Screening trials to determine the presence of two-dimensional (2D) protein crystals suitable for three-dimensional structure determination using electron crystallography is a very labor-intensive process. Methods compatible with fully automated screening have been developed for the process of crystal production by dialysis and for producing negatively stained grids of the resulting trials. Further automation via robotic handling of the EM grids, and semi-automated transmission electron microscopic imaging and evaluation of the trial grids is also possible. We, and others, have developed working prototypes for several of these tools and tested and evaluated them in a simple screen of 24 crystallization conditions. While further development of these tools is certainly required for a turn-key system, the goal of fully automated screening appears to be within reach.
TEM; 2D crystallization; automation; membrane proteins; electron crystallography
Tyrosyl-tRNA synthetase from the hyperthermophilic archaeon A. pernix K1 was cloned, purified and crystallized. The crystals belonged to the tetragonal space group P43212, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K.
Hyperthermophilic archaeal tyrosyl-tRNA synthetase from Aeropyrum pernix K1 was cloned and overexpressed in Escherichia coli. The expressed protein was purified by Cibacron Blue affinity chromatography following heat treatment at 363 K. Crystals suitable for X-ray diffraction studies were obtained under optimized crystallization conditions in the presence of 1.5 M ammonium sulfate using the hanging-drop vapour-diffusion method. The crystals belonged to the tetragonal space group P43212, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K.
aminoacyl-tRNA synthetase; tyrosine tRNA; Aeropyrum pernix K1
Crystals of human mitochondrial tyrosyl-tRNA synthetase lacking the C-terminal S4-like domain diffract to 2.7 Å resolution and are suitable for structure determination.
Human mitochondrial tyrosyl-tRNA synthetase and a truncated version with its C-terminal S4-like domain deleted were purified and crystallized. Only the truncated version, which is active in tyrosine activation and Escherichia coli tRNATyr charging, yielded crystals suitable for structure determination. These tetragonal crystals, belonging to space group P43212, were obtained in the presence of PEG 4000 as a crystallizing agent and diffracted X-rays to 2.7 Å resolution. Complete data sets could be collected and led to structure solution by molecular replacement.
tyrosyl-tRNA synthetase; aminoacyl-tRNA synthetases
Crystallization of isoquinoline 1-oxidoreductase from B. diminuta was achieved using two different crystallization buffers. Streak-seeding and cross-linking were essential to obtain well diffracting crystals. Suitable cryo-conditions were found and a structure solution was obtained by molecular replacement.
Isoquinoline 1-oxidoreductase (IOR) from Brevundimonas diminuta is a mononuclear molybdoenzyme of the xanthine-dehydrogenase family of proteins and catalyzes the conversion of isoquinoline to isoquinoline-1-one. Its primary sequence and behaviour, specifically in its substrate specificity and lipophilicity, differ from other members of the family. A crystal structure of the enzyme is expected to provide an explanation for these differences. This paper describes the crystallization and preliminary X-ray diffraction experiments as well as an optimized purification protocol for IOR. Crystallization of IOR was achieved using two different crystallization buffers. Streak-seeding and cross-linking were essential to obtain well diffracting crystals. Suitable cryo-conditions were found and a structure solution was obtained by molecular replacement. However, phases need to be improved in order to obtain a more interpretable electron-density map.
isoquinoline 1-oxidoreductase; xanthine oxidase/xanthine dehydrogenase; oxidoreductases; molybdenum enzymes; molybdopterin; Brevundimonas diminuta
The expression, purification and crystallization of the small laccase from S. coelicolor are reported. Diffraction data were collected to 3 Å resolution.
The small bacterial laccase from the actinobacterium Streptomyces coelicolor which lacks the second of the three domains of the laccases structurally characterized to date was crystallized. This multi-copper phenol oxidase crystallizes in a primitive tetragonal lattice, with unit-cell parameters a = b = 179.8, c = 175.3 Å. The crystals belong to either space group P41212 or P43212. The self-rotation function shows the presence of a noncrystallographic threefold axis in the structure. Phases will be determined from the anomalous signal of the natively present copper ions.
laccases; oxidoreductases; multicopper blue proteins
A water-soluble mutant of the outer membrane lipoprotein NlpE has been overexpressed, purified and crystallized. Diffraction data from two crystal forms obtained under two different conditions were collected to 2.8 and 3.0 Å resolution and processed in space groups P43212 and C2, respectively.
