| 1. |
- Addario, Barbara, et al.
(författare)
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Crystallization and preliminary X-ray analysis of the Entamoeba histolytica α-actinin-2 rod domain
- 2011
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Ingår i: Acta Crystallographica. Section F. - : International Union of Crystallography. - 1744-3091 .- 1744-3091. ; 67:10, s. 1214-1217
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Tidskriftsartikel (refereegranskat)abstract
- -Actinins form antiparallel homodimers that are able to cross-link actin filaments. The protein contains three domains: an N-terminal actin-binding domain followed by a central rod domain and a calmodulin-like EF-hand domain at the C-terminus. Here, crystallization of the rod domain of Entamoeba histolytica -actinin-2 is reported; it crystallized in space group P212121, with unit-cell parameters a = 47.8, b = 79.1, c = 141.8 Å. A Matthews coefficient VM of 2.6 Å3 Da-1 suggests that there are two molecules and 52.5% solvent content in the asymmetric unit. A complete native data set extending to a d-spacing of 2.8 Å was collected on beamline I911-2 at MAX-lab, Sweden.
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| 2. |
- Aguda, Adeleke Halilu, et al.
(författare)
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Expression, crystallization and preliminary crystallographic data analysis of filamin A repeats 14-16
- 2007
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 63:4, s. 291-293
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Tidskriftsartikel (refereegranskat)abstract
- Human filamin A is a 280 kDa protein involved in actin-filament cross-linking. It is structurally divided into an actin-binding headpiece (ABD) and a rod domain containing 24 immunoglobulin-like (Ig) repeats. A fragment of human filamin A (Ig repeats 14-16) was cloned and expressed in Escherichia coli and the purified protein was crystallized in 1.6 M ammonium sulfate, 2% PEG 1000 and 100 mM HEPES pH 7.5. The crystals diffracted to 1.95 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.63, b = 52.10, c = 98.46 A, alpha = beta = gamma = 90 degrees.
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| 3. |
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| 4. |
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| 5. |
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| 6. |
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| 7. |
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| 8. |
- Dobritzsch, Doreen, 1972-, et al.
(författare)
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Crystallization and X-ray diffraction analysis of dihydropyrimidinase from Saccharomyces kluyveri
- 2005
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 61, s. 359-362
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Tidskriftsartikel (refereegranskat)abstract
- Dihydropyrimidinase (EC 3.5.2.2) catalyzes the second step in the reductive pathway of pyrimidine degradation, the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated beta-amino acids. Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri diffracting to 2.6 A at a synchrotron-radiation source have been obtained by the hanging-drop vapour-diffusion method. They belong to space group P2(1) (unit-cell parameters a = 91.0, b = 73.0, c = 161.4 A, beta = 91.4 degrees), with one homotetramer per asymmetric unit.
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| 9. |
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| 10. |
- Feil, S. C., et al.
(författare)
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Crystallization and preliminary X-ray analysis of glutathione transferases from cyanobacteria
- 2009
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 65, s. 475-477
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Tidskriftsartikel (refereegranskat)abstract
- Glutathione S-transferases (GSTs) are a group of multifunctional enzymes that are found in animals, plants and microorganisms. Their primary function is to remove toxins derived from exogenous sources or the products of metabolism from the cell. Mammalian GSTs have been extensively studied, in contrast to bacterial GSTs which have received relatively scant attention. A new class of GSTs called Chi has recently been identified in cyanobacteria. Chi GSTs exhibit a high glutathionylation activity towards isothiocyanates, compounds that are normally found in plants. Here, the crystallization of two GSTs are presented: TeGST produced by Thermosynechococcus elongates BP-1 and SeGST from Synechococcus elongates PCC 6301. Both enzymes formed crystals that diffracted to high resolution and appeared to be suitable for further X-ray diffraction studies. The structures of these GSTs may shed further light on the evolution of GST catalytic activity and in particular why these enzymes possess catalytic activity towards plant antimicrobial compounds.
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| 11. |
- Forsgren, Nina, 1979-, et al.
