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1.  Purification, crystallization and structure determination of native GroEL from Escherichia coli lacking bound potassium ions 
Acta Crystallographica Section F  2007;63(Pt 6):457-461.
A 3.02 Å crystal structure of native GroEL from E. coli is presented.
GroEL is a member of the ATP-dependent chaperonin family that promotes the proper folding of many cytosolic bacterial proteins. The structures of GroEL in a variety of different states have been determined using X-ray crystallography and cryo-electron microscopy. In this study, a 3.02 Å crystal structure of the native GroEL complex from Escherichia coli is presented. The complex was purified and crystallized in the absence of potassium ions, which allowed evaluation of the structural changes that may occur in response to cognate potassium-ion binding by comparison to the previously determined wild-type GroEL structure (PDB code 1xck), in which potassium ions were observed in all 14 subunits. In general, the structure is similar to the previously determined wild-type GroEL crystal structure with some differences in regard to temperature-factor distribution.
doi:10.1107/S1744309107020295
PMCID: PMC2335072  PMID: 17554162
GroEL; chaperone proteins; ATP-binding proteins
2.  The structure of melon necrotic spot virus determined at 2.8 Å resolution 
Acta Crystallographica Section F  2007;64(Pt 1):8-13.
The structure of melon necrotic spot virus is reported.
The structure of melon necrotic spot virus (MNSV) was determined at 2.8 Å resolution. Although MNSV is classified into the genus Carmovirus of the family Tombusviridae, the three-dimensional structure of MNSV showed a higher degree of similarity to tomato bushy stunt virus (TBSV), which belongs to the genus Tombusvirus, than to carnation mottle virus (CMtV), turnip crinkle virus (TCV) or cowpea mottle virus (CPMtV) from the genus Carmovirus. Thus, the classification of the family Tombusviridae at the genus level conflicts with the patterns of similarity among coat-protein structures. MNSV is one of the viruses belonging to the genera Tombusvirus or Carmovirus that are naturally transmitted in the soil by zoospores of fungal vectors. The X-ray structure of MNSV provides us with a representative structure of viruses transmitted by fungi.
doi:10.1107/S1744309107066481
PMCID: PMC2374003  PMID: 18097092
Tombusviridae; tombusviruses; carmoviruses; coat proteins
3.  Structure of the minimized α/β-hydrolase fold protein from Thermus thermophilus HB8 
Acta Crystallographica Section F  2007;63(Pt 12):993-997.
The crystal structure of the minimized α/β-hydrolase fold protein encoded by the gene TTHA1544 from T. thermophilus HB8 has been determined at 2.0 Å resolution.
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 Å 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 α-helices and six β-strands, with the β-strands composing a central β-sheet. A structural homology search revealed that the overall structure of TTHA1544 resembles the α/β-hydrolase fold, although TTHA1544 lacks the catalytic residues of a hydrolase. These results suggest that TTHA1544 represents the minimized α/β-hydrolase fold and that an additional component would be required for its activity.
doi:10.1107/S1744309107061106
PMCID: PMC2344104  PMID: 18084077
T. thermophilus HB8; TTHA1544; α/β-hydrolase fold; singleton
4.  High-resolution structures of bacterially expressed soluble human CD59 
Acta Crystallographica Section F  2007;63(Pt 8):648-652.
The X-ray crystallographic structure of the complement regulator CD59 at 1.15 Å resolution hints at modes of interaction with C8/C9.
