spatial distribution of WBD outbreaks. Specifically, we illustrate that higher resolution sampling resulted in more realistic disease estimates. This should assist in selecting appropriate sampling designs for future outbreak investigations. The spatial techniques used here can be used to facilitate other coral disease studies, as well as, improve reef conservation and management.
doi:10.1371/journal.pone.0021830
PMCID: PMC3139597  PMID: 21818271
6.  Fortuitous structure determination of ‘as-isolated’ Escherichia coli bacterioferritin in a novel crystal form 
E. coli bacterioferritin was crystallized in a novel crystal form from different conditions and the structure was solved. The crystals belonged to space group P213 and diffracted to a resolution of 2.5 Å.
Escherichia coli bacterioferritin was serendipitously crystallized in a novel cubic crystal form and its structure could be determined to 2.5 Å resolution despite a high degree of merohedral twinning. This is the first report of crystallographic data on ‘as-isolated’ E. coli bacterioferritin. The ferroxidase active site contains positive difference density consistent with two metal ions that had co-purified with the protein. X-ray fluorescence studies suggest that the metal composition is different from that of previous structures and is a mix of zinc and native iron ions. The ferroxidase-centre configuration displays a similar flexibility as previously noted for other bacterioferritins.
doi:10.1107/S1744309106039583
PMCID: PMC2225212  PMID: 17077480
Escherichia coli bacterioferritin; iron storage and homeostasis; ferroxidase; merohedral twinning
7.  Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate 
Glucose-1-phosphate uridylyltransferase (UgpG) from S. elodea ATCC 31461 has been expressed, purified and crystallized. Seven crystal forms were obtained and characterized to a maximum resolution of 2.65 Å.
The cloning, expression, purification, crystallization and preliminary crystallographic analysis of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate are reported. Diffraction data sets were obtained from seven crystal forms in five different space groups, with highest resolutions ranging from 4.20 to 2.65 Å. The phase problem was solved for a P21 crystal form using multiple isomorphous replacement with anomalous scattering from an osmium derivative and a SeMet derivative. The best native crystal in space group P21 has unit-cell parameters a = 105.5, b = 85.7, c = 151.8 Å, β = 105.2°. Model building and refinement are currently under way.
doi:10.1107/S1744309106030107
PMCID: PMC2242879  PMID: 16946483
GalU; UgpG; RmlA; pyrophosphorylase; sugar activation
8.  Purification, crystallization and X-ray diffraction analysis of pavine N-methyltransferase from Thalictrum flavum  
The plant enzyme pavine N-methyltransferase from T. flavum has been produced in E. coli, purified and crystallized and its structure has been solved.
A cDNA from the plant Thalictrum flavum encoding pavine N-methyltrans­ferase, an enzyme belonging to a novel class of S-adenosylmethionine-dependent N-methyltransferases specific for benzylisoquinoline alkaloids, has been heterologously expressed in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatography and was crystallized in space group P21. The structure was solved at 2.0 Å resolution using a xenon derivative and the single isomorphous replacement with anomalous scattering method.
doi:10.1107/S1744309108033046
PMCID: PMC2581683  PMID: 18997344
pavine N-methyltransferase; Thalictrum flavum
9.  Purification, crystallization and X-ray diffraction analysis of the C-terminal protease domain of Venezuelan equine encephalitis virus nsP2 
The C-terminal protease domain of Venezuelan equine encephalitis virus (VEEV) nsP2 has been overexpressed in E. coli, purified and successfully crystallized. Native crystals diffract to beyond 2.5 Å resolution and isomorphous heavy-atom derivatives suitable for phase analysis have been identified.
