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ABSTRACT: The crystal structure of the 11.14 kDa orphan ORF 1382 from Archaeoglobus fulgidus (AF1382) has been determined by sulfur SAD phasing using a moderately diffracting crystal and 1.9 Å wavelength synchrotron X-rays. AF1382 was selected as a structural genomics target by the Southeast Collaboratory for Structural Genomics (SECSG) since sequence analyses showed that it did not belong to the Pfam-A database and thus could represent a novel fold. The structure was determined by exploiting longer wavelength X-rays and data redundancy to increase the anomalous signal in the data. AF1382 is a 95-residue protein containing five S atoms associated with four methionine residues and a single cysteine residue that yields a calculated Bijvoet ratio (ΔF(anom)/F) of 1.39% for 1.9 Å wavelength X-rays. Coupled with an average Bijvoet redundancy of 25 (two 360° data sets), this produced an excellent electron-density map that allowed 69 of the 95 residues to be automatically fitted. The S-SAD model was then manually completed and refined (R = 23.2%, R(free) = 26.8%) to 2.3 Å resolution (PDB entry 3o3k). High-resolution data were subsequently collected from a better diffracting crystal using 0.97 Å wavelength synchrotron X-rays and the S-SAD model was refined (R = 17.9%, R(free) = 21.4%) to 1.85 Å resolution (PDB entry 3ov8). AF1382 has a winged-helix-turn-helix structure common to many DNA-binding proteins and most closely resembles the N-terminal domain (residues 1-82) of the Rio2 kinase from A. fulgidus, which has been shown to bind DNA, and a number of MarR-family transcriptional regulators, suggesting a similar DNA-binding function for AF1382. The analysis also points out the advantage gained from carrying out data reduction and structure determination on-site while the crystal is still available for further data collection.
Acta crystallographica. Section D, Biological crystallography 09/2012; 68(Pt 9):1242-52. · 12.67 Impact Factor
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ABSTRACT: A multi-dataset (MDS) data-collection strategy is proposed and analyzed for macromolecular crystal diffraction data acquisition. The theoretical analysis indicated that the MDS strategy can reduce the standard deviation (background noise) of diffraction data compared with the commonly used single-dataset strategy for a fixed X-ray dose. In order to validate the hypothesis experimentally, a data-quality evaluation process, termed a readiness test of the X-ray data-collection system, was developed. The anomalous signals of sulfur atoms in zinc-free insulin crystals were used as the probe to differentiate the quality of data collected using different data-collection strategies. The data-collection results using home-laboratory-based rotating-anode X-ray and synchrotron X-ray systems indicate that the diffraction data collected with the MDS strategy contain more accurate anomalous signals from sulfur atoms than the data collected with a regular data-collection strategy. In addition, the MDS strategy offered more advantages with respect to radiation-damage-sensitive crystals and better usage of rotating-anode as well as synchrotron X-rays.
Acta crystallographica. Section A, Foundations of crystallography 11/2011; 67(Pt 6):544-9. · 49.93 Impact Factor
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Hongfei Wang,
Chie Takemoto,
Ryogo Akasaka,
Tomomi Uchikubo-Kamo,
Seiichiro Kishishita,
Kazutaka Murayama,
Takaho Terada, Lirong Chen,
Zhi-Jie Liu,
Bi-Cheng Wang,
Sumio Sugano,
Akiko Tanaka,
Makoto Inoue,
Takanori Kigawa,
Mikako Shirouzu,
Shigeyuki Yokoyama
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ABSTRACT: Interactions of Bcl-2 family proteins play a regulatory role in mitochondrial apoptosis. The pro-apoptotic protein Bak resides in the outer mitochondrial membrane, and the formation of Bak homo- or heterodimers is involved in the regulation of apoptosis. The previously reported structure of the human Bak protein (residues Glu16-Gly186) revealed that a zinc ion was coordinated with two pairs of Asp160 and His164 residues from the symmetry-related molecules. This zinc-dependent homodimer was regarded as an anti-apoptotic dimer. In the present study, we determined the crystal structure of the human Bak residues Ser23-Asn185 at 2.5A, and found a distinct type of homodimerization through Cys166 disulfide bridging between the symmetry-related molecules. In the two modes of homodimerization, the molecular interfaces are completely different. In the membrane-targeted model of the S-S bridged dimer, the BH3 motifs are too close to the membrane to interact directly with the anti-apoptotic relatives, such as Bcl-x(L). Therefore, the Bak dimer structure reported here may represent a pro-apoptotic mode under oxidized conditions.