CD59 is a membrane-bound glycoprotein that protects host cells from lysis by inhibiting the terminal pathway of complement, preventing the formation and insertion of the membrane attack complex (MAC). Crystals of bacterially expressed and nonglycosylated recombinant soluble human CD59 have been obtained from three crystallization conditions, each of which gave rise to a distinct crystal form. Each crystal form led to a crystal structure at high resolution (1.15, 1.35 and 1.8 Å). In one of these structures the electron-density map shows an as yet unidentified small molecule in the predicted C8/C9-binding site. The presence/absence of this ligand is linked to alternate conformations of the amino acids implicated in C8/C9 binding.
doi:10.1107/S1744309107033477
PMCID: PMC2335151  PMID: 17671359
CD59; complement regulator; MAC
5.  Oligomerization of BenM, a LysR-type transcriptional regulator: structural basis for the aggregation of proteins in this family 
Acta Crystallographica Section F  2007;63(Pt 5):361-368.
A general tetramerization and oligomerization scheme is proposed for BenM and other LysR-type transcriptional regulators based on two newly characterized structures of the effector-binding domain of BenM. An analysis of these structures in the light of other LysR-type structures may explain the general solubility problems associated with this protein family.
LysR-type transcriptional regulators comprise the largest family of homologous regulatory DNA-binding proteins in bacteria. A problematic challenge in the crystallization of LysR-type regulators stems from the insolubility and precipitation difficulties encountered with high concentrations of the full-length versions of these proteins. A general oligomerization scheme is proposed for this protein family based on the structures of the effector-binding domain of BenM in two different space groups, P4322 and C2221. These structures used the same oligomerization scheme of dimer–dimer interactions as another LysR-type regulator, CbnR, the full-length structure of which is available [Muraoka et al. (2003 ▶), J. Mol. Biol. 328, 555–566]. Evaluation of packing relationships and surface features suggests that BenM can form infinite oligomeric arrays in crystals through these dimer–dimer interactions. By extrapolation to the liquid phase, such dimer–dimer interactions may contribute to the significant difficulty in crystallizing full-length members of this family. The oligomerization of dimeric units to form biologically important tetramers appears to leave unsatisfied oligomerization sites. Under conditions that favor association, such as neutral pH and concentrations appropriate for crystallization, higher order oligomerization could cause solubility problems with purified proteins. A detailed model by which BenM and other LysR-type transcriptional regulators may form these arrays is proposed.
doi:10.1107/S1744309107019185
PMCID: PMC2334995  PMID: 17565172
oligomerization; protein–protein interactions; LysR-type transcriptional regulators; BenM
6.  The use of Co2+ for crystallization and structure determination, using a conventional mono­chromatic X-ray source, of flax rust avirulence protein  
Acta Crystallographica Section F  2007;63(Pt 3):209-213.
It is demonstrated that anomalous diffraction based on the signal from a cobalt ion measured on a conventional monochromatic X-ray source can be used to determine the structure of a protein with a novel fold (M. lini avirulence protein AvrL567-A). The approach could be applicable to many metal-binding proteins, particularly when synchrotron radiation is not readily available.
Metal-binding sites are ubiquitous in proteins and can be readily utilized for phasing. It is shown that a protein crystal structure can be solved using single-wavelength anomalous diffraction based on the anomalous signal of a cobalt ion measured on a conventional monochromatic X-ray source. The unique absorption edge of cobalt (1.61 Å) is compatible with the Cu Kα wavelength (1.54 Å) commonly available in macromolecular crystallography laboratories. This approach was applied to the determination of the structure of Melampsora lini avirulence protein AvrL567-A, a protein with a novel fold from the fungal pathogen flax rust that induces plant disease resistance in flax plants. This approach using cobalt ions may be applicable to all cobalt-binding proteins and may be advantageous when synchrotron radiation is not readily available.
doi:10.1107/S1744309107004599
PMCID: PMC2330185  PMID: 17329816
AvrL567-A; cobalt; plant disease resistance; single-wavelength anomalous diffraction
7.  Structure of the hypothetical protein PF0899 from Pyrococcus furiosus at 1.85 Å resolution 
Acta Crystallographica Section F  2007;63(Pt 7):549-552.
The crystal structure of the hypothetical protein PF0899 from P. furiosus has been determined to 1.85 Å resolution.
