The crystal structure of the archaeal peroxiredoxin from P. horikoshii was determined at a resolution of 2.25 Å and compared with that of a homologous protein from A. pernix.
The crystal structure of peroxiredoxin from the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii (PhPrx) was determined at a resolution of 2.25 Å. The overall structure was a ring-type decamer consisting of five homodimers. Citrate, which was included in the crystallization conditions, was bound to the peroxidatic cysteine of the active site, with two O atoms of the carboxyl group mimicking those of the substrate hydrogen peroxide. PhPrx lacked the C-terminal tail that forms a 32-residue extension of the protein in the homologous peroxiredoxin from Aeropyrum pernix (ApPrx).
peroxiredoxins; Pyrococcus horikoshii
The structure of the cellulose-binding module of the C. thermocellum cellulosome-integrating protein A scaffoldin with portions of the C- and N-terminal flanking linker regions has been determined at 1.98 Å resolution.
The cellulosome of the cellulolytic bacterium Clostridium thermocellum has a structural multi-modular protein called CipA (cellulosome-integrating protein A) that includes nine enzyme-binding cohesin modules and a family 3 cellulose-binding module (CBM3a). In the CipA protein, the CBM3a module is located between the second and third cohesin modules and is connected to them via proline/threonine-rich linkers. The structure of CBM3a with portions of the C- and N-terminal flanking linker regions, CBM3a-L, has been determined to a resolution of 1.98 Å. The structure is a β-sandwich with a structural Ca2+ ion. The structure is consistent with the previously determined CipA CBM structure; however, the structured linker regions provide a deeper insight into the overall cellulosome structure and assembly.
family 3 cellulose-binding module; CipA; Clostridium thermocellum; cellulosome
The structures of two Tiam1 PHn-CC-Ex domain variants have been determined to resolutions of 1.98 and 2.15 Å. Biochemical data indicate that this domain binds the coiled-coil domain of Par3b. SAXS data are in excellent agreement with the structures, indicating that the solution and crystal structures are very similar.
The T-lymphoma and metastasis gene 1 (TIAM1) encodes a guanine nucleotide-exchange factor protein (Tiam1) that is specific for the Rho-family GTPase Rac1 and is important for cell polarity, migration and adhesion. Tiam1 is a large multi-domain protein that contains several protein–protein binding domains that are important for regulating cellular function. The PHn-CC-Ex domain is critical for plasma-membrane association and interactions with protein-scaffold proteins (e.g. Par3b, spinophilin, IRSp53 and JIP2) that direct Tiam1–Rac1 signaling specificity. It was determined that the coiled-coil domain of Par3b binds the PHn-CC-Ex domain with a dissociation constant of ∼30 µM. Moreover, the structures of two variants of the Tiam1 PHn-CC-Ex domain were solved at resolutions of 1.98 and 2.15 Å, respectively. The structures indicate that the PHn, CC and Ex regions form independent subdomains that together provide an integrated platform for binding partner proteins. Small-angle X-ray scattering (SAXS) data indicate that the Tiam1 PHn-CC-Ex domain is monomeric in solution and that the solution and crystal structures are very similar. Together, these data provide the foundation necessary to elucidate the structural mechanism of the PHn-CC-Ex/scaffold interactions that are critical for Tiam1–Rac1 signaling specificity.
guanine nucleotide-exchange factors; Tiam1; PHn-CC-Ex domain; small-angle X-ray scattering
The RNA pseudouridine synthase TruB catalyses the isomerization of uridine to pseudouridine at residue 55 of elongator tRNAs. Here, the expression, purification, crystallization and preliminary crystallographic analysis of TruB from Streptococcus pneumoniae are reported.