Journal of Structural Biology 01/2009; 166(1):32-7. · 3.41 Impact Factor
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Yong Xie,
Chie Takemoto,
Seiichiro Kishishita,
Tomomi Uchikubo-Kamo,
Kazutaka Murayama, Lirong Chen,
Zhi Jie Liu,
Bi Cheng Wang,
Miho Manzoku,
Akio Ebihara,
Seiki Kuramitsu,
Mikako Shirouzu,
Shigeyuki Yokoyama
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ABSTRACT: The gene encoding TTHA1544 is a singleton found in the Thermus thermophilus HB8 genome and encodes a 131-amino-acid protein. The crystal structure of TTHA1544 has been determined at 2.0 A resolution by the single-wavelength anomalous dispersion method in order to elucidate its function. There are two molecules in the asymmetric unit. Each molecule consists of four alpha-helices and six beta-strands, with the beta-strands composing a central beta-sheet. A structural homology search revealed that the overall structure of TTHA1544 resembles the alpha/beta-hydrolase fold, although TTHA1544 lacks the catalytic residues of a hydrolase. These results suggest that TTHA1544 represents the minimized alpha/beta-hydrolase fold and that an additional component would be required for its activity.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 01/2008; 63(Pt 12):993-7. · 0.51 Impact Factor
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Noriko Handa,
Seiichiro Kishishita,
Satoshi Morita,
Ryogo Akasaka,
Zhongmin Jin,
John Chrzas, Lirong Chen,
Zhi-Jie Liu,
Bi-Cheng Wang,
Sumio Sugano,
Akiko Tanaka,
Takaho Terada,
Mikako Shirouzu,
Shigeyuki Yokoyama
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ABSTRACT: Familial oncocytic thyroid carcinoma is associated with a missense mutation, P308Q, in the C-terminal domain of Tim44. Tim44 is the mitochondrial inner-membrane translocase subunit and it functions as a membrane anchor for the mitochondrial heat-shock protein 70 (mtHsp70). Here, the crystal structure of the human Tim44 C-terminal domain complexed with pentaethylene glycol has been determined at 1.9 A resolution. The overall structure resembles that of the nuclear transport factor 2-like domain. In the crystal structure, pentaethylene glycol molecules are associated at two potential membrane-binding sites: the large hydrophobic cavity and the highly conserved loop between the alpha1 and alpha2 helices near Pro308. A comparison with the yeast homolog revealed that lipid binding induces conformational changes around the alpha1-alpha2 loop, leading to slippage of the alpha1 helix along the large beta-sheet. These changes may play important roles in the translocation of polypeptides across the mitochondrial inner membrane.
Acta Crystallographica Section D Biological Crystallography 01/2008; 63(Pt 12):1225-34. · 12.62 Impact Factor
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Laura-Lee Clancy Kelley,
Bret D Dillard,
Wolfram Tempel, Lirong Chen,
Neil Shaw,
Doowon Lee,
M Gary Newton,
Frank J Sugar,
Francis E Jenney,
Han Seung Lee,
Claudia Shah,
Farris L Poole,
Michael W W Adams,
Jane S Richardson,
David C Richardson,
Zhi-Jie Liu,
Bi-Cheng Wang,
John Rose
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ABSTRACT: The hypothetical protein PF0899 is a 95-residue peptide from the hyperthermophilic archaeon Pyrococcus furiosus that represents a gene family with six members. P. furiosus ORF PF0899 has been cloned, expressed and crystallized and its structure has been determined by the Southeast Collaboratory for Structural Genomics (http://www.secsg.org). The structure was solved using the SCA2Structure pipeline from multiple data sets and has been refined to 1.85 A against the highest resolution data set collected (a presumed gold derivative), with a crystallographic R factor of 21.0% and R(free) of 24.0%. The refined structure shows some structural similarity to a wedge-shaped domain observed in the structure of the major capsid protein from bacteriophage HK97, suggesting that PF0899 may be a structural protein.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2007; 63(Pt 7):549-52. · 0.51 Impact Factor
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ABSTRACT: The initial critical step of reduction of the azo bond during the metabolism of azo dyes is catalyzed by a group of NAD(P)H dependant enzymes called azoreductases. Although several azoreductases have been identified from microorganisms and partially characterized, very little is known about the structural basis for substrate specificity and the nature of catalysis. Enterococcus faecalis azoreductase A (AzoA) is a highly active azoreductase with a broad spectrum of substrate specificity and is capable of degrading a wide variety of azo dyes. Here, we report the crystal structure of the AzoA from E. faecalis determined at 2.07 A resolution with bound FMN ligand. Phases were obtained by single wavelength anomalous scattering of selenomethionine labeled protein crystals. The asymmetric unit consisted of two dimers with one FMN molecule bound to each monomer. The AzoA monomer takes a typical NAD(P)-binding Rossmann fold with a highly conserved FMN binding pocket. A salt bridge between Arg18 and Asp184 restricts the size of the flavin binding pocket such that only FMN can bind. A putative NADH binding site could be identified and a plausible mechanism for substrate reduction is proposed. Expression studies revealed azoA gene to be expressed constitutively in E. faecalis.
