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Shotgun crystallization strategy for structural genomics:: an optimized two-tiered crystallization screen against the Thermotoga maritima proteome

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ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 2003年第6期59卷 1028-1037页

作者： Page, R Grzechnik, SK Canaves, JM Spraggon, G Kreusch, A Kuhn, P Stevens, RC Lesley, SA Scripps Res Inst Dept Mol Biol La Jolla CA 92037 USA Univ Calif San Diego La Jolla CA 92093 USA Novartis Res Fdn Genom Inst San Diego CA 92121 USA Scripps Res Inst Dept Cell Biol La Jolla CA 92037 USA

As the field of structural genomics continues to grow and new technologies are developed, novel strategies are needed to efficiently crystallize large numbers of protein targets, thus increasing output, not just throughput [Chayen & Saridakis (2002). Acta Cryst. D58, 921-927]. One strategy, developed for the high-throughput structure determination of the Thermotoga maritima proteome, is to quickly determine which proteins have a propensity for crystal formation followed by focused SeMet-incorporated protein crystallization attempts. This experimental effort has resulted in over 320 000 individual crystallization experiments. As such, it has provided one of the most extensive systematic data sets of commonly used crystallization conditions against a wide range of proteins to date. Analysis of this data shows that many of the original screening conditions are redundant, as all of the T. maritima proteins that crystallize readily could be identified using just 23% of the original conditions. It also shows that proteins that contain selenomethionine and are more extensively purified often crystallize in distinctly different conditions from those of their native less pure counterparts. Most importantly, it shows that the two-tiered strategy employed here is extremely successful for predicting which proteins will readily crystallize, as greater than 99% of the proteins identified as having a propensity to crystallize under non-optimal native conditions did so again as selenomethionine derivatives during the focused crystallization trials. This crystallization strategy can be adopted for both large-scale genomics programs and individual protein studies with multiple constructs and has the potential to significantly accelerate future crystallographic efforts.

关键词：细菌/化学细菌蛋白质类/化学克隆分子结晶结晶学 X线扩散基因组学/方法甘油/化学氢离子浓度蛋白质组/化学蛋白质组/遗传学硒代甲硫氨酸/化学温度 X线衍射

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High-resolution structure of human phosphoserine phosphatase in open conformation

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ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY 2003年第6期59卷 971-977页

作者： Peeraer, Y Rabijns, A Verboven, C Collet, JF Van Schaftingen, E De Ranter, C Katholieke Univ Leuven Fac Pharmaceut Sci Lab Analyt Chem & Med Physicochem B-3000 Louvain Belgium Catholic Univ Louvain Christian de Duve Inst Cellular Pathol Physiol Chem Lab B-1200 Brussels Belgium

The crystal structure of human phosphoserine phosphatase (HPSP) in the open conformation has been determined at a resolution of 1.53 Angstrom. The crystals are orthorhombic, belonging to space group C222(1), with unit-cell parameters a = 49.03, b = 130.25, c = 157.29 Angstrom. The asymmetric unit contains two molecules. Phase information was derived from a multi-wavelength anomalous dispersion ( MAD) experiment conducted at three wavelengths using a selenomethionine-derivative crystal of HPSP. The structure was refined using CNS to a final crystallographic R value of 21.6% (R-free = 23.4%). HPSP is a dimeric enzyme responsible for the third and final step of the L-serine biosynthesis pathway. It catalyses the Mg2+-dependent hydrolysis of L-phosphoserine. Recently, the structure of HPSP in complex with an inhibitor bound to the active site has been reported to be the open conformation of the enzyme. Here, the structure of HPSP is reported in the absence of substrate in the active site. Evidence is presented that HPSP in an uncomplexed form is in an even more open conformation than in the inhibitor complex. In this state, the enzyme is partially unfolded to allow the substrate to enter the active site. Binding of the substrate causes HPSP to shift to the closed conformation by stabilizing the partially unfolded region. In the present structure a Ca2+ ion is bound to the active site and an explanation is given why HPSP is not active when in the active site Mg2+ is replaced by a Ca2+ ion.

关键词：氨基酸序列结合部位钙/化学结晶结晶学 X线模型分子磷酸单酯水解酶类/化学蛋白质构象硒代甲硫氨酸/化学水/化学人类

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Crystallization and preliminary X-ray crystallographic analysis of SP1, a novel chaperone-like protein

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ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 2003年第3期59卷 512-514页

作者： Wang, WX Dgany, O Dym, O Altman, A Shoseyov, O Almog, O Ben Gurion Univ Negev Fac Hlth Sci Dept Clin Biochem IL-84105 Beer Sheva Israel Hebrew Univ Jerusalem Inst Plant Sci & Genet Agr Fac Agr Food & Environm Qual Sci IL-76100 Rehovot Israel Hebrew Univ Jerusalem Inst Life Sci Wolfson Ctr Struct Biol IL-91904 Jerusalem Israel