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 Å 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.
doi:10.1107/S1744309107024049
PMCID: PMC2335137  PMID: 17620707
structural genomics; SECSG; Pfu-871755; PF0899; high-throughput structure
8.  The use of in situ proteolysis in the crystallization of murine CstF-77 
Acta Crystallographica Section F  2007;63(Pt 2):135-138.
In situ proteolysis with fungal protease or subtilisin is crucial for the crystallization of murine CstF-77.
The cleavage-stimulation factor (CstF) is required for the cleavage of the 3′-end of messenger RNA precursors in eukaryotes. During structure determination of the 77 kDa subunit of the murine CstF complex (CstF-77), it was serendipitously discovered that a solution infected by a fungus was crucial for the crystallization of this protein. CstF-77 was partially proteolyzed during crystallization; this was very likely to have been catalyzed by a protease secreted by the fungus. It was found that the fungal protease can be replaced by subtilisin and this in situ proteolysis protocol produced crystals of sufficient size for structural studies. After an extensive search, it was found that 55% glucose can be used as a cryoprotectant while maintaining the diffraction quality of the crystals; most other commonly used cryoprotectants were detrimental to the diffraction quality.
doi:10.1107/S1744309107002904
PMCID: PMC2330134  PMID: 17277459
CstF-77; in situ proteolysis
9.  Preliminary neutron and ultrahigh-resolution X-ray diffraction studies of the aspartic proteinase endothiapepsin cocrystallized with a gem-diol inhibitor 
Three data sets have been collected on endothiapepsin complexed with the gem-diol inhibitor PD-135,040: a high-resolution synchrotron X-ray data set, a room-temperature X-ray data set and a neutron diffraction data set. Until recently, it has been impossible to grow large protein crystals of endothiapepsin with any gem-diol inhibitor that are suitable for neutron diffraction.
Endothiapepsin has been cocrystallized with the gem-diol inhibitor PD-135,040 in a low solvent-content (39%) unit cell, which is unprecedented for this enzyme–inhibitor complex and enables ultrahigh-resolution (1.0 Å) X-ray diffraction data to be collected. This atomic resolution X-ray data set will be used to deduce the protonation states of the catalytic aspartate residues. A room-temperature neutron data set has also been collected for joint refinement with a room-temperature X-ray data set in order to locate the H/D atoms at the active site.
doi:10.1107/S1744309107061283
PMCID: PMC2344097  PMID: 18084100
endothiapepsin; gem-diol inhibitors; neutron diffraction
10.  Preliminary neutron and ultrahigh-resolution X-ray diffraction studies of the aspartic proteinase endothiapepsin cocrystallized with a gem-diol inhibitor 
Endothiapepsin has been cocrystallized with the gem-diol inhibitor PD-135,040 in a low solvent-content (39%) unit cell, which is unprecedented for this enzyme—inhibitor complex and enables ultrahigh-resolution (1.0 Å) X-ray diffraction data to be collected. This atomic resolution X-ray data set will be used to deduce the protonation states of the catalytic aspartate residues. A room-temperature neutron data set has also been collected for joint refinement with a room-temperature X-ray data set in order to locate the H/D atoms at the active site.
doi:10.1107/S1744309107061283
PMCID: PMC2344097  PMID: 18084100
11.  Purification, crystallization and structure determination of native GroEL from Escherichia coli lacking bound potassium ions 
GroEL is a member of the ATP-dependent chaperonin family that promotes the proper folding of many cytosolic bacterial proteins. The structures of GroEL in a variety of different states have been determined using X-ray crystallography and cryo-electron microscopy. In this study, a 3.02 Å crystal structure of the native GroEL complex from Escherichia coli is presented. The complex was purified and crystallized in the absence of potassium ions, which allowed evaluation of the structural changes that may occur in response to cognate potassium-ion binding by comparison to the previously determined wild-type GroEL structure (PDB code 1xck), in which potassium ions were observed in all 14 subunits. In general, the structure is similar to the previously determined wild-type GroEL crystal structure with some differences in regard to temperature-factor distribution.