Archives of Biochemistry and Biophysics 07/2007; 463(1):68-77. · 2.93 Impact Factor
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Atsushi Shimada,
Hideaki Niwa,
Kazuya Tsujita,
Shiro Suetsugu,
Koji Nitta,
Kyoko Hanawa-Suetsugu,
Ryogo Akasaka,
Yuri Nishino,
Mitsutoshi Toyama, Lirong Chen, [......],
Bi-Cheng Wang,
Masaki Yamamoto,
Takaho Terada,
Atsuo Miyazawa,
Akiko Tanaka,
Sumio Sugano,
Mikako Shirouzu,
Kuniaki Nagayama,
Tadaomi Takenawa,
Shigeyuki Yokoyama
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ABSTRACT: Pombe Cdc15 homology (PCH) proteins play an important role in a variety of actin-based processes, including clathrin-mediated endocytosis (CME). The defining feature of the PCH proteins is an evolutionarily conserved EFC/F-BAR domain for membrane association and tubulation. In the present study, we solved the crystal structures of the EFC domains of human FBP17 and CIP4. The structures revealed a gently curved helical-bundle dimer of approximately 220 A in length, which forms filaments through end-to-end interactions in the crystals. The curved EFC dimer fits a tubular membrane with an approximately 600 A diameter. We subsequently proposed a model in which the curved EFC filament drives tubulation. In fact, striation of tubular membranes was observed by phase-contrast cryo-transmission electron microscopy, and mutations that impaired filament formation also impaired membrane tubulation and cell membrane invagination. Furthermore, FBP17 is recruited to clathrin-coated pits in the late stage of CME, indicating its physiological role.
Cell 06/2007; 129(4):761-72. · 32.40 Impact Factor
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Amaresh Das,
Zheng-Qing Fu,
Wolfram Tempel,
Zhi-Jie Liu,
Jessie Chang, Lirong Chen,
Doowon Lee,
Weihong Zhou,
Hao Xu,
Neil Shaw,
John P Rose,
Lars G Ljungdahl,
Bi-Cheng Wang
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ABSTRACT: The strict anaerobic, thermophilic bacterium Moorella thermoacetica metabolizes C1 compounds for example CO(2)/H(2), CO, formate, and methanol into acetate via the Wood/Ljungdahl pathway. Some of the key steps in this pathway include the metabolism of the C1 compounds into the methyl group of methylenetetrahydrofolate (MTHF) and the transfer of the methyl group from MTHF to the methyl group of acetyl-CoA catalyzed by methyltransferase, corrinoid protein and CO dehydrogenase/acetyl CoA synthase. Recently, we reported the crystallization of a 25 kDa methanol-induced corrinoid protein from M. thermoacetica (Zhou et al., Acta Crystallogr F 2005; 61:537-540). In this study we analyzed the crystal structure of the 25 kDa protein and provide genetic and biochemical evidences supporting its role in the methanol metabolism of M. thermoacetia. The 25 kDa protein was encoded by orf1948 of contig 303 in the M. thermoacetica genome. It resembles similarity to MtaC the corrinoid protein of the methanol:CoM methyltransferase system of methane producing archaea. The latter enzyme system also contains two additional enzymes MtaA and MtaB. Homologs of MtaA and MtaB were found to be encoded by orf2632 of contig 303 and orf1949 of contig 309, respectively, in the M. thermoacetica genome. The orf1948 and orf1949 were co-transcribed from a single polycistronic operon. Metal analysis and spectroscopic data confirmed the presence of cobalt and the corrinoid in the purified 25 kDa protein. High resolution X-ray crystal structure of the purified 25 kDa protein revealed corrinoid as methylcobalamin with the imidazole of histidine as the alpha-axial ligand replacing benziimidazole, suggesting base-off configuration for the corrinoid. Methanol significantly activated the expression of the 25 kDa protein. Cyanide and nitrate inhibited methanol metabolism and suppressed the level of the 25 kDa protein. The results suggest a role of the 25 kDa protein in the methanol metabolism of M. thermoacetica.