The RNA pseudouridine synthase TruB catalyses the isomerization of uridine to pseudouridine (Ψ) at residue 55 of elongator tRNAs. In order to better elucidate the functions of TruB in the formation of pseudouridine, the three-dimensional structure of full-length TruB was determined by X-ray crystallography. Here, the expression, purification, crystallization and preliminary crystallographic analysis of TruB from Streptococcus pneumoniae are reported. The crystal belonged to space group P2, with unit-cell parameters a = 37.65, b = 78.09, c = 56.33 Å, β = 102.05°, and diffracted to a resolution of 1.7 Å. The crystal is most likely to contain one molecule in the asymmetric unit, with a V
M value of 2.40 Å3 Da−1.
TruB; Streptococcus pneumoniae
The purification and crystallization of the isolated toxin VapC (MvpT) from the VapBC toxin–antitoxin complex of the pathogen S. flexneri are reported. The crystals belonged to space group H3 and diffracted to 1.9 Å resolution.
Upon release from the stable complex formed with its antitoxin VapB, the toxin VapC (MvpT) of the Gram-negative pathogen Shigella flexneri is capable of globally down-regulating translation by specifically cleaving initiator tRNAfMet in the anticodon region. Recombinant Shigella flexneri VapCD7A harbouring an active-site mutation was overexpressed in Escherichia coli, purified to homogeneity and crystallized by the vapour-diffusion technique. A preliminary X-ray crystallographic analysis shows that the crystals diffracted to at least 1.9 Å resolution at a synchrotron X-ray source and belonged to the trigonal space group in the hexagonal setting, H3, with unit-cell parameters a = b = 120.1, c = 52.5 Å, α = β = 90, γ = 120°. The Matthews coefficient is 2.46 Å3 Da−1, suggesting two molecules per asymmetric unit and corresponding to a solvent content of 50.0%.
toxin–antitoxin; ribonucleases; tRNA; PIN domain; Shigella flexneri; VapC
This report describes the crystallization and X-ray diffraction analysis of a regulatory domain of the Toll-like receptor signalling adaptor TRIF/TICAM-1 and its SeMet-labelled mutant containing two additional Met residues. This domain is unrelated in sequence to any protein of known structure.
As part of the mammalian innate immune response, Toll-like receptors 3 and 4 can signal via the adaptor protein TRIF/TICAM-1 to elicit the production of type-I interferons and cytokines. Recent studies have suggested an auto-inhibitory role for the N-terminal domain (NTD) of TRIF. This domain has no significant sequence similarity to proteins of known structure. In this paper, the crystallization and X-ray diffraction analysis of TRIF-NTD and its selenomethionine-labelled mutant TRIF-NTDA66M/L113M are reported. Thin plate-like crystals of native TRIF-NTD obtained using polyethylene glycol 3350 as precipitant diffracted X-rays to 1.9 Å resolution. To facilitate phase determination, two additional methionines were incorporated into the protein at positions chosen based on the occurrence of methionines in TRIF homologues in different species. Crystals of the selenomethionine-labelled protein were obtained under conditions similar to the wild-type protein; these crystals diffracted X-rays to 2.5 Å resolution. The TRIF-NTD and TRIF-NTDA66M/L113M crystals have the symmetry of space groups P212121 and P1, and most likely contain two and four molecules in the asymmetric unit, respectively. These results provide a sound foundation for the future structure determination of this novel domain.
Toll-like receptors; adaptor proteins; innate immunity; selenomethionine labelling; introduction of additional methionine residues
Robo receptors participate in the orchestration of several developmental responses, most notably axonal guidance in the central nervous system (CNS). Here, the expression, derivatization, crystallization, and experimental phasing of the extracellular juxtamembrane segment of the human Robo1 receptor are described.
Robo receptors participate in the orchestration of several developmental responses, most notably axonal guidance in the central nervous system. Robo1 contains five tandem Ig-like and three fibronectin type-III (FnIII) domains in its ectodomain, followed by a single-pass transmembrane segment and an intracellular region. A human Robo1 construct that includes the two extracellular membrane-proximal fibronectin (Fn) domains and the juxtamembrane linker was overexpressed in Escherichia coli and purified. Crystals were obtained using the vapour-diffusion method at 293 K and X-ray diffraction data were collected. Molecular-replacement attempts using related Fn domains as search models did not result in a solution. After introducing two additional methionine residues using PCR site-directed mutagenesis, selenomethionine-derivative crystals were produced. These crystals belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 27.24, b = 77.64, c = 91.91 Å. Assuming the presence of a monomer in the asymmetric unit gave a crystal volume per protein weight (V
M) of 1.97 Å3 Da−1 and a solvent content of 37.6%. Anisotropic diffraction data and a fragmented single-wavelength anomalous dispersion electron-density map, to which homology-modelled domains were docked, were obtained.