The outer membrane lipoprotein NlpE functions in stress response by activating the Cpx signal transduction pathway. The nonlipidated Cys1Ala mutant of NlpE with a C-terminal His tag from Escherichia coli was constructed, overexpressed and purified. Crystals of NlpE were grown in two distinct forms by the sitting-drop vapour-diffusion method at 298 K. The tetragonal crystals diffracted to 2.8 Å resolution and belong to space group P43212. The monoclinic crystals diffracted to 3.0 Å resolution and belong to space group C2. Initial phases were obtained from a tetragonal crystal of selenomethionylated protein by the MAD method.
Cpx pathway; NlpE; outer membrane lipoproteins; periplasm; stress response
Wzi is a membrane protein from E. coli thought to be involved in the attachment of capsular polysaccharides to the bacterial surface. This reports describes recombinant Wzi’s purification, crystallization and the results of initial diffraction studies.
External polysaccharide capsules provide a physical barrier that is employed by many species of bacteria for the purposes of host evasion and persistence. Wzi is a 53 kDa outer membrane β-barrel protein that is thought to play a role in the attachment of group 1 capsular polysaccharides to the cell surface. The purification and crystallization of an Escherichia coli homologue of Wzi is reported and diffraction data from native and selenomethionine-incorporated protein crystals are presented. Crystals of C-terminally His6-tagged Wzi diffracted to 2.8 Å resolution. Data processing showed that the crystals belonged to the orthorhombic space group C222, with unit-cell parameters a = 128.8, b = 152.8, c = 94.4 Å, α = β = γ = 90°. A His-tagged selenomethionine-containing variant of Wzi has also been crystallized in the same space group and diffraction data have been recorded to 3.8 Å resolution. Data processing shows that the variant crystal has similar unit-cell parameters to the native crystal.
Wzi; polysaccharide capsules; Escherichia coli
Strongly diffracting crystals of a methanol-induced corrinoid protein from M. thermoacetica have been obtained.
A corrinoid protein was induced and overexpressed in methanol-grown cells of the thermophilic anaerobic bacterium Moorella thermoacetica. The protein was purified from cytosolic extracts. After screening for crystallization conditions and optimization, crystals were obtained that diffracted strongly on a rotating-anode X-ray source. A diffraction data set was collected and processed including reflections to 1.9 Å resolution. Reflections were indexed in a primitive orthorhombic cell with unit-cell parameters a = 55.69, b = 62.74, c = 34.54 Å. N-terminal amino-acid sequencing indicates that the crystals contain a C-terminal fragment of the protein.
Two sample-scanning features have been implemented for the macromolecular crystallography beamlines at APS sector 23: automated diffraction-based rastering employing multiple polygon-shaped two-dimensional grids overlaid on a sample to locate and center small and invisible crystals or to find the best-diffracting regions in a larger crystal, and automated data collection along a three-dimensional vector to mitigate the effects of radiation damage.
Automated scanning capabilities have been added to the data acquisition software, JBluIce-EPICS, at the National Institute of General Medical Sciences and the National Cancer Institute Collaborative Access Team (GM/CA CAT) at the Advanced Photon Source. A ‘raster’ feature enables sample centering via diffraction scanning over two-dimensional grids of simple rectangular or complex polygonal shape. The feature is used to locate crystals that are optically invisible owing to their small size or are visually obfuscated owing to properties of the sample mount. The raster feature is also used to identify the best-diffracting regions of large inhomogeneous crystals. Low-dose diffraction images taken at grid positions are automatically processed in real time to provide a quick quality ranking of potential data-collection sites. A ‘vector collect’ feature mitigates the effects of radiation damage by scanning the sample along a user-defined three-dimensional vector during data collection to maximize the use of the crystal volume and the quality of the collected data. These features are integrated into the JBluIce-EPICS data acquisition software developed at GM/CA CAT where they are used in combination with a robust mini-beam of rapidly changeable diameter from 5 µm to 20 µm. The powerful software–hardware combination is being applied to challenging problems in structural biology.
macromolecular crystallography; beamline automation; data acquisition; high-throughput crystallography; crystal centering; radiation damage; rastering
Native and selenomethionine-labeled crystals of Ebola VP35 interferon inhibitory domain were obtained by the hanging-drop vapor-diffusion method.
Ebola VP35 is a multifunctional protein that is important for host immune suppression and pathogenesis. VP35 contains an N-terminal oligomerization domain and a C-terminal interferon inhibitory domain (IID). Mutations within the VP35 IID result in loss of host immune suppression. Here, efforts to crystallize recombinantly overexpressed VP35 IID that was purified from Escherichia coli are described. Native and selenomethionine-labeled crystals belonging to the orthorhombic space group P212121 were obtained by the hanging-drop vapor-diffusion method and diffraction data were collected at the ALS synchrotron.