(författare)
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A crystallizable form of the Streptococcus gordonii surface antigen SspB C-domain obtained by limited proteolysis
- 2009
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 65:7, s. 712-714
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Tidskriftsartikel (refereegranskat)abstract
- SspB is a 1500-residue adhesin expressed on the surface of the oral bacterium Streptococcus gordonii. Its interaction with other bacteria and host cells initiates the development of dental plaque. The full-length C-terminal domain of SspB was cloned, overexpressed in Escherichia coli and purified. However, the protein could not be crystallized. Limited proteolysis of the full-length C-domain identified a core fragment. The proteolysis product was cloned, expressed and purified. The protein was crystallized using the hanging-drop vapour-diffusion method. X-ray data were collected and processed to a maximum resolution of 2.1 A with 96.4% completeness. The crystals belonged to space group P2(1), with one molecule in the asymmetric unit, a solvent content of 33.7% and a corresponding Matthews coefficient of 1.85 A(3) Da(-1).
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| 12. |
- Gabrielsen, Mads, et al.
(författare)
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Expression, purification, crystallization and initial X-ray diffraction analysis of thiol peroxidase from Yersinia pseudotuberculosis
- 2010
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Ingår i: Acta Crystallographica. Section F. - : International Union of Crystallography. - 1744-3091 .- 1744-3091. ; 66:Pt 12, s. 1606-1609
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Tidskriftsartikel (refereegranskat)abstract
- Thiol peroxidase is an atypical 2-Cys peroxiredoxin that reduces alkyl hydroperoxides. Wild-type and C61S mutant protein have been recombinantly expressed in Escherichia coli and purified using nickel-affinity chromatography. Initial crystallization trials yielded three crystal forms in three different space groups (P2(1), P6(4) and P2(1)2(1)2(1)) both in the presence and the absence of DTT.
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| 13. |
- Gurmu, Daniel, et al.
(författare)
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Expression, purification, crystallization and preliminary X-ray analysis of ORF60, the small subunit (R2) of ribonucleotide reductase from Kaposi's sarcoma-associated herpesvirus (KSHV)
- 2010
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 66, s. 734-737
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RNR) is responsible for converting ribonucleotides to deoxyribonucleotides, which are the building blocks of DNA. The enzyme is present in all life forms as well as in some large DNA viruses such as herpesviruses. The alpha-herpesviruses and gamma-herpesviruses encode two class Ia RNR subunits, R1 and R2, while the beta-herpesvirus subfamily only encode an inactive R1 subunit. Here, the crystallization of the R2 subunit of RNR encoded by the ORF60 gene from the oncovirus Kaposi's sarcoma-associated gamma-herpesvirus (KSHV) is reported. These are the first crystals of a viral R2 subunit; the use of in situ proteolysis with chymotrypsin and the addition of hexamine cobalt(III) chloride that were necessary to obtain crystals are described. Optimization of the crystallization conditions yielded crystals that diffracted to 2.0 angstrom resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 63.9, b = 71.2, c = 71.8 angstrom, alpha = 90, beta = 106.7, gamma = 90 degrees. The data set collected was 95.3% complete, with an R-merge of 9.6%. There are two molecules in the asymmetric unit, corresponding to a solvent content of 43.4%.
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| 14. |
- Hall, Michael, 1980-, et al.
(författare)
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Purification, crystallization and preliminary X-ray analysis of PPD6, a PsbP-domain protein from Arabidopsis thaliana
- 2012
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 68:3, s. 278-280
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Tidskriftsartikel (refereegranskat)abstract
- The PsbP protein is an extrinsic component of photosystem II that together with PsbO and PsbQ forms the thylakoid lumenal part of the oxygen-evolving complex in higher plants. In addition to PsbP, the thylakoid lumen contains two PsbP-like proteins (PPLs) and six PsbP-domain proteins (PPDs). While the functions of the PsbP-like proteins PPL1 and PPL2 are currently under investigation, the function of the PsbP-domain proteins still remains completely unknown. PPD6 is unique among the PsbP family of proteins in that it contains a conserved disulfide bond which can be reduced in vitro by thioredoxin. The crystal structure determination of the PPD6 protein has been initiated in order to elucidate its function and to gain deeper insights into redox-regulation pathways in the thylakoid lumen. PPD6 has been expressed, purified and crystallized and preliminary X-ray diffraction data have been collected. The crystals belonged to space group P2(1), with unit-cell parameters a = 47.0, b = 64.3, c = 62.0 Å, β = 94.2°, and diffracted to a maximum d-spacing of 2.1 Å.
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| 15. |
- Hasse, Dirk, et al.
(författare)
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Crystallization and preliminary X-ray diffraction analyses of the homodimeric glycine decarboxylase (P-protein) from the cyanobacterium Synechocystis sp. PCC 6803.