Robo1; Slit; selenomethionine; SAD
As a thermostable esterase with cold adaptation, EstL5 might be of significant industrial interest and value in scientific research. In order to provide new insights into the structure–function relationship of EstL5 and to help better understand the biochemical data, a project to determine the three-dimensional structure of this enzyme was initiated.
The novel thermostable esterase EstL5 belonging to the GDSL family exhibits a unique cold-adaptation feature at low temperatures. To better understand its biochemical and enzymatic properties, recombinant EstL5 protein was purified and crystallized using the vapour-diffusion method. The EstL5 crystals diffracted X-rays to 2.79 Å resolution using a synchrotron-radiation source, belonged to the tetragonal space group P41212 or P43212, with unit-cell parameters a = b = 101.51, c = 124.22 Å, and are expected to contain two molecules in each asymmetric unit. To obtain initial phases, selenomethionyl-substituted protein was overproduced. Purified SeMet-labelled EstL5 protein was crystallized and formed crystals that diffracted to a resolution of 3.0 Å.
EstL5; thermostability; GDSL esterases; Geobacillus thermodenitrificans
The N-terminal carbohydrate-binding domains of Flo1p and Lg-Flo1p from two different Saccharomyces spp. were overexpressed in E. coli, purified and crystallized in different conditions. A preliminary X-ray analysis of the crystallized flocculin–protein complexes is reported in this work.
Flo1p and Lg-Flo1p are two cell-wall adhesins belonging to the Flo (flocculation) protein family from the yeasts Saccharomyces cerevisiae and S. pastorianus. The main function of these modular proteins endowed with calcium-dependent lectin activity is to mediate cell–cell adhesion events during yeast flocculation, a process which is well known at the cellular level but still not fully characterized from a molecular perspective. Recently, structural features of the N-terminal Flo lectin domains, including the N-terminal domain of Lg-Flo1p (N-Lg-Flo1p), and their interactions with carbohydrate molecules have been investigated. However, structural data concerning the N-terminal domain of Flo1p (N-Flo1p), which is the most specific among the Flo proteins, are missing and information about the N-Lg-Flo1p–carbohydrate interaction still lacks detailed structural insight. Here, the crystallization and preliminary X-ray characterization of the apo form and the mannose complex of N-Flo1p and X-ray analysis of N-Lg-Flo1p crystals soaked in α-1,2-mannobiose are reported. The N-Flo1p crystals diffracted to a resolution of 1.43 Å in the case of the apo form and to 2.12 Å resolution for the mannose complex. Both crystals were orthorhombic and belonged to space group P212121, with one molecule in the asymmetric unit. The N-Lg-Flo1p–α-1,2-mannobiose complex crystal diffracted to 1.73 Å resolution and belonged to the monoclinic space group P1211 with two molecules in the asymmetric unit.
Saccharomyces cerevisiae; Saccharomyces pastorianus; yeast flocculation; flocculins; Flo proteins; lectins; Flo1p; Lg-Flo1p
Human histidine triad nucleotide-binding protein 2 (hHINT2) was cloned, expressed in E. coli, purified and crystallized. The initially obtained crystals of hHINT2 were used for a diffraction experiment and data were collected to 2.83 Å resolution.