doi:10.1107/S1744309107020295
PMCID: PMC2335072  PMID: 17554162
GroEL; 2nwc; r2nwcsf
12.  Overproduction, purification, crystallization and preliminary X-ray analysis of the peroxiredoxin domain of a larger natural hybrid protein from Thermotoga maritima  
Crystals of the peroxiredoxin domain of a larger natural hybrid protein from T. maritima were obtained which diffracted to 2.9 Å resolution on a synchrotron source.
Thermotoga maritima contains a natural hybrid protein constituted of two moieties: a peroxiredoxin domain at the N-terminus and a nitroreductase domain at the C-terminus. The peroxiredoxin (Prx) domain has been overproduced and purified from Escherichia coli cells. The recombinant Prx domain, which is homologous to bacterial Prx BCP and plant Prx Q, folds properly into a stable protein that possesses biological activity. The recombinant protein was crystallized and synchrotron data were collected to 2.9 Å resolution. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 176.67, c = 141.20 Å.
doi:10.1107/S1744309107064391
PMCID: PMC2374002  PMID: 18097097
natural hybrid proteins; Thermotoga maritima; peroxiredoxin domain
13.  Crystallization and preliminary crystallographic studies of an active-site mutant hydantoin racemase from Sinorhizobium meliloti CECT4114 
Crystals of an active-site mutated hydantoin racemase from S. meliloti have been obtained in the presence and absence of d,l-5-isopropyl-hydantoin and characterized by X-ray diffraction.
A recombinant active-site mutant of hydantoin racemase (C76A) from Sinorhizobium meliloti CECT 4114 (SmeHyuA) has been crystallized in the presence and absence of the substrate d,l-5-isopropyl hydantoin. Crystals of the SmeHyuA mutant suitable for data collection and structure determination were grown using the counter-diffusion method. X-ray data were collected to resolutions of 2.17 and 1.85 Å for the free and bound enzymes, respectively. Both crystals belong to space group R3 and contain two molecules of SmeHyuA per asymmetric unit. The crystals of the free and complexed SmeHyuA have unit-cell parameters a = b = 85.43, c = 152.37 Å and a = b = 85.69, c = 154.38 Å, crystal volumes per protein weight (V M) of 1.94 and 1.98 Å3 Da−1 and solvent contents of 36.7 and 37.9%, respectively.
doi:10.1107/S1744309107066122
PMCID: PMC2374001  PMID: 18097103
hydantoin racemase; Sinorhizobium meliloti; d,l-5-isopropyl hydantoin
14.  Overproduction, crystallization and preliminary X-­ray analysis of the putative l-ascorbate-6-phosphate lactonase UlaG from Escherichia coli  
UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in E. coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized in a monoclinic space group. Crystals were obtained by the sitting-drop vapour-diffusion method at 293 K. A data set diffracting to 3 Å resolution was collected from a single crystal at 100 K.
UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in Escherichia coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized. Crystals were obtained by sitting-drop vapour diffusion at 293 K. Preliminary X-ray diffraction analysis revealed that the UlaG crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 104.52, b = 180.69, c = 112.88 Å, β = 103.26°. The asymmetric unit is expected to contain six copies of UlaG, with a corresponding volume per protein weight of 2.16 Å3 Da−1 and a solvent content of 43%.
doi:10.1107/S1744309107065256
PMCID: PMC2373999  PMID: 18097099
UlaG; l-ascorbate metabolism; enterobacterial metabolism
15.  Expression, crystallization and preliminary X-ray diffraction analysis of human paired Ig-like type 2 receptor α (PILRα) 
Human paired Ig-like type 2 receptor α (PILRα) has been expressed, purified and crystallized. A diffraction data set has been collected to 1.3 Å resolution.