- 2010
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 66:Pt 2, s. 187-191
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Tidskriftsartikel (refereegranskat)abstract
- Glycine decarboxylase, or P-protein, is a major enzyme that is involved in the C(1) metabolism of all organisms and in the photorespiratory pathway of plants and cyanobacteria. The protein from Synechocystis sp. PCC 6803 is a homodimer with a mass of 215 kDa. Recombinant glycine decarboxylase was expressed in Escherichia coli and purified by metal-affinity, ion-exchange and gel-filtration chromatography. Crystals of P-protein that diffracted to a resolution of 2.1 A were obtained using the hanging-drop vapour-diffusion method at 291 K. X-ray diffraction data were collected from cryocooled crystals using synchrotron radiation. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 96.30, b = 135.81, c = 179.08 A.
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| 16. |
- Herman, Maria Dolores, et al.
(författare)
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Structures of BIR domains from human NAIP and cIAP2
- 2009
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 65, s. 1091-1096
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Tidskriftsartikel (refereegranskat)abstract
- The inhibitor of apoptosis (IAP) family of proteins contains key modulators of apoptosis and inflammation that interact with caspases through baculovirus IAP-repeat (BIR) domains. Overexpression of IAP proteins frequently occurs in cancer cells, thus counteracting the activated apoptotic program. The IAP proteins have therefore emerged as promising targets for cancer therapy. In this work, X-ray crystallography was used to determine the first structures of BIR domains from human NAIP and cIAP2. Both structures harbour an N-terminal tetrapeptide in the conserved peptide-binding groove. The structures reveal that these two proteins bind the tetrapeptides in a similar mode as do other BIR domains. Detailed interactions are described for the P1'-P4' side chains of the peptide, providing a structural basis for peptide-specific recognition. An arginine side chain in the P3' position reveals favourable interactions with its hydrophobic moiety in the binding pocket, while hydrophobic residues in the P2' and P4' pockets make similar interactions to those seen in other BIR domain-peptide complexes. The structures also reveal how a serine in the P1' position is accommodated in the binding pockets of NAIP and cIAP2. In addition to shedding light on the specificity determinants of these two proteins, the structures should now also provide a framework for future structure-based work targeting these proteins.
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| 17. |
- Ingvarsson, Henrik, et al.
(författare)
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Crystallization of Mycobacterium smegmatis methionyl-tRNA synthetase in the presence of methionine and adenosine
- 2009
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 65:Part 6, s. 618-620
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Tidskriftsartikel (refereegranskat)abstract
- Methionyl-tRNA synthetase (MetRS) from Mycobacterium smegmatis was recombinantly expressed in Escherichia coli and purified using Ni(2+)-affinity and size-exclusion chromatography. Crystals formed readily in the presence of the ligands methionine and adenosine. These two ligands are components of an intermediate in the two-step catalytic mechanism of MetRS. The crystals were produced using the vapour-diffusion method and a full data set to 2.1 A resolution was collected from a single crystal. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 155.9, b = 138.9, c = 123.3 A, beta = 124.8 degrees . The presence of three molecules in the asymmetric unit corresponded to a solvent content of 60% and a Matthews coefficient of 3.1 A(3) Da(-1). Structure determination is in progress.
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| 18. |
- Jakobsson, Emma, et al.
(författare)
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Crystallization of a truncated soluble human semicarbazide-sensitive amine oxidase.
- 2005
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Ingår i: Acta Crystallograph Sect F Struct Biol Cryst Commun. - 1744-3091. ; 61:Pt 3, s. 274-8
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Tidskriftsartikel (refereegranskat)abstract
- Human semicarbazide-sensitive amine oxidase (SSAO) is a homodimeric copper-containing monoamine oxidase that occurs in both a membrane-bound and a soluble form. SSAO is also known as vascular adhesion protein-1 (VAP-1). A truncated soluble form of human SSAO (comprising residues 29-763) was expressed in human embryonic kidney 293 cells and purified to homogeneity. Tetragonal crystals were obtained and a data set extending to 2.5 A was collected. The crystals are merohedrally twinned and the estimation of the twinning fraction was complicated by pseudo-symmetry and the anisotropic character of the crystals. Using a recently developed method for twinning detection that is insensitive to phenomena such as anisotropy or pseudo-symmetry [Padilla & Yeates (2003), Acta Cryst. D59, 1124-1130], the twinning fraction was estimated to be 0.3. The structure was eventually solved by molecular replacement in space group P4(3).
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| 19. |
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| 20. |
- Karlsson, Anders, et al.
(författare)
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Heating of proteins as a means of improving crystallization : a successful case study on a highly amyloidogenic triple mutant of human transthyretin.