Proteins Structure Function and Bioinformatics 05/2007; 67(1):167-76. · 3.39 Impact Factor
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Brian Gerwe,
Laura-Lee Clancy Kelley,
Bret D Dillard,
Thomas Lai,
Zhi-Jie Liu,
Wolfram Tempel, Lirong Chen,
Jeff Habel,
Doowon Lee,
Francis E Jenney,
Frank J Sugar,
Jane S Richardson,
David C Richardson,
M Gary Newton,
Bi-Cheng Wang,
Michael W W Adams,
John P Rose
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ABSTRACT: The open-reading frame PF0895 in the genome of the hyperthermophilic archaeon, Pyrococcus furiosus, encodes a 206-residue protein (M(R )23,152). The structure of the recombinant protein was solved by single isomorphous replacement with anomalous scattering (SIRAS) using a mercury derivative. It has been refined to 1.70 A with a crystallographic R and R(free )values of 19.7% and 22.3%, respectively. The PF0895 structure is similar to those of the ATP binding cassettes observed in the ABC transporter family. However, bioinformatics and molecular analyses indicate that PF0895 is not part of the expected five-gene operon that encodes a typical prokaryotic solute-binding ABC transporter. Rather, transcriptional profiling data show that PF0895 is part of a novel four-gene operon (PF0895-PF0896-PF0897-PF0897.1) where only PF0895 has homologs in other organisms. Interestingly, from genome analysis, P. furiosus itself contains a second version of this complex, encoded by PF1090-PF1093. From the structural studies we can only conclude that one of the subunits of this novel membrane complex, PF0895, and its homolog PF1090, likely bind a purine nucleotide. PF0895 is therefore predicted to be part of a membrane-bound multiprotein complex unrelated to ABC transporters that is so far unique to P. furiosus. It appears to play a role in the stress response, as its expression is down regulated when the organism is subjected to cold-shock, where cells are transferred from 95 degrees C, near the optimal growth temperature, to 72 degrees C, near the minimal growth temperature. The related PF1090-containing operon is unaffected by cold-shock and is independently regulated.
Journal of Structural and Functional Genomics 04/2007; 8(1):1-10.
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ABSTRACT: Deoxyribonucleoside triphosphate triphosphohydrolase from Thermus thermophilus (Tt-dNTPase) has a unique regulatory mechanism for the degradation of deoxyribonucleoside triphosphates (dNTPs). Whereas the Escherichia coli homologue specifically hydrolyzes dGTP alone, dNTPs act as both substrate and activator for Tt-dNTPase. Here, the crystal structure of Tt-dNTPase has been determined at 2.2 A resolution, representing the first report of the tertiary structure of a dNTPase homologue belonging to the HD superfamily, a diverse group of metal-dependent phosphohydrolases that includes a variety of uncharacterized proteins. This enzyme forms a homohexamer as a double ring of trimers. The subunit is composed of 19 alpha-helices; the inner six helices include the region annotated as the catalytic domain of the HD superfamily. Structural comparison with other HD-superfamily proteins indicates that a pocket at the centre of the inner six helices, formed from highly conserved charged residues clustered around a bound magnesium ion, constitutes the catalytic site. Tt-dNTPase also hydrolyzed noncanonical dNTPs, but hardly hydrolyzed dNDP and dNMP. The broad substrate specificity for different dNTPs might be rationalized by the involvement of a flexible loop during molecular recognition of the base moiety. Recognition of the triphosphate moiety crucial for the activity might be attained by highly conserved positively charged residues. The possible mode of dNTP binding is discussed in light of the structure.