The C-terminal region of Venezuelan equine encephalitis virus (VEEV) nsP2 is responsible for proteolytic processing of the VEEV polyprotein replication complex. This action regulates the activity of the replication complex and is essential for viral replication, thus making nsP2 a very attractive target for development of VEEV therapeutics. The 338-amino-acid C-terminal region of VEEV nsP2 has been overexpressed in Escherichia coli, purified and crystallized. Crystals diffract to beyond 2.5 Å resolution and belong to the orthorhombic space group P212121. Isomorphous heavy-atom derivatives suitable for phase analysis have been obtained and work on building a complete structural model is under way.
doi:10.1107/S1744309106014667
PMCID: PMC2243096  PMID: 16754969
alphaviruses; Venezuelan equine encephalitis virus; cysteine proteases; nsP2
10.  Crystallization and preliminary crystallographic analysis of recombinant VSP1 from Arabidopsis thaliana  
VSP1 from Arabidopsis thaliana was expressed in E. coli, purified and crystallized. X-ray diffraction data were collected to 1.9 Å resolution.
VSP1 is a defence protein in Arabidopsis thaliana that may also be involved in control of plant development. The recombinant protein has been overexpressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal diffracted to 1.9 Å resolution and a complete X-­ray data set was collected at 100 K using Cu Kα radiation from a rotating-anode X-ray source. The crystals belonged to space group C2. As there are no related structures that could be used as a search model for molecular replacement, work is in progress on experimental phasing using heavy-atom derivatives and selenomethionine derivatives.
doi:10.1107/S1744309109053688
PMCID: PMC2815693  PMID: 20124723
VSP1; Arabidopsis thaliana; defence proteins
11.  Crystallization and initial X-ray diffraction analysis of the tellurite-resistance S-adenosyl-l-methionine transferase protein TehB from Escherichia coli  
The E. coli TehB methyltransferase has been purified and crystallized in the presence of SAM and sinefungin. Diffraction data have been collected to 1.9 Å resolution for both complexes.
TehB is an S-adenosyl-l-methionine (SAM) dependent methyltransferase that detoxifies tellurite in bacteria. The Escherichia coli TehB protein was purified and crystallized in the presence of both SAM and sinefungin. The TehB–SAM and TehB–sinefungin crystals both diffracted X-rays to 1.9 Å resolution. The TehB–SAM crystals belonged to space group C2, with unit-cell parameters a = 60.0, b = 56.1, c = 130.6 Å, β = 97.9°. The TehB–sinefungin crystals belonged to space group P21, with unit-cell parameters a = 59.1, b = 55.5, c = 129.7 Å, β = 95.9°.
doi:10.1107/S1744309110036043
PMCID: PMC3001658  PMID: 21045305
tellurite; methyltransferases; S-adenosyl-l-methionine; sinefungin; seeding
12.  Purification, crystallization and preliminary X-ray analysis of bifunctional isocitrate dehydrogenase kinase/phosphatase in complex with its substrate, isocitrate dehydrogenase, from Escherichia coli  
The protein complex of bifunctional isocitrate dehydrogenase kinase/phosphatase with its substrate, isocitrate dehydrogenase, has been crystallized for structural analysis. A complete data set was collected from the complex crystal and processed to 2.9 Å resolution.
Escherichia coli isocitrate dehydrogenase (ICDH) can be phosphorylated and dephosphorylated by a single bifunctional protein, isocitrate dehydrogenase kinase/phosphatase (AceK), which is encoded by the aceK gene. In order to investigate the regulatory mechanism of (de)phosphorylation of ICDH by AceK, AceK was successfully cocrystallized in complex with its intact protein substrate, ICDH, in the presence of ATP. The complex crystal was obtained by the hanging-drop vapour-diffusion technique using PEG 300 as a precipitant and magnesium sulfate as an additive. SDS–PAGE analysis of dissolved crystals showed that the crystals contained both AceK and ICDH proteins. The complex crystals diffracted to a resolution of 2.9 Å in space group P63, with unit-cell parameters a = b = 196.80, c = 156.46 Å.
doi:10.1107/S1744309109038718
PMCID: PMC2777047  PMID: 19923739
isocitrate dehydrogenases; Ser/Thr protein kinases; protein phosphatases; citric acid cycle; glyoxylate bypass; AceK; protein–protein complexes
13.  Moniliophthora perniciosa Necrosis- and Ethylene-Inducing Protein 2 (MpNep2) as a Metastable Dimer in Solution: Structural and Functional Implications 
PLoS ONE  2012;7(9):e45620.
