Isomorphous Heavy-atom Derivatives Research Articles

Purification, crystallization and X-ray diffraction analysis of the C-terminal protease domain of Venezuelan equine encephalitis virus nsP2

The C-terminal region of Venezuelan equine encephalitis virus (VEEV) nsP2 is responsible for proteolytic processing of the VEEV polyprotein replication complex. This action regulates the activity of the replication complex and is essential for viral replication, thus making nsP2 a very attractive target for development of VEEV therapeutics. The 338-amino-acid C-terminal region of VEEV nsP2 has been overexpressed in Escherichia coli, purified and crystallized. Crystals diffract to beyond 2.5 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1). Isomorphous heavy-atom derivatives suitable for phase analysis have been obtained and work on building a complete structural model is under way.

Generating isomorphous heavy-atom derivatives by a quick-soak method. Part II: phasing of new structures.

A quick-soak method has been applied to generate de novo heavy-atom phasing to solve two new protein structures, a type II transforming growth factor beta receptor (TBRII) and a natural killer cell receptor-ligand complex, NKG2D-ULBP3. In the case of TBRII, a crystal derivatized for only 10 min in saturated HgCl(2) provided adequate phasing for structure determination. Comparison between HgCl(2) derivatives generated by 10 min soaking and by 12 h soaking revealed similar phasing statistics. The shorter soak, however, resulted in a derivative more isomorphous to the native than the longer soak as judged by changes in the unit-cell parameter a upon derivatization as well as by the quality of a combined SIRAS electron-density map. In the case of the NKG2D-ULBP3 structure, all overnight soaks in heavy-atom solutions resulted in crystal lattice disorder and only the quick soaks preserved diffraction. Despite fragile lattice packing, the quick-soaked K(2)PtCl(4) derivative was isomorphous with the native crystal and the electron-density map calculated from combined SIR and MAD phases is better than that calculated from MAD phases alone. Combined with mass-spectrometry-assisted solution heavy-atom derivative screening and the use of synchrotron radiation, the quick-soak derivatization has the potential to transform the time-consuming conventional heavy-atom search into a real-time 'on-the-fly' derivatization process that will benefit high-throughput structural genomics.

Generating isomorphous heavy-atom derivatives by a quick-soak method. Part I: test cases.

Screening for heavy-atom derivatives remains a time-consuming and cumbersome process that often results in non-isomorphous derivatives whose phases cannot be combined. Using lysozyme and FcgammaRIII receptor crystals as test cases, an improved soaking method for the generation of conventional heavy-atom derivatives has been developed. The method is based on soaking crystals in heavy-atom compounds for a very brief time at near-saturation concentrations. Compared with the current heavy-atom soaking method, which often takes days to achieve a derivatization, the quick-soak method completes a derivatization within 10 min to 2 h. The bound heavy-atom sites display higher peak heights from quick soaks than from overnight soaks in all cases tested. The quick-soak derivatives also preserved native-like diffraction resolution and data quality that was better than the prolonged-soak derivatives. Furthermore, derivatives generated by brief soaks are more isomorphous to the native than those generated by overnight soaks. Short soaks not only increase the likelihood of success in heavy-atom screening by reducing the pitfalls associated with prolonged soaks, such as lack of isomorphism and overall lattice disorder, but also have the potential to transform a time-consuming derivative screening into an 'on-the-fly' real-time derivatization process.

Crystallization and preliminary X-ray diffraction analysis of the seed lectin from Parkia platycephala.

The crystallization and preliminary X-ray diffraction analysis of the seed lectin of Parkia platycephala, a Mimosoideae, regarded as the most primitive group of the Leguminosae plants, are reported. Its amino-acid sequence consists of three tandemly arranged jacalin-related beta-prism domains, which is a novel fold for a leguminous lectin. Furthermore, no other lectin structure with this arrangement of domains has been described. P2(1)2(1)2(1) crystals (unit-cell parameters a = 63.6, b = 68.5, c = 208.5 A), which diffract to a maximum resolution of 2.2 A, were obtained in hanging drops at pH 8 and 293 K by the vapor-diffusion method using 10% 2-propanol and 20% polyethylene glycol 4000 as precipitants. The asymmetric unit contains two lectin molecules and has a solvent content of 46%. Only a single beta-prism domain could be located by molecular replacement using the structure of the Helianthus tuberosus lectin (PDB code 1c3k) as the search model. Isomorphous heavy-atom derivatives are currently being produced to solve the complete structure of the P. platycephala seed lectin.

Implementation of cluster analysis for ab initio phasing using the molecular envelope from solution X-ray scattering.