SP1 (108 amino acids) is a boiling-stable stress-responsive protein. It has no significant sequence homology to other stress-related proteins or to small heat-shock proteins (sHsps). SP1 activity is ATP-independent, similar to other small heat-shock proteins. Based on these features, it is expected that the structure-function relationship of SP1 will be unique. In this work, the crystallization and preliminary crystallographic data of native SP1 and its selenomethionine derivative are described. Recombinant SP1 and its selenomethionine derivative were expressed in Escherichia coli and used for crystallization experiments. SP1 crystals were grown from 0.1 M HEPES pH 7.5, 20% PEG 3K, 0.2 M NaCl. One to four single crystals appeared in each droplet within a few days and grew to dimensions of about 0.5 x 0.5 x 0.8 mm after about two weeks. Diffraction studies of these crystals at low temperature indicated that they belong to space group I422, with unit-cell parameters a = 89, b = 89, c = 187 Angstrom. Efforts to crystallize the selenomethionine derivative of SP1 are in progress.

关键词：结晶结晶学 X线电泳聚丙烯酰氨凝胶内肽酶K/化学大肠杆菌/代谢热休克蛋白质类/化学水解作用模型分子分子伴侣/分析硒代甲硫氨酸/化学

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Crystallization and preliminary X-ray analysis of native and selenomethionine 2-hydroxybiphenyl 3-monooxygenase

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ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY 2003年第4期59卷 741-743页

作者： Meyer, A Tanner, D Schmid, A Sargent, DF Kohler, HPE Witholt, B ETH Honggerberg ETHZ Swiss Fed Inst Technol Inst Biotechnol CH-8093 Zurich Switzerland ETH Honggerberg ETHZ Swiss Fed Inst Technol Inst Mol Biol & Biophys CH-8093 Zurich Switzerland Swiss Fed Inst Environm Sci & Technol EAWAG CH-8600 Dubendorf Switzerland

2-Hydroxybiphenyl 3-monooxygenase (HbpA;EC 1.14.13.44) from Pseudomonas azelaica HBP1 was produced in Escherichia coli both as native and SeMet-labelled protein. The two enzymes were purified to homogeneity and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 108.6, b = 196.8, c = 79.3 Angstrom, beta = 97.7degrees for the native protein and a = 108.3, b = 196.8, c = 79.0 Angstrom, beta = 97.8degrees for SeMet HbpA. Crystal-packing considerations led to the assumption of two HbpA subunits per asymmetric unit, which corresponds to a V-M value of 3.3 Angstrom(3) Da(-1) and a solvent content of 62%. The crystals were radiation-sensitive and only had a lifespan of about 120 s when exposed to synchrotron radiation on an undulator beamline. To obtain complete data sets, data were collected from 23 native and 26 derivative crystals. The high-resolution limit was 2.0 Angstrom for native and 2.25 Angstrom for SeMet HbpA.

关键词：结晶结晶学 X线大肠杆菌/代谢混合功能氧化酶类/化学假单胞菌属/化学硒代甲硫氨酸/化学硒代甲硫氨酸/代谢 X线衍射

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Crystallization and preliminary crystallographic analysis of extracellular fragment X3 of YWK-II/APPH:: a human sperm membrane protein related to the Alzheimer βA4-amyloid precursor protein

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ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY 2003年第3期59卷 506-508页

作者： Yang, MJ Zhou, WH Zhang, XD Hu, WJ Gao, F Pang, H Miao, SY Wang, LF Rao, ZH Tsinghua Univ MOE Prot Sci Lab Dept Biol Sci & Biotechnol Beijing 100084 Peoples R China Tsinghua Univ Struct Biol Lab Dept Biol Sci & Biotechnol Beijing 100084 Peoples R China Natl Human Genome Ctr Beijing Peoples R China Chinese Acad Med Sci Natl Lab Med Mol Biol Inst Basic Med Sci Peking Union Med Coll Beijing 100005 Peoples R China

Crystals of extracellular fragment X3 of a human sperm membrane protein YWK-II/APPH have been grown at 291 K using 8% PEG 4000 as precipitant by the vapour-diffusion method. The diffraction pattern of the crystal extends to 2.9 Angstrom resolution at 100 K using Cu Kalpha radiation in-house. The crystals belong to space group P2(1), with unit-cell parameters a = 46.0, b = 43.7, c = 90.2 Angstrom, alpha = gamma = 90.0, beta = 106.6degrees. Furthermore, a selenomethionine (SeMet) derivative of the protein was overexpressed in the same expression system and was purified in a reducing environment. The derivative crystals were obtained under similar conditions. Subsequently, a single-wavelength data set was collected to 2.38 Angstrom resolution from the derivative crystal at ESRF. The crystals belong to space group P2(1), with unit-cell parameters a = 46.2, b = 44.0, c = 88.3 Angstrom, alpha = gamma = 90.0, beta = 103.6degrees. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (V-M) of 2.8 Angstrom(3) Da(-1) and a solvent content of 56.4% by volume.