Understanding how Nep-like proteins (NLPs) behave during the cell cycle and disease progression of plant pathogenic oomycetes, fungi and bacteria is crucial in light of compelling evidence that these proteins play a role in Witches` Broom Disease (WBD) of Theobroma cacao, one of the most important phytopathological problems to afflict the Southern Hemisphere. The crystal structure of MpNep2, a member of the NLP family and the causal agent of WBD, revealed the key elements for its activity. This protein has the ability to refold after heating and was believed to act as a monomer in solution, in contrast to the related homologs MpNep1 and NPP from the oomyceteous fungus Phytophthora parasitica. Here, we identify and characterize a metastable MpNep2 dimer upon over-expression in Escherichia coli using different biochemical and structural approaches. We found using ultra-fast liquid chromatography that the MpNep2 dimer can be dissociated by heating but not by dilution, oxidation or high ionic strength. Small-angle X-ray scattering revealed a possible tail-to-tail interaction between monomers, and nuclear magnetic resonance measurements identified perturbed residues involved in the putative interface of interaction. We also explored the ability of the MpNep2 monomer to refold after heating or chemical denaturation. We observed that MpNep2 has a low stability and cooperative fold that could be an explanation for its structure and activity recovery after stress. These results can provide new insights into the mechanism for MpNep2′s action in dicot plants during the progression of WBD and may open new avenues for the involvement of NLP- oligomeric species in phytopathological disorders.
doi:10.1371/journal.pone.0045620
PMCID: PMC3454426  PMID: 23029140
14.  Crystallization and preliminary X-ray analysis of the inducible lysine decarboxylase from Escherichia coli  
The structure of the decameric inducible lysine decarboxylase from E. coli was determined by SIRAS using a hexatantalum dodecabromide (Ta6Br12 2+) derivative. Model building and refinement are under way.
The decameric inducible lysine decarboxylase (LdcI) from Escherichia coli has been crystallized in space groups C2 and C2221; the Ta6Br12 2+ cluster was used to derivatize the C2 crystals. The method of single isomorphous replacement with anomalous scattering (SIRAS) as implemented in SHELXD was used to solve the Ta6Br12 2+-derivatized structure to 5 Å resolution. Many of the Ta6Br12 2+-binding sites had twofold and fivefold noncrystallographic symmetry. Taking advantage of this feature, phase modification was performed in DM. The electron-density map of LdcI displays many features in agreement with the low-resolution negative-stain electron-density map [Snider et al. (2006 ▶), J. Biol. Chem. 281, 1532–1546].
doi:10.1107/S1744309108018757
PMCID: PMC2494963  PMID: 18678936
inducible lysine decarboxylase; hexatantalum dodecabromide; Escherichia coli; SIRAS
15.  Intermolecular interactions and water structure in a condensed phase B-DNA crystal 
Nucleic Acids Research  2000;28(5):1259-1265.
By controlled dehydration, the unit cells of dodecamer DNA–drug crystals have been shrunk from 68 000 (normal state) to 60 000 (partially dehydrated intermediate state) to 51 000 Å3 (fully dehydrated state), beyond which no further solvent loss occurs. The total solvent content in the normal crystals is ~40% by volume, reducing to ~20% in the fully dehydrated phase. The 25% reduction in cell volume induced a dramatic enhancement in the resolution of the X-ray diffraction data (from 2.6 to beyond 1.5 Å). We have determined the structures of the normal, partially dehydrated and fully dehydrated crystals. Details of the ligand binding have been presented in the preceding article. The present paper describes the unique features of the structure of the fully dehydrated phase. This structure was refined with 9015 unique observed reflections to R = 14.9%, making it one of the most reliable models of B-form DNA available. The crystals exist as infinite polymeric networks, in which neighbouring dodecamer duplexes are crosslinked through phosphate oxygens via direct bonding to magnesium cations. The DNA is packed so tightly that there is essentially only a single layer of solvent between adjacent molecules. The details of the crystal packing, magnesium bridging, DNA hydration and DNA conformation are described and compared with other experimental evidence related to DNA condensation.