- 2007
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 63:Pt 8, s. 695-700
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Tidskriftsartikel (refereegranskat)abstract
- The use of high temperatures in the purification procedures of heat-stable proteins is a well established technique. Recently, rapid pre-heat treatment of protein samples prior to crystallization trials was described as a final polishing step to improve the diffraction properties of crystals [Pusey et al. (2005), Prog. Biophys. Mol. Biol. 88, 359-386]. The present study demonstrates that extended high-temperature incubation (328 K for 48 h) of the highly amyloidogenic transthyretin mutant TTR G53S/E54D/L55S successfully removes heterogeneities and allows the reproducible growth of well diffracting crystals. Heat treatment might be applied as an optimization method to other cases in which the protein/biomolecule fails to form diffracting crystals.
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| 21. |
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| 22. |
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| 23. |
- Krengel, Ute, 1964, et al.
(författare)
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Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: A tricky crystallization problem solved
- 2006
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Ingår i: Acta Crystallographica Section F: Structural Biology and Crystallization Communications. - 1744-3091. ; 62:5, s. 441-445
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Tidskriftsartikel (refereegranskat)abstract
- Chorismate mutase catalyzes the conversion of chorismate to prephenate in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine in bacteria, fungi and plants. Here, the crystallization of the unusual secreted chorismate mutase from Mycobacterium tuberculosis (encoded by Rv1885c), a 37.2 kDa dimeric protein belonging to the AroQγ subclass of mutases, is reported. Crystal optimization was non-trivial and is discussed in detail. To obtain crystals of sufficient quality, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5-15 min, in an adaptation of a previously described technique [Saridakis & Chayen (2000), Protein Sci. 9, 755-757]. As a result of the optimization, diffraction improved from 3.5 to 1.3 Å resolution. The crystals belong to space group P21, with unit-cell parameters a = 42.6, b = 72.6, c = 62.0 Å., β = 104.5°. The asymmetric unit contains one biological dimer, with 167 amino acids per protomer. A soak with a transition-state analogue is also described.
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| 24. |
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| 25. |
- Lejon, Sara, et al.
(författare)
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Structural basis for the binding of naproxen to human serum albumin in the presence of fatty acids and the GA module
- 2008
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 64:Pt 2, s. 64-69
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Tidskriftsartikel (refereegranskat)abstract
- The previously determined crystal structure of the bacterial albumin-binding GA module in complex with human serum albumin (HSA) suggested the possibility of utilizing the complex in the study of ligand binding to HSA. As a continuation of these studies, the crystal structure of the HSA-GA complex with the drug molecule naproxen and the fatty acid decanoate bound to HSA has been determined to a resolution of 2.5 A. In terms of drug binding, the structure suggests that the binding of decanoate to the albumin molecule may play a role in making the haemin site in subdomain IB of the albumin molecule available for the binding of naproxen. In addition, structure comparisons with solved structures of HSA and of the HSA-GA complex show that the GA module is capable of binding to different conformations of HSA. The HSA-GA complex therefore emerges as a possible platform for the crystallographic study of specific HSA-drug interactions and of the influence exerted by the presence of fatty acids.
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| 26. |
- Lohkamp, B, et al.
(författare)
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Purification, crystallization and X-ray diffraction analysis of dihydropyrimidinase from Dictyostelium discoideum
- 2006
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Ingår i: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications. - : International Union of Crystallography (IUCr). - 2053-230X .- 1744-3091. ; 62:1, s. 36-38
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Tidskriftsartikel (refereegranskat)abstract
- Dihydropyrimidinase (EC 3.5.2.2) is the second enzyme in the reductive pyrimidine-degradation pathway and catalyses the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated beta-amino acids. The recombinant enzyme from the slime mould Dictyostelium discoideum was overexpressed, purified and crystallized by the vapour-diffusion method. One crystal diffracted to better than 1.8 angstrom resolution on a synchrotron source and was shown to belong to space group I222, with unit-cell parameters a = 84.6, b = 89.6, c = 134.9 angstrom and one molecule in the asymmetric unit.
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| 27. |
- Lohkamp, Bernhard, et al.