Ebola VP35; interferon inhibitory domain; antiviral antagonists
A Kunitz-type trypsin inhibitor purified from the seeds of Murraya koenigii has been crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitating agent.
A Kunitz-type trypsin inhibitor purified from the seeds of Murraya koenigii has been crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitating agent. The crystals belong to the tetragonal space group P43212, with unit-cell parameters a = b = 75.8, c = 150.9 Å. The crystals contain two molecules in the asymmetric unit with a V
M value of 2.5 Å3 Da−1. Diffraction was observed to 2.65 Å resolution and a complete data set was collected to 2.9 Å resolution.
Kunitz-type trypsin inhibitor; Murraya koenigii
The two C-terminal domains of the cellulase ctCel9D-Cel44A from C. thermocellum cellulosome have been crystallized in tetragonal space group P43212 and X-ray diffraction data have been collected to 2.1 and 2.8 Å from native and seleno-l-methionine-derivative crystals, respectively.
Clostridium thermocellum produces a highly organized multi-enzyme complex of cellulases and hemicellulases for the hydrolysis of plant cell-wall polysaccharides, which is termed the cellulosome. The bifunctional multi-modular cellulase ctCel9D-Cel44A is one of the largest components of the C. thermocellum cellulosome. The enzyme contains two internal catalytic domains belonging to glycoside hydrolase families 9 and 44. The C-terminus of this cellulase, comprising a polycystic kidney-disease module (PKD) and a carbohydrate-binding module (CBM44), has been crystallized. The crystals belong to the tetragonal space group P43212, containing a single molecule in the asymmetric unit. Native and seleno-l-methionine-derivative crystals diffracted to 2.1 and 2.8 Å, respectively.
cellulase; carbohydrate-binding module; glycoside hydrolase; Clostridium thermocellum
The expression, purification and crystallization of the collagen-binding region of the E. rhusiopathiae surface protein RspB is described. The crystals diffracted to 2.2 Å resolution using synchrotron radiation.
RspB is a surface adhesin of Erysipelothrix rhusiopathiae. A recombinant form of the collagen-binding region of this protein, RspB(31–348), has been overexpressed in Escherichia coli in native and selenomethionine-derivative forms and purified using affinity and gel-permeation chromatography. Thin plate-like crystals were obtained by the hanging-drop vapour-diffusion method using the same condition for both forms. The native crystals diffracted to a resolution of 2.5 Å using an in-house X-ray source, while the selenomethionine-derivative crystals diffracted to a resolution of 2.2 Å using synchrotron radiation. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 46.19, b = 66.65, c = 101.72 Å, β = 94.11°.
RspB; Erysipelothrix rhusiopathiae; collagen binding
Single crystals have been grown of Cd,Zn metallothionein isoform II from rat liver. The space group is P41212(P43212) with unit cell dimensions a = b = 31.0 A and c = 120.0 A, and one molecule in the crystallographic asymmetric unit. The crystals are square bipyramids elongated on the tetragonal c-axis and are grown by repetitive seeding. The crystals are suitable for high resolution structure analysis. Assays of dissolved crystals show that the crystals have the same Cd and Zn content and amino acid composition as the native, as-isolated protein.
The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described.
The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6122 or P6522, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.
matrix proteins; Rhabdoviridae; lyssaviruses; Lagos bat virus; SUMO tag
Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein crystallography applications. The Escherichia coli proteome was fractionated, purified, crystallized, and structurally characterized. Macro-scale fermentation and fractionation were used to subdivide the soluble proteome into 408 unique fractions of which 295 fractions yielded crystals in microfluidic crystallization chips. Of the 295 crystals, 152 were selected for optimization, diffraction screening, and data collection. Twenty-three structures were determined, four of which were novel. This study demonstrates the utility of native source proteins for high-throughput crystallography.
The Escherichia coli gene encoding the transcription cleavage factor GreB and the Thermus thermophilus gene encoding the anti-GreA transcription factor Gfh1 were cloned and expressed and the purified proteins were crystallized by the sitting-drop vapor-diffusion technique. The GreB and Gfh1 crystals, which were improved by macroseeding, belong to space group P41212 (or P43212), with unit-cell parameters a = b = 148, c = 115.2 Å and a = b = 59.3, c = 218.9 Ǻ, respectively. Complete diffraction data sets were collected for the GreB and Gfh1 crystals to 2.6 and 2.8 Å resolution, respectively. Crystals of the selenomethionine proteins were obtained by microseeding using the native protein crystals and diffract as well as the native ones. The structure determination of these proteins is now in progress.