Solution of the phase problem is central to crystallographic structure determination. The conventional methods of isomorphous replacement (MIR or SIR) and molecular replacement are ineffective in the absence of a suitable isomorphous heavy-atom derivative or knowledge of the structure of a homologous protein. A recent method utilizing the low-resolution molecular shape determined from solution X-ray scattering data has shown to be successful in locating the molecular shape within the crystallographic unit cell in the case of the trimer nitrite reductase (NiR, 105 kDa) [Hao et al. (1999), Acta Cryst. D55, 243-246]. This was achieved by performing a direct real-space search for orientation and translation using knowledge of the orientation of the polar angles of the non-crystallographic axis obtained by performing a self-rotation on crystallographic data. This effectively reduces the potential six-dimensional search to a four-dimensional one (Eulerian angle gamma and three translational parameters). In the case of NiR, the direct four-dimensional search produced a clear solution that was in good agreement with the known structure. The program FSEARCH incorporating this method has been generalized to handle molecules from all space groups and in particular those in possession of non-crystallographic symmetry. However, the method employed was initially unsuccessful when applied to the small dimeric molecule superoxide dismutase (SOD, 32 kDa) owing to the absence of strong reflections at low resolution caused by saturation at the detector. The determined solution deviated greatly from that of the known structure [Hough & Hasnain (1999), J. Mol. Biol. 287, 579-592]. It was found that once these absent reflections were replaced by a series of randomly generated intensity values and cluster analysis was performed on the output, the signal-to-noise ratio was improved and a most probable solution was found. The electron-density map of the stochastically determined solution agrees well with the known structure; the phase error calculated from this map was 67 degrees within 14 A resolution.

X-ray crystallographic determination of the structure of the influenza C virus haemagglutinin-esterase-fusion glycoprotein.

The structure of the haemagglutinin‐esterase‐fusion (HEF) glycoprotein from influenza C virus has been determined to 3.2 Å resolution by X‐ray crystallography. A synthetic mercury‐containing esterase inhibitor and receptor analogue, 9‐acetamidosialic acid α‐thiomethylmercuryglycoside, was designed as the single isomorphous heavy‐atom derivative. The asymmetric unit of one crystal form (form I; P4322, a = b = 155.4, c = 414.4 Å) contained an HEF trimer. Six mercury sites identifying the three haemagglutination and three esterase sites were located by difference Patterson map analysis of a 6.5 Å resolution derivative data set. These positions defined the molecular threefold‐symmetry axis of the HEF trimer. A molecular envelope was defined by averaging a 7.0 Å resolution electron‐density map, phased by single isomorphous replacement (SIR), about the non‐crystallographic threefold‐symmetry axis. Iterative non‐crystallographic symmetry averaging in real space, solvent flattening and histogram matching were used to extend the phases to 3.5 Å resolution. Molecular replacement of the model into a second crystal form (form II; P43212, a = b = 217.4, c = 421.4 Å) containing two HEF trimers per asymmetric unit permitted iterative ninefold averaging of the electron density. The 3.5 Å electron‐density map allowed an unambiguous tracing of the polypeptide chain and identification of N‐linked carbohydrates. The model has been refined by least squares to 3.2 Å resolution (R free = 26.7%).

Crystallization and preliminary X-ray analysis of alpha-D-glucuronidase from Bacillus stearothermophilus T-6.

alpha-D-Glucuronidases cleave the alpha-1,2-glycosidic bond of the 4-O-methyl-alpha-D-glucuronic acid side chain in xylan. Of the xylan-debranching hydrolases, these enzymes are the least studied and characterized. The alpha-glucuronidase gene (aguA) from Bacillus stearothermophilus T-6 has been cloned, sequenced and overproduced in Escherichia coli. The gene encodes for a protein of 679 amino acids with a calculated molecular weight of 78480 and a pI of 5.42. alpha-Glucuronidase T-6 shows high homology to the alpha-glucuronidases of Thermotoga maritima (60% identity) and of Tri-choderma reesei (44% identity). Based on the amino-acid sequence similarity, it is likely that these enzymes represent a new class of glycosyl hydrolases. Crystallographic studies of alpha-glucuronidase T-6 were initiated to study the mechanism of catalysis, as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis. In this report, the crystallization and preliminary crystallographic characterization of the native alpha-glucuronidase T-6 enzyme is described. Two crystal forms were found suitable for detailed crystal structure analysis. The T1 form was obtained by the vapour-diffusion method using PEG 4000 as a precipitant and 2-propanol as an organic additive. The crystals belong to a primitive tetragonal crystal system (space group P41212 or P43212) with unit-cell dimensions a = b = 76.1 and c = 331.2 A. These crystals are mechanically strong, are stable in the X--ray beam and diffract X-rays to better than 2.4 A resolution. A full 3.0 A resolution diffraction data set (97.3% completeness, Rmerge 9.8%) has recently been collected on one crystal at room temperature using a rotating-anode X-ray source and an R-AXIS IIc imaging-plate detector. The M1 form was obtained and characterized by similar techniques. The best crystallization occurred at a slightly lower pH and a lower concentration of 2-propanol. The crystals belong to a primitive monoclinic crystal system (space group P21) with unit-cell dimensions a = 65.8, b = 127.4, c = 96.6 A and beta = 97.9 degrees. These crystals are also quite strong and stable, and diffract to better than 2.8 A resolution. A full 2.8 A resolution diffraction data set (96.2% completeness, Rmerge 7.6%) has recently been collected on one crystal at room temperature using the same R-AXIS IIc setup. Both forms are currently being used to obtain crystallographic phasing via isomorphous heavy-atom derivatives and selenomethionine MAD experiments.