PMCID: PMC102594  PMID: 10666471
16.  Crystallization and preliminary diffraction analysis of Wzi, a member of the capsule export and assembly pathway in Escherichia coli  
Wzi is a membrane protein from E. coli thought to be involved in the attachment of capsular polysaccharides to the bacterial surface. This reports describes recombinant Wzi’s purification, crystallization and the results of initial diffraction studies.
External polysaccharide capsules provide a physical barrier that is employed by many species of bacteria for the purposes of host evasion and persistence. Wzi is a 53 kDa outer membrane β-barrel protein that is thought to play a role in the attachment of group 1 capsular polysaccharides to the cell surface. The purification and crystallization of an Escherichia coli homologue of Wzi is reported and diffraction data from native and selenomethionine-incorporated protein crystals are presented. Crystals of C-terminally His6-tagged Wzi diffracted to 2.8 Å resolution. Data processing showed that the crystals belonged to the orthorhombic space group C222, with unit-cell parameters a = 128.8, b = 152.8, c = 94.4 Å, α = β = γ = 90°. A His-tagged selenomethionine-containing variant of Wzi has also been crystallized in the same space group and diffraction data have been recorded to 3.8 Å resolution. Data processing shows that the variant crystal has similar unit-cell parameters to the native crystal.
doi:10.1107/S1744309110040546
PMCID: PMC2998369  PMID: 21139210
Wzi; polysaccharide capsules; Escherichia coli
17.  Expression, purification, crystallization and preliminary crystallographic study of isolated modules of the mouse coactivator-associated arginine methyltransferase 1 
Isolated modules of mouse coactivator-associated arginine methyltransferase 1 encompassing the protein arginine N-methyltransferase catalytic domain have been overexpressed, purified and crystallized. X-ray diffraction data have been collected and have enabled determination of the structures by multiple isomorphous replacement using anomalous scattering.
Coactivator-associated arginine methyltransferase 1 (CARM1) plays a crucial role in gene expression as a coactivator of several nuclear hormone receptors and also of non-nuclear receptor systems. Its recruitment by the transcriptional machinery induces protein methylation, leading to chromatin remodelling and gene activation. CARM128–507 and two structural states of CARM1140–480 were expressed, purified and crystallized. Crystals of CARM128–507 belong to space group P6222, with unit-cell parameters a = b = 136.0, c = 125.3 Å; they diffract to beyond 2.5 Å resolution using synchrotron radiation and contain one monomer in the asymmetric unit. The structure of CARM128–507 was solved by multiple isomorphous replacement and anomalous scattering methods. Crystals of apo CARM1140–480 belong to space group I222, with unit-cell parameters a = 74.6, b = 99.0, c = 207.4 Å; they diffract to beyond 2.7 Å resolution and contain two monomers in the asymmetric unit. Crystals of CARM1140–480 in complex with S-­adenosyl-l-homocysteine belong to space P21212, with unit-cell parameters a = 74.6, b = 98.65, c = 206.08 Å; they diffract to beyond 2.6 Å resolution and contain four monomers in the asymmetric unit. The structures of apo and holo CARM1140–480 were solved by molecular-replacement techniques from the structure of CARM128–507.
doi:10.1107/S1744309107011785
PMCID: PMC2330207  PMID: 17401209
CARM1; gene expression
18.  A Single Amino Acid Substitution in a Mannosyltransferase, WbdA, Converts the Escherichia coli O9 Polysaccharide into O9a: Generation of a New O-Serotype Group 
Journal of Bacteriology  2000;182(9):2567-2573.
wbdA is a mannosyltransferase gene that is involved in synthesis of the Escherichia coli O9a polysaccharide, a mannose homopolymer with a repeating unit of 2-αMan-1,2-αMan-1,3-αMan-1,3-αMan-1. The equivalent structural O polysaccharide in the E. coli O9 and Klebsiella O3 strains is 2-αMan-1,2-αMan-1,2-αMan-1,3-αMan-1,3-αMan-1, with an excess of one mannose in the 1,2 linkage. We have cloned wbdA genes from these O9 and O3 strains and shown by genetic and functional studies that wbdA is the only gene determining the O-polysaccharide structure of O9 or O9a. Based on functional analysis of chimeric genes and site-directed mutagenesis, we showed that a single amino acid substitution, C55R, in WbdA of E. coli O9 converts the O9 polysaccharide into O9a. DNA sequencing revealed the substitution to be conserved in other E. coli O9a strains. The reverse substitution, R55C, in WbdA of E. coli O9a resulted in lipopolysaccharide synthesis showing no ladder profile instead of the conversion of O9a to O9. This suggests that more than one amino acid substitution in WbdA is required for conversion from O9a to O9.