Crystals of S. elodea ATCC 31461 UDP-glucose dehydrogenase (EC 18.104.22.168) were obtained in space groups P622 and P43212 and diffracted to 2.4 and 3.4 Å resolution, respectively.
Gellan gum, a commercial gelling agent produced by Sphingomonas elodea ATCC 31461, is a high-value microbial exopolysaccharide. UDP-glucose dehydrogenase (UGD; EC 22.214.171.124) is responsible for the NAD-dependent twofold oxidation of UDP-glucose to UDP-glucuronic acid, one of the key components for gellan biosynthesis. S. elodea ATCC 31461 UGD, termed UgdG, was cloned, expressed, purified and crystallized in native and SeMet-derivatized forms in hexagonal and tetragonal space groups, respectively; the crystals diffracted X-rays to 2.40 and 3.40 Å resolution, respectively. Experimental phases were obtained for the tetragonal SeMet-derivatized crystal form by a single-wavelength anomalous dispersion experiment. This structure was successfully used as a molecular-replacement probe for the hexagonal crystal form of the native protein.
Sphingomonas elodea; gellan gum; exopolysaccharides; UDP-glucose dehydrogenases
The C-terminal domain protein of the PB2 subunit of influenza A virus RNA-dependent RNA polymerase was expressed and crystallized and diffraction data were obtained from the crystals.
The C-terminal domain protein (amino-acid residues 535–759) of the PB2 subunit of the RNA-dependent RNA polymerase from the highly pathogenic influenza A virus was expressed as a soluble protein in Escherichia coli and crystallized using sodium formate as a precipitant. Data sets were collected from crystals of native and selenomethionine-substituted protein on the KEK NW12 beamline at the Photon Factory and the crystals diffracted to a maximum resolution of 2.44 Å for the SeMet-derivative crystal. The native crystals were found to belong to space group P3221, with unit-cell parameters a = b = 52.5, c = 156.3 Å. The Matthews value (V
M) was 2.7 Å3 Da−1, assuming the presence of one molecule in the asymmetric unit. The SeMet-derivative crystals were found to belong to the same space group, with unit-cell parameters a = b = 52.6, c = 156.4 Å. Attempts are being made to solve the structure by multi-wavelength anomalous dispersion phasing.
influenza A virus; RNA-dependent RNA polymerase; PB2
The crystallization and preliminary X-ray analysis of a β-d-xylosidase from G. stearothermophilus T-6, a family 43 glycoside hydrolase, is described. Native and catalytic inactive mutants of the enzymes were crystallized in two different space groups, orthorhombic P21212 and tetragonal P41212 (or the enantiomorphic space group P43212), using a sensitive cryoprotocol. The latter crystal form diffracted X-rays to a resolution of 2.2 Å.
β-d-Xylosidases (EC 126.96.36.199) are hemicellulases that cleave single xylose units from the nonreducing end of xylooligomers. In this study, the crystallization and preliminary X-ray analysis of a β-d-xylosidase from Geobacillus stearothermophilus T-6 (XynB3), a family 43 glycoside hydrolase, is described. XynB3 is a 535-amino-acid protein with a calculated molecular weight of 61 891 Da. Purified recombinant native and catalytic inactive mutant proteins were crystallized and cocrystallized with xylobiose in two different space groups, P21212 (unit-cell parameters a = 98.32, b = 99.36, c = 258.64 Å) and P41212 (or the enantiomorphic space group P43212; unit-cell parameters a = b = 140.15, c = 233.11 Å), depending on the detergent. Transferring crystals to cryoconditions required a very careful protocol. Orthorhombic crystals diffract to 2.5 Å and tetragonal crystals to 2.2 Å.
family 43 glycosidase hydrolases; xylosidases; hemicellulases; Geobacillus stearothermophilus; xylan; xylose
The cloning, overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of the transcriptional repressor SirR (staphylococcal iron regulator) from M. tuberculosis are reported.
SirR, a metal-dependent transcriptional repressor from Mycobacterium tuberculosis (Rv2788), was cloned in pQE30 expression vector with an N-terminal His6 tag for heterologous overexpression in Escherichia coli M15 (pREP4) cells and purified to homogeneity using chromatographic procedures. The purified protein was crystallized using the sitting-drop vapour-diffusion technique. The crystals belonged to the tetragonal space group P41212/P43212, with unit-cell parameters a = 105.21, b = 105.21, c = 144.85 Å. The X-ray diffraction data were processed to a maximum resolution of 2.5 Å. The Matthews coefficient suggests the presence of two (V
M = 4.01 Å3 Da−1) to four (V
M = 2.0 Å3 Da−1) molecules in the asymmetric unit. Calculation of the self-rotation function shows a crystallographic fourfold symmetry axis along the z axis (χ = 90°) and also a twofold symmetry axis around the z axis (χ = 180°).