Histidine triad nucleotide-binding protein 2 (HINT2) is a mitochondrial adenosine phosphoramidase mainly expressed in the pancreas, liver and adrenal gland. HINT2 possibly plays a role in apoptosis, as well as being involved in steroid biosynthesis, hepatic lipid metabolism and regulation of hepatic mitochondria function. The expression level of HINT2 is significantly down-regulated in hepatocellular carcinoma patients. To date, endogenous substrates for this enzyme, as well as the three-dimensional structure of human HINT2, are unknown. In this study, human HINT2 was cloned, overexpressed in Escherichia coli and purified. Crystallization was performed at 278 K using PEG 4000 as the main precipitant; the crystals, which belonged to the tetragonal space group P41212 with unit-cell parameters a = b = 76.38, c = 133.25 Å, diffracted to 2.83 Å resolution. Assuming two molecules in the asymmetric unit, the Matthews coefficient and the solvent content were calculated to be 2.63 Å3 Da−1 and 53.27%, respectively.
HINT; histidine triad nucleotide-binding protein
The kinase domain of AtMAP4Kalpha2 was cloned, expressed, purified and crystallized. The crystals diffracted to 1.9 Å resolution and belonged to space group C2221, with unit-cell parameters a = 55.27, b = 82.93, c = 133.15 Å.
Arabidopsis thaliana (At) MAP4Kalpha2, a member of the Ste20/PAK-like protein kinase family, is an essential component of the septum initiation network involved in cell division. To better understand the mode of action of AtMAP4Kalpha2, a structural biology approach has been pursued. In this study, the kinase domain of AtMAP4Kalpha2 was cloned, expressed, purified and crystallized. The crystals diffracted to 1.9 Å resolution and belonged to space group C2221, with unit-cell parameters a = 55.27, b = 82.93, c = 133.15 Å.
AtMAP4Kalpha2; Arabidopsis thaliana
The GRASP65 GRASP domain from R. norvegicus has been expressed, purified and crystallized. The crystals belonged to space group P21212, with unit-cell parameters a = 44.99, b = 104.29, c = 37.93 Å, and diffracted to 2.0 Å resolution.
GRASP65 and GRASP55 were classified as Golgi reassembly stacking proteins which play crucial and complementary roles in the stacking of Golgi cisternae. They also participate in vesicle tethering, mitotic progression, the disassembly and reassembly of the Golgi apparatus during mitosis and unconventional secretory pathway regulation. In this study, the expression, crystallization and preliminary crystallographic analysis of the GRASP65 GRASP domain from Rattus norvegicus are presented. The crystals diffracted to 2.0 Å resolution and belonged to space group P21212, with unit-cell parameters a = 44.99, b = 104.29, c = 37.93 Å, α = β = γ = 90°. Furthermore, molecular replacement was employed to determine the structure of the GRASP65 GRASP domain from R. norvegicus.
Golgi; GRASP65; GRASP domain; membrane stacking; Rattus norvegicus
The AAA+ σ54 interaction domain of FlrC from V. cholerae has been cloned, overexpressed, purified and crystallized in a heptameric form. Crystals belonging to space group P212121 produced diffraction data to 2.8 Å resolution.
A σ54-dependent transcriptional activator FlrC containing an N-terminal regulatory domain, a central AAA+ domain and a C-terminal DNA-binding domain has been implicated both in flagellar synthesis and enhanced intestinal colonization. FlrC is phosphorylated by the kinase FlrB at the regulatory domain and both nonphosphorylated and phosphorylated states of FlrC seem to be important for its functions. Oligomerization plays a key role in the functions of such transcriptional activators and the AAA+ σ54 interaction domain is critical in deciding the oligomerization state. Therefore, to obtain structural insights into FlrC at the atomic level, the AAA+ σ54 interaction domain of FlrC was cloned, overexpressed and crystallized using PEG 6000 as precipitant at pH 6.0, and diffraction data were collected to 2.8 Å resolution. Molecular-replacement calculations and subsequent refinement confirmed the presence of a closed heptamer in the asymmetric unit.
Vibrio cholerae; flagellar synthesis; motility and colonization; AAA+ domain; FlrC
The N-terminal PDZ-like domain of the human peripheral nervous system myelin protein periaxin has been crystallized and preliminary diffraction data are reported. The periaxin crystals diffracted X-rays to 2.85 Å resolution using synchrotron radiation.