Ab initio phasing using molecular envelope from solution X-ray scattering.

Solving the phase problem is the crucial and quite often the most difficult and time-consuming step in crystallographic structure determination. The traditional methods of isomorphous replacement (MIR or SIR) and molecular replacement require the availability of an isomorphous heavy-atom derivative or the structure of a homologous protein, respectively. Here, a method is presented which utilizes the low-resolution molecular shape determined from solution X-ray scattering data for the molecular search. The molecular shape of a protein is an important structural property and can be determined directly by the small-angle scattering technique. The idea of locating this molecular shape in the crystallographic unit cell has been tested with experimental diffraction data from nitrite reductase (NiR). The conventional Patterson search proved to be unsuccessful, as the intra-envelope vectors are uniformly distributed and do not match those of intra-molecular (atom-to-atom) vectors. A direct real-space search for orientation and translation was then performed. A self-rotation function using 2.8 A crystallographic data yielded the polar angles of the non-crystallographic threefold axis. Knowledge of the orientation of this axis reduces the potential six-dimensional search to four (Eulerian angle gamma and three translational parameters). The direct four-dimensional search within the unit cell produced a clear solution. The electron-density map based on this solution agrees well with the known structure, and the phase error calculated from the map was 61 degrees within 20 A resolution. It is anticipated that the low-resolution envelope can be used as a starting model for phase extension by the maximum-entropy and density-modification method.

Structure determination of a 16.8 kDa copper protein at 2.1 A resolution using anomalous scattering data with direct methods.

The structure of rusticyanin, an acid-stable copper protein, has been determined at 2.1 A resolution by direct methods combined with the single-wavelength anomalous scattering (SAS) of copper (f" = 3.9 e-) and then conventionally refined (Rcryst = 18.7%, Rfree = 21.9%). This is the largest unknown protein structure (Mr approximately /= 16.8 kDa) to be determined using the SAS and direct-methods approach and demonstrates that by exploiting the anomalous signal at a single wavelength, direct methods can be used to determine phases at typical (approximately 2 A) macromolecular crystallographic resolutions. Extrapolating from the size of the anomalous signal for copper (f" approximately 4 e-), this result suggests that the approach could be used for proteins with molecular weights of up to 33 kDa per Se (f"max++ = 8 e- at the 'white line') and 80 kDa for a Pt derivative (f"max = 19 e- at the 'white line', L3 edge). The method provides a powerful alternative in solving a de novo protein structure without either preparing multiple crystals (i.e. isomorphous heavy-atom derivative plus native crystals) or collecting multi-wavelength anomalous diffraction (MAD) data.

Low-resolution structural characterization of the arginine repressor/activator from Bacillus subtilis: a combined X-ray crystallographic and electron microscopical approach.

Attempts to determine the X-ray crystal structure of the intact homohexameric arginine repressor/activator from B. subtilis have so far been unsuccessful. The major problem appears to be the lack of an isomorphous heavy-atom derivative with a manageable number of substitution sites. Here it is shown how electron microscopy of thin three-dimensional crystals, the same as those used for the X-ray crystallographic studies, made it possible (i) to obtain experimental support for some conclusions drawn on the basis of X-ray data alone, (ii) to determine the low-resolution distribution of electron density in several different crystallographic projections, and (iii) to obtain a tentative low-resolution model of the whole hexamer.