PMCID: PMC111322  PMID: 10762260
19.  Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli  
The 23S rRNA methyltransferase RlmJ from E. coli has been cloned, expressed, purified and crystallized. X-ray diffraction data to 1.85 Å resolution have been collected from the apo RlmJ crystals.
Methyltransferase RlmJ uses the cofactor S-adenosylmethionine to methylate the exocyclic nitrogen N6 of nucleotide A2030 in 23S rRNA during ribosome assembly in Escherichia coli. RlmJ with a C-terminal hexahistidine tag was overexpressed in E. coli and purified as a monomer using Ni2+-affinity and size-exclusion chromatography. The recombinant RlmJ was crystallized using the sitting-drop vapour-diffusion method and a full data set was collected to 1.85 Å resolution from a single apo crystal. The crystals belonged to space group P21, with unit-cell parameters a = 46.9, b = 77.8, c = 82.5 Å, β = 104°. Data analysis suggested two molecules per asymmetric unit and a Matthews coefficient of 2.20 Å3 Da−1.
doi:10.1107/S1744309113020289
PMCID: PMC3758148  PMID: 23989148
RlmJ; S-adenosylmethionine; methyltransferases; 23S rRNA; m6A2030; ribosome assembly; Escherichia coli
20.  Imperfect pseudo-merohedral twinning in crystals of fungal fatty acid synthase 
A case of imperfect pseudo-merohedral twinning in monoclinic crystals of fungal fatty acid synthase is discussed. A space-group transition during crystal dehydration resulted in a Moiré pattern-like interference of the twinned diffraction patterns.
The recent high-resolution structures of fungal fatty acid synthase (FAS) have provided new insights into the principles of fatty acid biosynthesis by large multifunctional enzymes. The crystallographic phase problem for the 2.6 MDa fungal FAS was initially solved to 5 Å resolution using two crystal forms from Thermomyces lanuginosus. Monoclinic crystals in space group P21 were obtained from orthorhombic crystals in space group P212121 by dehydration. Here, it is shown how this space-group transition induced imperfect pseudo-merohedral twinning in the monoclinic crystal, giving rise to a Moiré pattern-like interference of the two twin-related reciprocal lattices. The strategy for processing the twinned diffraction images and obtaining a quantitative analysis is presented. The twinning is also related to the packing of the molecules in the two crystal forms, which was derived from self-rotation function analysis and molecular-replacement solutions using a low-resolution electron microscopy map as a search model.
doi:10.1107/S0907444909000778
PMCID: PMC2631638  PMID: 19171964
imperfect pseudo-merohedral twinning; fungal fatty acid synthase
21.  Crystallization and preliminary crystallographic studies of UbiG, an O-methyltransferase from Escherichia coli  
An O-methyltransferase from the ubiquinone-biosynthesis pathway in Escherichia coli, UbiG, with an N-­terminal hexahistidine tag has been expressed and crystallized. Crystals grown by the hanging-drop vapour-diffusion method diffracted to 2.00 Å resolution.
UbiG, an O-methyltransferase from the ubiquinone-biosynthesis pathway in Escherichia coli, catalyzes two O-methyl transfer steps. The primary structures of the O-methyltransferase enzyme family used in ubiquinone synthesis are conserved in both prokaryotes and eukaryotes, but their tertiary structures and catalytic mechanisms are not yet known. Here, UbiG with an N-­terminal hexahistidine tag was expressed and crystallized. Crystals grown by the hanging-drop vapour-diffusion method diffracted to 2.00 Å resolution and belonged to space group C121, with unit-cell parameters a = 119.8, b = 58.6, c = 40.2 Å, β = 105.3°. Both Matthews coefficient analysis and the self-rotation function suggested the presence of one molecule per asymmetric unit in the crystal, with a solvent content of 50.52% (V M = 2.48 Å3 Da−1).
doi:10.1107/S1744309111014278
PMCID: PMC3107154  PMID: 21636923
lysine methylation; O-methyltransferases; UbiG; ubiquinone
22.  Crystallization and preliminary crystallographic analysis of tRNA (m7G46) methyltransferase from Escherichia coli  
tRNA (m7G46) methyltransferase from E. coli was overexpressed, purified and crystallized. Diffraction data were collected to 2.04 Å resolution.