(författare)
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Purification, crystallization and X-ray diffraction analysis of dihydropyrimidinase from Dictyostelium discoideum
- 2006
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 62:Pt 1, s. 36-38
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Tidskriftsartikel (refereegranskat)abstract
- Dihydropyrimidinase (EC 3.5.2.2) is the second enzyme in the reductive pyrimidine-degradation pathway and catalyses the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated beta-amino acids. The recombinant enzyme from the slime mould Dictyostelium discoideum was overexpressed, purified and crystallized by the vapour-diffusion method. One crystal diffracted to better than 1.8 A resolution on a synchrotron source and was shown to belong to space group I222, with unit-cell parameters a = 84.6, b = 89.6, c = 134.9 A and one molecule in the asymmetric unit.
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| 28. |
- Lundgren, Stina, et al.
(författare)
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Crystallization and preliminary X-ray data analysis of beta-alanine synthase from Drosophila melanogaster
- 2007
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 63, s. 874-877
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Tidskriftsartikel (refereegranskat)abstract
- Beta-alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine, which represents the main clearance route for the widely used anticancer drug 5-fluorouracil. Crystals of the recombinant enzyme from Drosophila melanogaster, which is closely related to the human enzyme, were obtained by the hanging-drop vapour-diffusion method. They diffracted to 3.3 A at a synchrotron-radiation source, belong to space group C2 (unit-cell parameters a = 278.9, b = 95.0, c = 199.3 A, beta = 125.8 degrees) and contain 8-10 molecules per asymmetric unit.
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| 29. |
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| 30. |
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| 31. |
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| 32. |
- Mishra, Yogesh, et al.
(författare)
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Expression, purification, crystallization and preliminary X-ray crystallographic studies of alkyl hydroperoxide reductase (AhpC) from the cyanobacterium Anabaena sp. PCC 7120
- 2011
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Ingår i: Acta Crystallographica. Section F. - : International Union of Crystallography. - 1744-3091 .- 1744-3091. ; 67:10, s. 1203-1206
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Tidskriftsartikel (refereegranskat)abstract
- Alkyl hydroperoxide reductase (AhpC) is a key component of a large family of thiol-specific antioxidant (TSA) proteins distributed among prokaryotes and eukaryotes. AhpC is involved in the detoxification of reactive oxygen species (ROS) and reactive sulfur species (RSS). Sequence analysis of AhpC from the cyanobacterium Anabaena sp. PCC 7120 shows that this protein belongs to the 1-Cys class of peroxiredoxins (Prxs). It has recently been reported that enhanced expression of this protein in Escherichia coli offers tolerance to multiple stresses such as heat, salt, copper, cadmium, pesticides and UV-B. However, the structural features and the mechanism behind this process remain unclear. To provide insights into its biochemical function, AhpC was expressed, purified and crystallized by the hanging-drop vapour-diffusion method. Diffraction data were collected to a maximum d-spacing of 2.5 Å using synchrotron radiation. The crystal belonged to space group P212121, with unit-cell parameters a = 80, b = 102, c = 109.6 Å. The structure of AhpC from Anabaena sp. PCC 7120 was determined by molecular-replacement methods using the human Prx enzyme hORF6 (PDB entry1prx) as the template.
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| 33. |
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| 34. |
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| 35. |
- Nylander, Åsa, 1974-, et al.
(författare)
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Structure of the C-terminal domain of the surface antigen SpaP from the caries pathogen Streptococcus mutans
- 2011
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Ingår i: Acta Crystallographica. Section F. - : International Union of Crystallography. - 1744-3091 .- 1744-3091. ; 67, s. 23-26
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Tidskriftsartikel (refereegranskat)abstract
- SpaP is a 1500-residue adhesin expressed on the surface of the caries-implicated bacterium Streptococcus mutans. SpaP is a member of the antigen I/II (AgI/II) family of proteins expressed by oral streptococci. These surface proteins are crucial for the incorporation of streptococci into dental plaque. The structure of the C-terminal domain of SpaP (residues 1136-1489) was solved and refined to 2.2 Å resolution with six molecules in the asymmetric unit. Similar to a related AgI/II structure, SpaP is stabilized by isopeptide bonds between lysine and asparagine side chains.
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| 36. |
- Persson, Karina, 1969-
(författare)
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Crystallization of the fimbrial protein FimP from Actinomyces oris and of a triple Ile-to-Met mutant engineered to facilitate selenomethionine labelling
- 2011
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Ingår i: Acta Crystallographica. Section F. - : International Union of Crystallography. - 1744-3091 .- 1744-3091. ; F67, s. 1207-1210
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Tidskriftsartikel (refereegranskat)abstract
- Actinomyces oris is an oral bacterium important for the development of dental plaque. It expresses two forms of fimbriae: type 1 and type 2. FimP, which is the fimbrial protein that is polymerized into the stalk of the type 1 fimbriae, was cloned, overexpressed and crystallized. X-ray data were collected and processed to 2.2 Å resolution. The crystals belonged to space group P21212, with one molecule in the asymmetric unit. To facilitate structure determination using single anomalous dispersion, three methionines were introduced by site-directed mutagenesis. Crystals of selenomethionine-labelled protein were obtained by streak-seeding and diffracted to 2.0 Å resolution.