Human paired immunoglobulin-like (Ig-like) type 2 receptor α (PILRα) is a type I membrane protein that is mainly expressed in immune-related cells such as monocytes, granulocytes and dendritic cells. PILRα can suppress the functions of such immune cells because it has the immunoreceptor tyrosine-based inhibitory motif (ITIM) in the intracellular region, which recruits the phosphatase Src homology-2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP-­2) to inhibit phophorylations induced by activation signals. The extracellular region of human PILRα comprises one immuno­globulin superfamily V-set domain and a stalk region. The V-set domain (residues 13–131) of human PILRα was overexpressed in Escherichia coli as inclusion bodies, refolded by rapid dilution and purified. The PILRα protein was successfully crystallized at 293 K using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.3 Å resolution at SPring-8 BL41XU; they belong to space group P212121, with unit-cell parameters a = 40.4, b = 45.0, c = 56.9 Å, and contain one molecule per asymmetric unit.
doi:10.1107/S1744309107065384
PMCID: PMC2373998  PMID: 18097101
paired Ig-like type 2 receptor α; membrane proteins
16.  Characterization and crystallization of a recombinant IgE Fab fragment in complex with the bovine β-lactoglobulin allergen 
The high-resolution mass-spectrometric characterization, crystallization and X-ray diffraction studies of a recombinant IgE Fab fragment in complex with bovine β-lactoglobulin are reported.
A D1 Fab fragment containing the allergen-binding variable domains of the IgE antibody was characterized by ESI FT–ICR mass spectrometry and crystallized with bovine β-lactoglobulin (BLG) using the hanging-drop vapour-diffusion method at 293 K. X-ray data suitable for structure determination were collected to 2.8 Å resolution using synchrotron radiation. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 67.0, b = 100.6, c = 168.1 Å. The three-dimensional structure of the D1 Fab fragment–BLG complex will provide the first insight into IgE antibody–allergen interactions at the molecular level.
doi:10.1107/S174430910706160X
PMCID: PMC2373997  PMID: 18097096
antibodies; IgE; food allergens; mass spectrometry
17.  Purification, identification and preliminary crystallographic studies of Pru du amandin, an allergenic protein from Prunus dulcis  
The purification, identification, crystallization and preliminary crystallographic studies of an allergy-related protein, Pru du amandin, from P. dulcis nuts are reported.
Food allergies appear to be one of the foremost causes of hypersensitivity reactions. Nut allergies account for most food allergies and are often permanent. The 360 kDa hexameric protein Pru du amandin, a known allergen, was purified from almonds (Prunus dulcis) by ammonium sulfate fractionation and ion-exchange chromatography. The protein was identified by a BLAST homology search against the nonredundant sequence database. Pru du amandin belongs to the 11S legumin family of seed storage proteins characterized by the presence of a cupin motif. Crystals were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P41 (or P43), with unit-cell parameters a = b = 150.7, c = 164.9 Å.
doi:10.1107/S1744309107064615
PMCID: PMC2373995  PMID: 18097098
Pru du amandin; 11S legumins; cupin motif
18.  Crystallization and preliminary crystallographic analysis of the catalytic domain of human flap endonuclease 1 in complex with a nicked DNA product: use of a DPCS kit for efficient protein–DNA complex crystallization 
Human flap endonuclease 1 complexed with nicked DNA has been crystallized. A diffraction data set was collected to a resolution of 2.75 Å.