关键词：淀粉样β蛋白前体/化学结晶结晶学 X线膜蛋白质类/生物合成膜蛋白质类/化学膜蛋白质类/分离和提纯膜/化学神经组织蛋白质类肽碎片/化学蛋白质结构二级重组蛋白质类/化学硒代甲硫氨酸/化学精子/化学人类男(雄)性

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Crystallization and preliminary X-ray studies of the glutaredoxin from poplar in complex with glutathione

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ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY 2003年第6期59卷 1043-1045页

作者： D'Ambrosio, K Kauffmann, BA Rouhier, N Benedetti, E Jacquot, JP Aubry, A Corbier, C Univ Nancy 1 Fac Sci Grp Biocristallog LCM3BCNRSUMR 7036 F-54506 Vandoeuvre Les Nancy France Univ Naples Federico II Dipartimento Chim Biol CNR Ist Biostrutture & Bioimmagini I-80134 Naples Italy Univ Nancy 1 Fac Sci INRA Interact Arbres Microorganismes UMR 1136 F-54506 Vandoeuvre Les Nancy France

A monocysteinic mutant of poplar glutaredoxin (C30S) has been overproduced and purified. The protein has been crystallized in complex with glutathione using the hanging-drop vapour-diffusion technique in the presence of PEG 4000 as a precipitating agent. A native data set was collected at 1.55 Angstrom resolution. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 45.7, b = 49.1, c = 104.8 Angstrom. Isomorphous crystals of a selenomethionine derivative were grown under the same conditions. Three data sets were collected at 1.73 Angstrom using the FIP synchrotron beamline at the ESRF. The positions of the Se atoms were determined and model rebuilding and refinement are in progress.

关键词：克隆分子结晶结晶学 X线电泳聚丙烯酰氨凝胶谷氧还蛋白质类谷胱甘肽/化学诱变定点氧化还原酶类蛋白质类/化学硒代甲硫氨酸/化学树

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Crystallization and preliminary X-ray crystallographic analysis of the trimer core from measles virus fusion protein

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ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 2003年第3期59卷 587-590页

作者： Zhu, JQ Ding, Y Gao, F Wu, TH Zhang, CWH Tien, P Rao, ZH Gao, GF Chinese Acad Sci Inst Microbiol Dept Mol Virol Beijing 100080 Peoples R China Tsing Hua Univ Sch Life Sci & Bioengn Struct Biol Lab Beijing 100084 Peoples R China Tsing Hua Univ Sch Life Sci & Bioengn MOE Lab Prot Sci Beijing 100084 Peoples R China Oxagen Ltd Oxford OX14 4RY England Univ Oxford John Radcliffe Hosp Nuffield Dept Clin Med Oxford OX3 9DU England

Two heptad-repeat regions (HR1 and HR2) are highly conserved in paramyxovirus fusion proteins and form a stable helical trimer of heterodimers [(HR1-HR2)(3)] after the fusion between viral and cellular membranes. In this study, two HR regions of the fusion protein of measles virus, a member of the paramyxoviruses, were selected and overexpressed as a single chain (named 2-Helix) connected by an amino-acid linker using a GST-fusion expression system in Escherichia coli. Crystals of 2-Helix protein (GST removed) could be obtained from many conditions using the sitting- or hanging-drop vapour-diffusion method. A complete data set was collected in-house to 1.9 Angstrom resolution from a single crystal. The crystal belongs to space group P6, with unit-cell parameters a = b = 51.637, c = 67.058 Angstrom. To facilitate the crystal structure solution, SeMet-substituted 2-Helix crystals, grown under similar conditions to the native, were also obtained and diffracted X-rays to 1.8 Angstrom using synchrotron radiation.

关键词：结晶结晶学 X线大肠杆菌/代谢基因表达调控病毒/遗传学蛋白质构象重组蛋白质类/生物合成重组蛋白质类/化学重组蛋白质类/分离和提纯硒代甲硫氨酸/化学病毒融合蛋白质类/化学病毒融合蛋白质类/遗传学病毒融合蛋白质类/分离和提纯 X线衍射

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Crystallization of the actin-binding domain of human α-actinin:: analysis of microcrystals of SeMet-labelled protein

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ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY 2003年第4期59卷 724-726页