Acta Crystallographica Section D Biological Crystallography 03/2007; 63(Pt 2):230-9. · 12.62 Impact Factor
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Shankar Prasad Kanaujia,
Chellamuthu Vasuki Ranjani,
Jeyaraman Jeyakanthan,
Seiki Baba, Lirong Chen,
Zhi-Jie Liu,
Bi-Cheng Wang,
Masami Nishida,
Akio Ebihara,
Akeo Shinkai,
Seiki Kuramitsu,
Yoshitsugu Shiro,
Kanagaraj Sekar,
Shigeyuki Yokoyama
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ABSTRACT: The gram-negative aerobic eubacterium Thermus thermophilus is an extremely important thermophilic microorganism that was originally isolated from a thermal vent environment in Japan. The molybdenum cofactor in this organism is considered to be an essential component required by enzymes that catalyze diverse key reactions in the global metabolism of carbon, nitrogen and sulfur. The molybdenum-cofactor biosynthesis protein C derived from T. thermophilus was crystallized in two different space groups. Crystals obtained using the first crystallization condition belong to the monoclinic space group P2(1), with unit-cell parameters a = 64.81, b = 109.84, c = 115.19 A, beta = 104.9 degrees; the crystal diffracted to a resolution of 1.9 A. The other crystal form belonged to space group R32, with unit-cell parameters a = b = 106.57, c = 59.25 A, and diffracted to 1.75 A resolution. Preliminary calculations reveal that the asymmetric unit contains 12 monomers and one monomer for the crystals belonging to space group P2(1) and R32, respectively.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 02/2007; 63(Pt 1):27-9. · 0.51 Impact Factor
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Mitsuo Kuratani,
Hiroaki Sakai,
Masahiro Takahashi,
Tatsuo Yanagisawa,
Takatsugu Kobayashi,
Kazutaka Murayama, Lirong Chen,
Zhi-Jie Liu,
Bi-Cheng Wang,
Chizu Kuroishi,
Seiki Kuramitsu,
Takaho Terada,
Yoshitaka Bessho,
Mikako Shirouzu,
Shun-ichi Sekine,
Shigeyuki Yokoyama
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ABSTRACT: Tyrosyl-tRNA synthetase (TyrRS) catalyzes the tyrosylation of tRNA(Tyr) in a two-step reaction. TyrRS has the "HIGH" and "KMSKS" motifs, which play essential roles in the formation of the tyrosyl-adenylate from tyrosine and ATP. Here, we determined the crystal structures of Archaeoglobus fulgidus and Pyrococcus horikoshii TyrRSs in the l-tyrosine-bound form at 1.8A and 2.2A resolutions, respectively, and that of Aeropyrum pernix TyrRS in the substrate-free form at 2.2 A. The conformation of the KMSKS motif differs among the three TyrRSs. In the A.pernix TyrRS, the KMSKS loop conformation corresponds to the ATP-bound "closed" form. In contrast, the KMSKS loop of the P.horikoshii TyrRS forms a novel 3(10) helix, which appears to correspond to the "semi-closed" form. This conformation enlarges the entrance to the tyrosine-binding pocket, which facilitates the pyrophosphate ion release after the tyrosyl-adenylate formation, and probably is involved in the initial tRNA binding. The KMSSS loop of the A.fulgidus TyrRS is somewhat farther from the active site and is stabilized by hydrogen bonds. Based on the three structures, possible structural changes of the KMSKS motif during the tyrosine activation reaction are discussed. We suggest that the insertion sequence just before the KMSKS motif, which exists in some archaeal species, enhances the binding affinity of the TyrRS for its cognate tRNA. In addition, a non-proline cis peptide bond, which is involved in the tRNA binding, is conserved among the archaeal TyrRSs.
Journal of Molecular Biology 02/2006; 355(3):395-408. · 4.00 Impact Factor
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Hao Xu,
Cheng Yang, Lirong Chen,
Irina A Kataeva,
Wolfram Tempel,
Doowon Lee,
Jeff E Habel,
Duong Nguyen,
James W Pflugrath,
Joseph D Ferrara,
W Bryan Arendall,
Jane S Richardson,
David C Richardson,
Zhi Jie Liu,
M Gary Newton,
John P Rose,
Bi Cheng Wang
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ABSTRACT: Recently, the demands of high-throughput macromolecular crystallography have driven continuous improvements in phasing methods, data-collection protocols and many other technologies. Single-wavelength anomalous scattering (SAS) phasing with chromium X-ray radiation opens a new possibility for phasing a protein with data collected in-house and has led to several successful examples of de novo structure solution using only weak anomalous scatterers such as sulfur. To further reduce data-collection time and make SAS phasing more robust, it is natural to combine selenomethionine-derivatized protein (SeMet protein) with Cr Kalpha radiation to take advantage of the larger anomalous scattering signal from selenium (f'' = 2.28 e(-)) compared with sulfur (f'' = 1.14 e(-)). As reported herein, the crystal structure of a putative chorismate mutase from Clostridium thermocellum was determined using Se-SAS with Cr Kalpha radiation. Each protein molecule contains eight selenomethionine residues in 148 amino-acid residues, providing a calculated Bijvoet ratio of about 3.5% at the Cr Kalpha wavelength. A single data set to 2.2 A resolution with approximately ninefold redundancy was collected using an imaging-plate detector coupled with a Cr source. Structure solution, refinement and deposition to the Protein Data Bank were performed within 9 h of the availability of the scaled diffraction data. The procedure used here is applicable to many other proteins and promises to become a routine pathway for in-house high-throughput crystallography.
