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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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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
[show abstract]
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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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[show abstract]
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ABSTRACT: The first X-ray crystal structure of a non-natural dinucleotide, 5'-O-phosphoryl-1'-deoxy-2'-isoadenylyl-(3' --> 5')-cytidine 6.5-hydrate (pIsodApC), C19H26N8O13P2 x 6.5H2O, belonging to a family of dinucleotides that contain an isomeric nucleoside component, is described. A complex system of hydrogen bonds between water molecules and various sites on the dinucleotide was found. All H atoms were located from electron-density difference maps, which allowed identification of protonation sites. Compounds of this family have been found to bind at the active site of HIV integrase and to be inhibitors of this key viral enzyme. These dinucleotides are completely resistant to cleavage by exonucleases; an abnormal dihedral angle twist in an internucleotide phosphate bond revealed in the X-ray crystal structure may be contributing to this unusual stability towards nucleases.
Acta Crystallographica Section C Crystal Structure Communications 09/2005; 61(Pt 8):o518-20. · 0.52 Impact Factor
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Bi-Cheng Wang,
Michael W.W. Adams,
Harry Dailey,
Larry DeLucas,
Ming Luo,
John Rose,
Robert Bunzel,
Tamara Dailey,
Jeff Habel,
Peter Horanyi, [......],
Chi-Hao Luan,
Michael Mayer,
Lisa Nagy, M. Gary Newton,
Joseph Ng,
Farris L. Poole,
Ashit Shah,
Claudia Shah,
Frank J. Sugar,
Hao Xu
[show abstract]
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ABSTRACT: Using a high degree of automation, the Southeast Collaboratory for Structural Genomics (SECSG) has developed high throughput
pipelines for protein production, and crystallization using a two-tiered approach. Primary, or tier-1, protein production
focuses on producing proteins for members of large Pfam families that lack a representative structure in the Protein Data
Bank. Target genomes are Pyrococcus furiosus and Caenorhabditis elegans. Selected human proteins are also under study. Tier-2 protein production, or target rescue, focuses on those tier-1 proteins,
which either fail to crystallize or give poorly diffracting crystals. This two tier approach is more efficient since it allows
the primary protein production groups to focus on the production of new targets while the tier-2 efforts focus on providing
additional sample for further studies and modified protein for structure determination. Both efforts feed the SECSG high throughput
crystallization pipeline, which is capable of screening over 40 proteins per week. Details of the various pipelines in use
by the SECSG for protein production and crystallization, as well as some examples of target rescue are described.
Journal of Structural and Functional Genomics 08/2005; 6(2):233-243.
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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
[show abstract]
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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
[show abstract]
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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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Wolfram Tempel,
Zhi-Jie Liu,
Peter S Horanyi,
Lu Deng,
Doowon Lee, M Gary Newton,
John P Rose,
Hisashi Ashida,
Su-Chen Li,
Yu-Teh Li,
Bi-Cheng Wang
Proteins Structure Function and Bioinformatics 05/2005; 59(1):141-4. · 3.39 Impact Factor
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[show abstract]
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ABSTRACT: Two recent developments, a novel screening/optimization strategy that considerably reduces the number of trials required to produce diffraction-size crystals and a simple modification that doubles the screening capacity of the Douglas Instruments ORYX 1-6 protein-crystallization robot, have been implemented into a structural genomics project. The new two-step screening/optimization strategy yields diffraction-quality crystals directly from the screening process, reducing the need for further optimization. The ORYX modification involves the addition of extensions to the sample- and oil-delivery arms and software modifications that allow two plates to be set up simultaneously.
Acta Crystallographica Section D Biological Crystallography 03/2005; 61(Pt 2):123-9. · 12.62 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
[show abstract]
[hide abstract]
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.
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Wolfram Tempel,
Zhi-Jie Liu,
Peter S. Horanyi,
Lu Deng,
Doowon Lee, M. Gary Newton,
John P. Rose,
Hisashi Ashida,
Su-Chen Li,
Yu-Teh Li,
Bi-Cheng Wang
Proteins Structure Function and Bioinformatics 01/2005; 59(1):141 - 144. · 3.39 Impact Factor
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[show abstract]
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ABSTRACT: The NAD(+)-dependent glycerol dehydrogenase (EC 1.1.1.6) from the extremely thermophilic bacterium Thermotoga maritima has been crystallized in the presence of glycerol by the hanging-drop vapour-diffusion method using 2-methyl-2,4-pentanediol (MPD) as the precipitating agent. Crystals of the enzyme complexed with NAD(+) have also been obtained. The crystals belong to the tetragonal system with space group I422 and unit-cell parameters a = 105.3, c = 134.5 A. They diffract to a maximum resolution of 1.4 A using synchrotron radiation (lambda = 0.838 A). Crystals of the enzyme-NAD(+) complex diffract to 2.5 A resolution using in-house Cu Kalpha radiation.
Acta Crystallographica Section D Biological Crystallography 06/2002; 58(Pt 5):867-9. · 12.62 Impact Factor