SirR; Mycobacterium tuberculosis; transcriptional repressors; tuberculosis
ϕ29 bacteriophage scaffolding protein (gp7) has been overproduced in E. coli, purified, crystallized and characterized by X-ray diffraction. Two distinct crystal forms were obtained and a diffraction data set was collected to 1.8 Å resolution.
The Bacillus subtilis bacteriophage ϕ29 scaffolding protein (gp7) has been crystallized by the hanging-drop vapour-diffusion method at 293 K. Two new distinct crystal forms that both differed from a previously crystallized and solved scaffolding protein were grown under the same conditions. Form I belongs to the primitive tetragonal space group P41212, with unit-cell parameters a = b = 77.13, c = 37.12 Å. Form II crystals exhibit an orthorhombic crystal form, with space group C222 and unit-cell parameters a = 107.50, b = 107. 80, c = 37.34 Å. Complete data sets have been collected to 1.78 and 1.80 Å for forms I and II, respectively, at 100 K using Cu Kα X-rays from a rotating-anode generator. Calculation of a V
M value of 2.46 Å3 Da−1 for form I suggests the presence of one molecule in the asymmetric unit, corresponding to a solvent content of 50.90%, whereas form II has a V
M of 4.80 Å3 Da−1 with a solvent content of 48.76% and two molecules in the asymmetric unit. The structures of both crystal forms are being determined by the molecular-replacement method using the coordinates of the published crystal structure of gp7.
scaffolding protein; bacteriophage ϕ29
Good-quality crystals of selenomethionine-substituted Msmeg_3380 were obtained by the hanging-drop vapour-diffusion technique and diffracted to 1.2 Å using synchrotron radiation.
Pyridoxine 5′-phosphate oxidases (PNPOxs) are known to catalyse the terminal step in pyridoxal 5′-phosphate biosynthesis in a flavin mononucleotide-dependent manner in humans and Escherichia coli. Recent reports of a putative PNPOx from Mycobacterium tuberculosis, Rv1155, suggest that the cofactor or catalytic mechanism may differ in Mycobacterium species. To investigate this, a putative PNPOx from M. smegmatis, Msmeg_3380, has been cloned. This enzyme has been recombinantly expressed in E. coli and purified to homogeneity. Good-quality crystals of selenomethionine-substituted Msmeg_3380 were obtained by the hanging-drop vapour-diffusion technique and diffracted to 1.2 Å using synchrotron radiation.
pyridoxine 5′-phosphate oxidases; Mycobacterium smegmatis; FMN
Various N-terminally truncated forms of the S-layer protein SbsC were crystallized and the form rSbsC(755–1099), which was obtained by unintentional in situ proteolysis, was used for preliminary structure determination.
The S-layer protein SbsC from Geobacillus stearothermophilus ATCC 12980 is the most prevalent single protein produced by the bacterium and covers the complete bacterial surface in the form of a two-dimensional crystalline monolayer. In order to elucidate the structural features of the assembly domains, several N-terminally truncated fragments of SbsC have been crystallized. Crystals obtained from recombinant fragments showed anisotropic diffraction to a maximum of 3.5 Å resolution using synchrotron radiation. The best diffracting crystals were obtained from rSbsC(755–1099), an unintentional in situ proteolytic degradation product of rSbsC(447–1099). Crystals were obtained in two different space groups, P21 and P41212, and diffracted to 2.6 and 3 Å resolution, respectively. Native and heavy-atom derivative data have been collected. The structure of the C-terminal part will yield atomic resolution information for the domains that are crucial for the assembly of the two-dimensional lattice.
S-layer proteins; SbsC
The fungal β-N-acetylhexosaminidase from A. oryzae was crystallized and diffraction data were collected from two crystal forms to 3.2 and 2.4 Å resolution, respectively.
Fungal β-N-acetylhexosaminidases are enzymes that are used in the chemoenzymatic synthesis of biologically interesting oligosaccharides. The enzyme from Aspergillus oryzae was produced and purified from its natural source and crystallized using the hanging-drop vapour-diffusion method. Diffraction data from two crystal forms (primitive monoclinic and primitive tetragonal) were collected to resolutions of 3.2 and 2.4 Å, respectively. Electrophoretic and quantitative N-terminal protein-sequencing analyses confirmed that the crystals are formed by a complete biologically active enzyme consisting of a glycosylated catalytic unit and a noncovalently attached propeptide.
fungal hexosaminidases; glycoproteins; quantitative Edman degradation