Acta Crystallographica Section D Biological Crystallography 08/2005; 61(Pt 7):960-6. · 12.62 Impact Factor
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Zhi-Jie Liu,
Wolfram Tempel,
Joseph D Ng,
Dawei Lin,
Ashit K Shah, Lirong Chen,
Peter S Horanyi,
Jeff E Habel,
Irina A Kataeva,
Hao Xu, [......],
Shu-Huey Chang,
Weihong Zhou,
Doowon Lee,
Jeremy L Praissman,
Hua Zhang,
M Gary Newton,
John P Rose,
Jane S Richardson,
David C Richardson,
Bi-Cheng Wang
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ABSTRACT: Using a high degree of automation, the crystallography core at the Southeast Collaboratory for Structural Genomics (SECSG) has developed a high-throughput protein-to-structure pipeline. Various robots and automation procedures have been adopted and integrated into a pipeline that is capable of screening 40 proteins for crystallization and solving four protein structures per week. This pipeline is composed of three major units: crystallization, structure determination/validation and crystallomics. Coupled with the protein-production cores at SECSG, the protein-to-structure pipeline provides a two-tiered approach for protein production at SECSG. In tier 1, all protein samples supplied by the protein-production cores pass through the pipeline using standard crystallization screening and optimization procedures. The protein targets that failed to yield diffraction-quality crystals (resolution better than 3.0 A) become tier 2 or salvaging targets. The goal of tier 2 target salvaging, carried out by the crystallomics core, is to produce the target proteins with increased purity and homogeneity, which would render them more likely to yield well diffracting crystals. This is performed by alternative purification procedures and/or the introduction of chemical modifications to the proteins (such as tag removal, methylation, surface mutagenesis, selenomethionine labelling etc.). Details of the various procedures in the pipeline for protein crystallization, target salvaging, data collection/processing and high-throughput structure determination/validation, as well as some examples, are described.
Acta Crystallographica Section D Biological Crystallography 07/2005; 61(Pt 6):679-84. · 12.62 Impact Factor
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Weihong Zhou,
Amaresh Das,
Jeff E Habel,
Zhi-Jie Liu,
Jessie Chang, Lirong Chen,
Doowon Lee,
Duong Nguyen,
Shu-Huey Chang,
Wolfram Tempel,
John P Rose,
Lars G Ljungdahl,
Bi-Cheng Wang
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ABSTRACT: A corrinoid protein was induced and overexpressed in methanol-grown cells of the thermophilic anaerobic bacterium Moorella thermoacetica. The protein was purified from cytosolic extracts. After screening for crystallization conditions and optimization, crystals were obtained that diffracted strongly on a rotating-anode X-ray source. A diffraction data set was collected and processed including reflections to 1.9 A resolution. Reflections were indexed in a primitive orthorhombic cell with unit-cell parameters a = 55.69, b = 62.74, c = 34.54 A. N-terminal amino-acid sequencing indicates that the crystals contain a C-terminal fragment of the protein.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 06/2005; 61(Pt 5):537-40. · 0.51 Impact Factor
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Zhi-Jie Liu,
Ashit K Shah,
Jeff E Habel,
Joseph D Ng,
Irina Kataeva,
Hao Xu,
Peter Horanyi,
Hua Yang,
Jessie Chang,
Min Zhao, [......],
Sue Chang,
Wolfram Tempel, Lirong Chen,
Weihong Zhou,
Doowon Lee,
Dawei Lin,
Hua Zhang,
M Gary Newton,
John Rose,
Bi-Cheng Wang
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ABSTRACT: Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2,200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.
Journal of Structural and Functional Genomics 02/2005; 6(2-3):121-7.