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| 37. |
- Punekar, Avinash S., et al.
(författare)
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Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli
- 2013
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 69, s. 1001-1003
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Tidskriftsartikel (refereegranskat)abstract
- 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 angstrom resolution from a single apo crystal. The crystals belonged to space group P2(1), with unit-cell parameters a = 46.9, b = 77.8, c = 82.5 angstrom, beta = 104 degrees. Data analysis suggested two molecules per asymmetric unit and a Matthews coefficient of 2.20 angstrom(3) Da(-1).
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| 38. |
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| 39. |
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| 40. |
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| 41. |
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| 42. |
- Stojanoff, Vivian, et al.
(författare)
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From screen to structure with a harvestable microfluidic device
- 2011
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 67, s. 971-975
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Tidskriftsartikel (refereegranskat)abstract
- Advances in automation have facilitated the widespread adoption of high-throughput vapour-diffusion methods for initial crystallization screening. However, for many proteins, screening thousands of crystallization conditions fails to yield crystals of sufficient quality for structural characterization. Here, the rates of crystal identification for thaumatin, catalase and myoglobin using microfluidic Crystal Former devices and sitting-drop vapour-diffusion plates are compared. It is shown that the Crystal Former results in a greater number of identified initial crystallization conditions compared with vapour diffusion. Furthermore, crystals of thaumatin and lysozyme obtained in the Crystal Former were used directly for structure determination both in situ and upon harvesting and cryocooling. On the basis of these results, a crystallization strategy is proposed that uses multiple methods with distinct kinetic trajectories through the protein phase diagram to increase the output of crystallization pipelines.
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| 43. |
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| 44. |
- Uchtenhagen, Hannes, et al.
(författare)
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Production, purification, crystallization and preliminary X-ray diffraction analysis of the HIV-2-neutralizing V3 loop-specific Fab fragment 7C8
- 2009
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 65, s. 705-708
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Tidskriftsartikel (refereegranskat)abstract
- 7C8 is a mouse monoclonal antibody that is specific for the third hypervariable loop (V3 loop) of the human immunodeficiency virus type 2 (HIV-2) associated protein gp125. Fab fragments of 7C8 effectively neutralize HIV-2. 7C8 was expressed and purified from a hybridoma cell line in order to establish the molecular basis underlying the specificity of the 7C8 antibody for the V3 loop as well as the specific role of the elongated third complementarity-determining region of the heavy chain (CDRH3). The antibody was digested with papain and Fab fragments were purified using size-exclusion chromatography. Hanging-drop vapour-diffusion crystallization techniques were employed and the protein was crystallized in 50 mM ammonium sulfate, 100 mM Tris-HCl pH 8.5, 25%(w/v) PEG 8000 and 2.5%(w/v) PEG 400 at 275 K. The analysed crystals belonged to the rhombohedral space group P3(2)21, with unit-cell parameters a = b = 100.1, c = 196.8 angstrom, and diffracted to 2.7 angstrom resolution.
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- Wikström Hultdin, Ulrika, et al.
(författare)
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Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli
- 2010
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Ingår i: Acta Crystallographica. Section F. - 1744-3091 .- 1744-3091. ; 66:Pt 3, s. 337-341
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Tidskriftsartikel (refereegranskat)abstract
- The transcription factor FocB belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic Escherichia coli. Recent findings suggest that FocB-family proteins may form different protein-protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. However, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. The 109-amino-acid FocB transcription factor from the foc gene cluster in E. coli strain J96 has been cloned, expressed and purified. The His6-tagged fusion protein was captured by Ni2+-affinity chromatography, cleaved with tobacco etch virus protease and purified by gel filtration. The purified protein is oligomeric, most likely in the form of dimers. NMR analysis guided the crystallization attempts by showing that probable conformational exchange or oligomerization is reduced at temperatures above 293 K and that removal of the highly flexible His6 tag is advantageous. The protein was crystallized using the hanging-drop vapour-diffusion method at 295 K. A native data set to 2.0 Å resolution was collected at 100 K using synchrotron radiation.
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