Periaxin (PRX) is an abundant protein in peripheral nerves and contains a predicted PDZ-like domain at its N-terminus. The large isoform, L-PRX, is required for the maintenance of myelin in the peripheral nervous system and its defects cause neurological disease. Here, the human periaxin PDZ-like domain was crystallized and X-ray diffraction data were collected to 2.85 Å resolution using synchrotron radiation. The crystal belonged to the primitive hexagonal space group P3121 or P3221, with unit-cell parameters a = b = 80.6, c = 81.0 Å, γ = 120° and either two or three molecules in the asymmetric unit. The structure of PRX will shed light on its poorly characterized function in the nervous system.
myelin; periaxin; PDZ-like domain; neuropathy
Rice l-galactose dehydrogenase was crystallized and X-ray diffraction data were collected to 1.2 Å resolution.
In plants, l-galactose dehydrogenase (l-GalDH) is a key enzyme in the biosynthesis of ascorbic acid (AsA), which is well known as a unique antioxidant compound and a cofactor for many enzymes. l-GalDH catalyses the oxidation of l-galactose to l-galactono-1,4-lactone. Rice l-GalDH was overexpressed in Escherichia coli, purified and crystallized. Diffraction-quality rod-shaped crystals were grown using a sitting-drop vapour-diffusion method. The l-GalDH crystals exhibited the symmetry of space group P21 and diffracted to a resolution of 1.2 Å. The crystals had unit-cell parameters a = 46.8, b = 54.9, c = 56.9 Å, β = 102.3°. On the basis of the Matthews coefficient (V
M = 2.1 Å3 Da−1, solvent content of 42.3%), it was estimated that one peptide was present in the asymmetric unit.
l-galactose dehydrogenase; ascorbic acid biosynthesis; Oryza sativa L.
The deaminase AmnE from Pseudomonas sp. AP-3 was expressed in E. coli and purified. Crystallization and preliminary X-ray crystallographic analysis were performed for this recombinant enzyme.
The amnE gene from Pseudomonas sp. AP-3 has been verified as encoding a deaminase with 142 amino-acid residues. In order to change the substrate specificity via structure-based protein engineering, the amnE gene, after gene-code optimization, was chemically synthesized and cloned into the expression vector pET-28a. The protein was expressed in Escherichia coli BL21 (DE3) and purified by Ni2+-chelating affinity chromatography. Diffraction-quality crystals were obtained using the hanging-drop vapour-diffusion method and diffracted to a resolution of 2.09 Å. The crystals belonged to the orthorhombic space group C2221, with unit-cell parameters a = 63.23, b = 88.93, c = 137.83 Å.
AmnE; Pseudomonas sp. AP-3
An automated spreadsheet-based solution for macromolecular crystallization screening is presented that allows easy formulation and optimization of crystal screens via multiple approaches, including incomplete-factorial and grid screening.
In this paper, a simple low-cost alternative to large commercial systems for preparing macromolecular crystallization conditions is described. Using an intuitive spreadsheet-based approach, the system allows the rapid calculation of relevant pipetting volumes given known stock-solution concentrations and incorporates the automatic design of custom crystallization screens via the incomplete-factorial and grid-screen approaches. Automated dispensing of the resulting crystallization screens is achieved using a generic and relatively inexpensive liquid handler.
protein crystallization; crystallization screening; spreadsheet; incomplete factorial; grid screen; software
The pH of a solution is an important parameter in crystallization that needs to be controlled in order to ensure success. In this study, the effects of the buffer and protein solution pH values on the results of screening are systematically investigated.