作者： Ekström, F Stier, G Sauer, UH Umea Univ UCMP Umea Ctr Mol Pathogenisis SE-90187 Umea Sweden EMBL D-69017 Heidelberg Germany

alpha-Actinin forms antiparallel homodimers that cross-link actin filaments from adjacent sarcomeres within the Z-discs of striated muscle. The N-terminal actin-binding domain (ABD) is composed of two calponin homology (CH) domains followed by four spectrin-like repeats and a calmodulin-like EF-hand domain at the C-terminus. The ABD of human alpha-actinin crystallizes in space group P2(1), with unit-cell parameters a=101.9, b=38.4, c=154.9 Angstrom, beta=109.2degrees. A complete native data set from a native crystal was collected extending to 2.0 Angstrom resolution and a single-wavelength anomalous dispersion (SAD) data set to 2.9 Angstrom resolution was collected from a selenomethionine-labelled microcrystal using the microfocusing beamline ID-13 at the ESRF. Analysis of the anomalous contribution shows a rapid decrease in the sigma(normal)/sigma(anomal) ratio owing to radiation damage.

关键词：辅肌动蛋白/化学辅肌动蛋白/分离和提纯辅肌动蛋白/代谢肌动蛋白类/代谢克隆分子结晶指示剂和试剂蛋白质结合硒代甲硫氨酸/化学人类

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Selenomethionine substitution of orotidine-5′-monophosphate decarboxylase causes a change in crystal contacts and space group

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ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY 2001年第9期57卷 1251-1259页

作者： Poulsen, JCN Harris, P Jensen, KF Larsen, S Univ Copenhagen Dept Chem Ctr Crystallog Studies DK-2100 Copenhagen O Denmark Univ Copenhagen Inst Mol Biol DK-1307 Copenhagen K Denmark

Orotidine 5'-monophosphate decarboxylase (ODCase) catalyses the decarboxylation of orotidine 5'-monophosphate to uridine 5'-monophosphate, the last step in the de novo biosynthesis of uridine 5'-monophosphate. In order to determine the structure of ODCase from Escherichia coli by the multi-wavelength anomalous dispersion technique, both native and SeMet-substituted proteins have been produced and purified. During the production of SeMet ODCase, it was observed that SeMet was the only amino acid that it was necessary to add to the defined medium during expression. SeMet-substituted ODCase in complex with the inhibitor 1-(5'-phospho-beta -D-ribofuranosyl)barbituric acid crystallizes under similar conditions as the native enzyme. In contrast to the native enzyme, where the crystals belong to the orthorhombic space group P2(1)2(1)2(1), the SeMet-substituted enzyme crystallizes in the monoclinic space group P2(1), with a quadrupling of the volume of the asymmetric unit. Despite the drastic difference in symmetry, the overall crystal packing is effectively identical in the two crystal forms. The change in space group appears to originate in differences in the crystal contacts near the SeMet and Met residues. These differences can be rationalized in terms of SeMet's larger size and hydrophobicity.

关键词：氨基酸取代结晶结晶学 X线大肠杆菌/酶学模型分子乳清酸核苷-5'-磷酸脱羧酶/化学乳清酸核苷-5'-磷酸脱羧酶/遗传学蛋白质构象硒代甲硫氨酸/化学

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Crystallization and preliminary X-ray crystallographic analysis of native and selenomethionyl recombinant tabtoxin-resistance protein complexed with acetyl-coenzyme A

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ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 2001年第11期57卷 1729-1731页

作者： He, HZ Ding, Y Cao, ZB Shao, Y Bartlam, M Tang, H Jiang, F Liu, YW Liu, JY Zhao, NM Rao, ZH Tsing Hua Univ Sch Life Sci & Engn Struct Biol Lab Beijing 100084 Peoples R China

Tabtoxin-resistance protein (TTR), an acetyltransferase from Pseudomonas syringae pv. tabaci, was overexpressed in Eschericha coli M15 and the TTR fusion protein complexed with acetyl-coenzyme A (AcCoA) was purified and crystallized. Diffraction data were collected to 3.0 Angstrom resolution in-house and the crystal was found to belong to space group P2(1), with unit-cell parameters a = 47.6, b = 66.6, c = 53.5 Angstrom, beta = 104.3 degrees. Furthermore, a selenomethionine (SeMet) TTR fusion protein derivative was overexpressed in the same expression system and its complex with AcCoA was purified in a reductive environment. The SeMet TTR derivative crystallized in two forms: the first was identical to that observed for native crystals and the second belonged to space group C2, with unit-cell parameters a = 101.7, b = 45.6, c = 84.2 Angstrom, beta = 105.8 degrees. Data from the P2(1) crystal form were collected in-house to 2.3 Angstrom resolution. Subsequently, three different wavelength data sets of the C2 crystal form to 1.55 Angstrom resolution were collected at the Advanced Photon Source at Argonne National Laboratory.

关键词：乙酰辅酶A/化学乙酰基转移酶类/化学细菌蛋白质类结晶结晶学 X线蛋白质构象假单胞菌属/酶学重组蛋白质类/化学硒代甲硫氨酸/化学

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