Flap endonuclease 1 (FEN1) is a structure-specific nuclease that removes the RNA/DNA primer associated with Okazaki fragments in DNA replication. Here, crystals of the complex between the catalytic domain of human FEN1 and a DNA product have been obtained. For efficient crystallization screening, a DNA–protein complex crystallization screening (DPCS) kit was designed based on commercial crystallization kits. The crystal was found to belong to space group P21, with unit-cell parameters a = 61.0, b = 101.3, c = 106.4 Å, β = 106.4°. The asymmetric unit is predicted to contain two complexes in the crystallo­graphic asymmetric unit. A diffraction data set was collected to a resolution of 2.75 Å.
doi:10.1107/S1744309107065372
PMCID: PMC2373994  PMID: 18097100
DNA–protein complexes; DNA repair; DNA replication; nucleases
19.  Crystallization and preliminary X-ray analysis of Acetivibrio cellulolyticus cellulosomal type II cohesin module: two versions having different linker lengths 
The cloning, expression, purification, crystallization and preliminary X-ray characterization of two protein constructs of the second type II cohesin module from A. cellulolyticus ScaB are described. Both constructs contain the native N-terminal linker, but only one of them contains the full-length 45-residue C-terminal linker; the other contains a five-residue segment of this linker.
The second type II cohesin module of the cellulosomal scaffoldin polypeptide ScaB from Acetivibrio cellulolyticus (CohB2) was cloned into two constructs: one containing a short (five-residue) C-terminal linker (CohB2_S) and the second incorporating the full native 45-residue linker (CohB2_L). Both constructs encode proteins that also include the full native six-residue N-­terminal linker. The CohB2_S and CohB2_L proteins were expressed, purified and crystallized in the orthorhombic crystal system, but with different unit cells and symmetries: space group P212121 with unit-cell parameters a = 90.36, b = 68.65, c = 111.29 Å for CohB2_S and space group P21212 with unit-cell parameters a = 68.76, b = 159.22, c = 44.21 Å for CohB2_L. The crystals diffracted to 2.0 and 2.9 Å resolution, respectively. The asymmetric unit of CohB2_S contains three cohesin molecules, while that of CohB2_L contains two molecules.
doi:10.1107/S1744309107066821
PMCID: PMC2373993  PMID: 18097105
cohesin type II; linkers; cellulosome
20.  Overexpression, purification and crystallization of the tetrameric form of SorC sorbitol operon regulator 
The sorbitol operon regulator from K. pneumoniae has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 3.2 Å.
The sorbitol operon regulator (SorC) regulates the metabolism of l-sorbose in Klebsiella pneumonia. SorC was overexpressed in Escherichia coli and purified, and crystals were obtained of a tetrameric form. A single crystal showed X-ray diffraction to 3.20 Å. The crystal belongs to space group P212121, with unit-cell parameters a = 91.6, b = 113.3, c = 184.1 Å. Analysis of the molecular-replacement solution indicates the presence of four SorC molecules in the asymmetric unit.
doi:10.1107/S1744309107060897
PMCID: PMC2373991  PMID: 18097095
sorbitol operon regulator
21.  Semi-automated microseeding of nanolitre crystallization experiments 
A procedure for microseeding into nanolitre crystallization drops is described with selected successful examples.
A simple semi-automated microseeding procedure for nanolitre crystallization experiments is described. Firstly, a microseed stock solution is made from microcrystals using a Teflon bead. A dilution series of this microseed stock is then prepared and dispensed as 100 nl droplets into 96-well crystallization plates, facilitating the incorporation of seeding into high-throughput crystallization pipelines. This basic microseeding procedure has been modified to include additive-screening and cross-seeding methods. Five examples in which these techniques have been used successfully are described.
doi:10.1107/S1744309107057260
PMCID: PMC2373990  PMID: 18097093
crystallization; crystal optimization; microseeding; additives
22.  Cleaved thioredoxin fusion protein enables the crystallization of poorly soluble ERα in complex with synthetic ligands 
A new crystallization strategy: the presence of cleaved thioredoxin fusion is critical for crystallization of the estrogen nuclear receptor ligand binding domain in complex with synthetic ligands. This novel technique should be regarded as an interesting alternative for crystallization of difficult proteins.