The pH of a solution is an important parameter in crystallization that needs to be controlled in order to ensure success. The actual pH of the crystallization droplet is determined by the combined contribution of the buffers in the screening and protein solutions, although the contribution of the latter to the pH is often ignored. In this study, the effects of the buffer and protein solution pH values on the results of screening are systematically investigated. It was found that these parameters significantly affected the results and thus the following strategy for the selection of appropriate pH values is proposed: (i) when screening with only one protein solution, the pH should be as low, as high or as divergent from the pI as possible for a basic, acidic or neutral protein, respectively, within its stable pH range; (ii) when screening with two protein solutions, the pH values should be well separated from one another; and (iii) when multiple pH values are utilized, an even distribution of pH values is the best approach to increase the success rate of crystallization.
protein crystallization; pH; crystallization screening
The crystal structure of Mast-M1 has been determined at 2.0 Å resolution and provides the first detailed structural description of a TOG-like domain.
Mast/Orbit is a nonmotor microtubule-associated protein (MAP) present in Drosophila melanogaster that reportedly binds microtubules at the plus end and is essential for mitosis. Sequence analysis has shown that the N-terminal domain (Mast-M1) resembles TOG domains from the Dis1-TOG family of proteins and stands as a representative of one of the three subclasses of divergent TOG-like domains (TOGL1) that includes human CLASP1. The crystal structure of Mast-M1 has been determined at 2.0 Å resolution and provides the first detailed structural description of any TOG-like domain. The structure confirms that Mast-M1 adopts a similar fold to the previously described Dis1-TOG domains of microtubule-binding proteins. A comparison with three known TOG-domain structures from XMAP215/Dis1 family members exposes significant differences between Mast-M1 and other TOG-domain structures in key residues at the proposed tubulin-binding edge.
Dis1-TOG domain; TOG-like domains; microtubules; CLASP; XMAP215/Dis1
The triclinic structure of the HIV-1 capsid protein contains four molecules in the asymmetric unit that form a novel packing interface that could conceivably resemble an intermediate structure that is involved in the early steps of HIV-1 assembly.
The Gag precursor is the major structural protein of the virion of human immunodeficiency virus-1 (HIV-1). Capsid protein (CA), a cleavage product of Gag, plays an essential role in virus assembly both in Gag-precursor multimerization and in capsid core formation. The carboxy-terminal domain (CTD) of CA contains 20 residues that are highly conserved across retroviruses and constitute the major homology region (MHR). Genetic evidence implies a role for the MHR in interactions between Gag precursors during the assembly of the virus, but the structural basis for this role remains elusive. This paper describes a novel triclinic structure of the HIV-1 CA CTD at 1.6 Å resolution with two canonical dimers of CA CTD in the asymmetric unit. The canonical dimers form a newly identified packing interface where interactions of four conserved MHR residues take place. This is the first structural indication that these MHR residues participate in the putative CTD–CTD interactions. These findings suggest that the molecules forming this novel interface resemble an intermediate structure that participates in the early steps of HIV-1 assembly. This interface may therefore provide a novel target for antiviral drugs.
HIV-1; capsid protein; Gag precursor
The crystal structure of the type VI secretion-system protein TssJ1 from P. aeruginosa was solved by iodide SAD at a resolution of 1.4 Å.
The type VI secretion system of Pseudomonas aeruginosa has been shown to be responsible for the translocation of bacteriolytic effectors into competing bacteria. A mechanistic understanding of this widely distributed secretion system is developing and structural studies of its components are ongoing. Two representative structures of one highly conserved component, TssJ, from Escherichia coli and Serratia marcescens have been published. Here, the X-ray crystal structure of TssJ1 from P. aeruginosa is presented at 1.4 Å resolution. The overall structure is conserved among the three proteins. This finding suggests that the homologues function in a similar manner and bolsters the understanding of the structure of this family of proteins.
T6SS; lipoproteins; Pseudomonas aeruginosa; TssJ1
The crystal structure of an A-form RNA duplex obtained by degradation of 6S RNA in a crystallization droplet has been solved. The role of a ribose-zipper motif in the intermolecular packing is presented.