Transfer RNA (tRNA) (m7G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N 7-­methylguanosine (m7G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His6 tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P21.
doi:10.1107/S1744309108020241
PMCID: PMC2494960  PMID: 18678947
tRNA (m7G46) methyltransferase; Rossmann-fold methyltransferase family
23.  Reductive methylation to improve crystallization of the putative oxidoreductase Rv0765c from Mycobacterium tuberculosis  
The M. tuberculosis protein Rv0765c was cloned, expressed, purified and crystallized. In an attempt to improve the quality of the crystals of Rv0765c, the protein was modified by reductive methylation. The methylated protein crystallized in a new crystal form with profoundly improved diffraction properties.
Rv0765c from Mycobacterium tuberculosis was cloned and heterologously expressed in Escherichia coli. It was purified using affinity and size-exclusion chromatographic techniques and crystallized. The native protein crystallized in a hexagonal crystal form which diffracted to 7 Å resolution. In an attempt to improve the quality of the Rv0765c crystals, the protein was modified by reductive methylation using dimethylaminoborane and formaldehyde. The modified protein crystallized under different conditions in a tetragonal crystal form, from which diffraction data could be collected to a resolution of 3.2 Å. In both crystal forms of Rv0765c, the asymmetric unit contained two copies of the protein molecule.
doi:10.1107/S1744309107022506
PMCID: PMC2335070  PMID: 17554174
reductive methylation; Rv0765c; Mycobacterium tuberculosis
24.  Expression, purification and preliminary X-ray analysis of proliferating cell nuclear antigen from the archaeon Thermococcus thioreducens  
The proliferating cell nuclear antigen (PCNA) from a novel hyperthermophilic archaeon Thermococcus thioreducens has been crystallized, and diffraction data have been collected to 1.86 Å.
Proliferating cell nuclear antigen (PCNA) is a DNA sliding clamp which confers processivity on replicative DNA polymerases. PCNA also acts as a sliding platform that enables the association of many DNA-processing proteins with DNA in a non-sequence-specific manner. In this investigation, the PCNA from the hyperthermophilic archaeon Thermococcus thioreducens (TtPCNA) was cloned, overexpressed in Escherichia coli and purified to greater than 90% homogeneity. TtPCNA crystals were obtained by sitting-drop vapor-diffusion methods and the best ordered crystal diffracted to 1.86 Å resolution using synchrotron radiation. The crystals belonged to the hexagonal space group P63, with unit-cell parameters a = b = 89.0, c = 62.8 Å. Crystals of TtPCNA proved to be amenable to complete X-ray analysis and future structure determination.
doi:10.1107/S174430910903036X
PMCID: PMC2795597  PMID: 19724129
proliferating cell nuclear antigen; Thermococcus thioreducens
25.  Crystallization of the focal adhesion kinase targeting (FAT) domain in a primitive orthorhombic space group 
X-ray diffraction data from the targeting (FAT) domain of focal adhesion kinase (FAK) were collected from a single crystal that diffracted to 1.99 Å resolution.
X-ray diffraction data from the targeting (FAT) domain of focal adhesion kinase (FAK) were collected from a single crystal that diffracted to 1.99 Å resolution and reduced to the primitive orthorhombic lattice. A single molecule was predicted to be present in the asymmetric unit based on the Matthews coefficient. The data were phased using molecular-replacement methods using an existing model of the FAK FAT domain. All structures of human focal adhesion kinase FAT domains solved to date have been solved in a C-centered orthorhombic space group.
doi:10.1107/S1744309108011421
PMCID: PMC2496861  PMID: 18540077
focal adhesion kinase; targeting domain

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