The ligand-binding domain (LBD) of human oestrogen receptor α was produced in Escherichia coli as a cleavable thioredoxin (Trx) fusion in order to improve solubility. Crystallization trials with either cleaved and purified LBD or with the purified fusion protein both failed to produce crystals. In another attempt, Trx was not removed from the LBD after endoproteolytic cleavage and its presence promoted nucleation and subsequent crystal growth, which allowed the structure determination of two different LBD–ligand–coactivator peptide complexes at 2.3 Å resolution. This technique is likely to be applicable to other low-solubility proteins.
doi:10.1107/S1744309107066444
PMCID: PMC2373989  PMID: 18097104
oestrogen receptor; thioredoxin fusion
23.  Crystallization and preliminary X-ray studies of SdiA from Escherichia coli  
E. coli SdiA was overexpressed, purified and crystallized. The crystals belonged to the hexagonal space group P6122 or P6522 and diffracted to 2.7 Å resolution.
SdiA enhances cell division by regulating the ftsQAZ operon in Escherichia coli as a transcription activator. In addition, SdiA is suggested to play a role in detecting quorum signals that emanate from other species. It is therefore a homologue of LuxR, a cognate quorum-sensing receptor that recognizes a quorum signal and activates the quorum responses. To elucidate the role of SdiA and its functional and structural relationship to LuxR, structural studies were performed on E. coli SdiA. Recombinant SdiA was overexpressed, purified and crystallized at 287 K using the hanging-drop vapour-diffusion method. X-ray diffraction data from a native crystal were collected with 99.7% completeness to 2.7 Å resolution with an R merge of 6.0%. The crystals belong to the hexagonal space group P6122 or P6522, with unit-cell parameters a = b = 130.47, c = 125.23 Å.
doi:10.1107/S1744309107059696
PMCID: PMC2373988  PMID: 18097094
SdiA; quorum sensing
24.  Preliminary X-ray crystallographic analysis of SMU.573, a putative sugar kinase from Streptococcus mutans  
SMU.573 from S. mutans was expressed in E. coli and crystallized. The crystals belong to space group I4 and 2.5 Å resolution diffraction data were collected at an in-house chromium radiation source.
SMU.573 from Streptococcus mutans is a structurally and functionally uncharacterized protein that was selected for structural biology studies. Native and SeMet-labelled proteins were expressed with an N-His tag in Escherichia coli BL21 (DE3) and purified by Ni2+-chelating and size-exclusion chromatography. Crystals of the SeMet-labelled protein were obtained by the hanging-drop vapour-diffusion method and a 2.5 Å resolution diffraction data set was collected using an in-house chromium radiation source. The crystals belong to space group I4, with unit-cell parameters a = b = 96.53, c = 56.26 Å, α = β = γ = 90°.
doi:10.1107/S1744309107065645
PMCID: PMC2373987  PMID: 18097102
SMU.573; Streptococcus mutans
25.  The structure of allophycocyanin from Thermosynechococcus elongatus at 3.5 Å resolution 
The crystal structure of a light-harvesting protein that interacts with photosystem II is reported.
Cyanobacteria and red algae use light-harvesting pigments bound by proteins to capture solar radiation and to channel excitation energy into their reaction centres. In most cyanobacteria, a multi-megadalton soluble structure known as the phycobilisome is a major light-harvesting system. Allophycocyanin is the main component of the phycobilisome core, forming a link between the rest of the phycobilisome and the reaction-centre core. The crystal structure of allophycocyanin from Thermosynechococcus elongatus (TeAPC) has been determined and refined at 3.5 Å resolution to a crystallographic R value of 26.0% (R free = 28.5%). The structure was solved by molecular replacement using the allophycocyanin structure from Spirulina platensis as the search model. The asymmetric unit contains an (αβ) monomer which is expanded by symmetry to a crystallographic trimer.
doi:10.1107/S1744309107050920
PMCID: PMC2344114  PMID: 18084078
photosynthesis; light-harvesting proteins

Results 1-25 (285)