In the course of a crystallographic study of a 132 nt variant of Aquifex aeolicus 6S RNA, a crystal structure of an A-form RNA duplex containing 12 base pairs was solved at a resolution of 2.6 Å. In fact, the RNA duplex is part of the 6S RNA and was obtained by accidental but precise degradation of the 6S RNA in a crystallization droplet. 6S RNA degradation was confirmed by microscopic observation of crystals and gel electrophoresis of crystallization droplets. The RNA oligomers obtained form regular A-form duplexes containing three GoU wobble-type base pairs, one of which engages in intermolecular contacts through a ribose-zipper motif at the crystal-packing interface.
A-form RNA; 6S RNA; RNA degradation
The crystal of E. coli 23S rRNA methyltransferase RlmM in this study belongs to a new space group (P21) and is different from the known P31 or P3121 space groups.
RlmM is an AdoMet-dependent methyltransferase that is responsible for 2′-O-methylation of C2498 in the peptidyl-transferase loop of bacterial 23S rRNA. This modification occurs before assembly of the 50S ribosomal subunit, and lack of C2498 methylation can cause a slight reduction in bacterial fitness. Here, the purification and crystallization of RlmM from Escherichia coli as well as its preliminary crystallographic analysis are presented. Cocrystallization of RlmM with AdoMet was carried out and X-ray diffraction data were collected to a resolution of 2.30 Å on beamline BL17U at the SSRF. However, electron density for AdoMet cannot be observed by comprehensive crystallographic analysis, indicating that it is not bound by RlmM during the cocrystallization process. The structure was solved by molecular replacement and refinement is in progress. The crystal contained one molecule in the asymmetric unit and belonged to space group P21, with unit-cell parameters a = 56.07, b = 59.38, c = 54.35 Å, β = 94.84°, which differs from the P31 or P3121 space groups of previously reported RlmM structures (PDB entries 4auk, 4atn and 4b17).
RNA modification; methyltransferases; RlmM; AdoMet
A primitive monoclinic crystal of the rhesus macaque MHC class I molecule Mamu-B*17 complexed with an SIVmac239 Env peptide was obtained and belonged to space group P2, with unit-cell parameters a = 68.3, b = 45.0, c = 81.5 Å, β = 96.5°. The crystal diffracted to 2.55 Å resolution.
Long-term nonprogression during simian immunodeficiency virus (SIV) infection has been strongly associated with the major histocompatibility complex (MHC) class I allele Mamu-B*17. Here, a complex of rhesus macaque Mamu-B*17 with rhesus macaque β2-microglobulin (β2m) and an immunodominant peptide (SIVmac239 Env241–251; LRCNDTNYSGF; Env LF11) derived from the SIV Env protein was crystallized by the hanging-drop method using PEG 3350 as a precipitating agent. The crystals belonged to the primitive monoclinic space group P2, with unit-cell parameters a = 68.3, b = 45.0, c = 81.5 Å, β = 96.5°. Assuming the presence of one molecule in the asymmetric unit, the Matthews coefficient and solvent content were calculated to be 2.96 Å3 Da−1 and 58.5%, respectively.
elite controllers; MHC; rhesus macaque; SIV
An osmotin from the latex of C. procera has been crystallized in both tetragonal and trigonal forms suitable for structure determination.
An osmotin (CpOsm) from the latex of Calotropis procera has been crystallized in both tetragonal and trigonal forms suitable for structure determination. Crystallographic studies of CpOsm are of great interest because limited information is available concerning the structure of latex proteins and CpOsm has previously been shown to interact with the spore membranes of some plant pathogenic fungi, thus impairing spore germination and hyphal growth. CpOsm crystals were grown using 0.1 M HEPES buffer pH 7.5, 26% PEG 4000, 0.2 M ammonium sulfate (space group P43) or using 0.1 M HEPES buffer pH 7.5, 35% MPD, 0.7 M ammonium sulfate (space group P3112). X-ray diffraction data were collected to 2.17 Å (P43) and 1.80 Å (P3112) resolution and molecular-replacement analyses produced initial phases for both crystal forms.
Calotropis procera; latex proteins; osmotins; PR-5; thaumatin-like proteins