Use of Chemically Modified Nucleotides to Determine a 62-Nucleotide RNA Crystal Structure: A Survey of Phosphorothioates, Br, Pt and Hg

Two important challenges confronting RNA crystallographers are producing crystals and finding isomorphous heavy-atom derivatives. Non-isomorphism can be addressed by determining the phases using the multiwavelength anomalous dispersion (MAD) method. These phases can be greatly improved by combining phases from MAD experiments done on different heavy-atom derivatives. Heavy-atom derivatives can be created by chemically modifying the RNA through covalent attachment of bromine or mercury to C5 of pyrimidines or [Pt(NH3)3]2+ to N7 of guanine. While phosphorothioates can provide mercury binding sites, disorder can reduce their value for phase determination. The location of these chemical modifications is critical since crystallization of these derivatized RNAs is sensitive to heavy atom induced conformational alterations and crystal packing.

Crystallization and preliminary structural studies of lactose-specific enzyme IIA from Lactococcus lactis.

Lactose-specific enzyme IIA of the phosphoenol:pyruvate-dependent sugar phosphotransferase system from Lactococcus lactis has been crystallized in phosphate buffer. The crystals belong to space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2 with unit-cell axes a = b = 90.9 and c = 82.4 A. The packing parameter (Matthews parameter) V(m) of 2.48 A(3) Da(-1) is consistent with one trimer per asymmetric unit and non-crystallographic threefold symmetry has been confirmed by calculating a self-rotation function. The crystals diffract X-rays to at least 2.3 A resolution, are stable in an X-ray beam and are therefore appropriate for structure determination. Native data to 2.3 A resolution have been collected using a MAR image-plate system at a synchrotron source. One isomorphous heavy-atom derivative has been identified and the presence of an isomorphous signal in the data has been confirmed by Patterson methods.

Three-dimensional structure of cytochrome c' from two Alcaligenes species and the implications for four-helix bundle structures.

The three-dimensional structures of two cytochromes c' have been determined in order to analyse the common features of proteins of this family and their relationship with other four-helix bundle structures. The structure of cytochrome c' from Alcaligenes sp was determined by molecular replacement supplemented with the iron anomalous scattering and the use of a single isomorphous heavy-atom derivative, and was refined using synchrotron data to 1.8 A resolution. The final model, comprising 956 protein atoms (one monomer) and 89 water molecules, has a final R value of 0.188 for all data in the range 20.0-1.8 A resolution (14 673 reflections). The structure of the cytochrome c' from Alcaligenes denitrificans is isomorphous and essentially identical (r.m.s. deviation for all atoms 0.36 A). Although its amino-acid sequence has not been determined chemically, only four differences from that of Alcaligenes sp cytochrome c' were identified by the X-ray analysis. The final model for Alcaligenes denitrificans cytochrome c', comprising 953 protein atoms and 75 water molecules, gave a final R factor of 0.167 for all data in the range 20.0-2.15 A (8220 reflections). The cytochrome c' monomer forms a classic four-helix bundle, determined by the packing of hydrophobic side chains around the enclosed haem group. There are very few cross-linking hydrogen bonds between the helices, the principal side-chain hydrogen bonding involving one of the haem propionates and a conserved Arg residue. The cytochrome c' dimer is created by a crystallographic twofold axis. Monomer-monomer contacts primarily involve the two A helices, with size complementarity of side chains in a central solvent-excluded portion of the interface and hydrogen bonding at the periphery. Both species have a pyroglutamic acid N-terminal residue. The haem iron is five-coordinate, 0.32 A out of the haem plane towards the fifth ligand, His120. The unusual magnetic properties of the Fe atom may be linked to a conserved basic residue, Arg124, adjacent to His120.

Structure and function of the DNA repair enzyme exonuclease III from E. coli.

The three-dimensional structure of exonuclease III, the major AP DNA repair endonuclease of Escherichia coli, has been determined using x-ray crystallographic methods at 2.7 A resolution. The atomic model was fit to an electron density map calculated with phases obtained from three isomorphous heavy atom derivatives. The overall chain fold of exonuclease III is that of a compact alpha,beta-protein of dimensions 55 by 50 by 45 A. The pair of extended beta-pleated sheets pack against each other in an approximately parallel fashion to form the hydrophobic core of a four-layered sandwich structure. These beta sheets are flanked by four alpha-helices that form the outer two layers of the fold. The individual strands of the beta-sheets are in a mostly antiparallel configuration and are linked by extensive loop regions that connect adjoining strands. The structure contains internal symmetry with the two extended beta-sheets and four alpha-helices related by a pseudo-twofold axis running approximately down the center of the two sheets. This internal symmetry is not mirrored in the structure of the loop regions, nor is it detectable within the amino acid sequence. There is a "groove" between the beta-sheets at one end of the molecule that is bordered by several of the exposed loop regions and may be significant for DNA binding.

A progress report on the crystallographic studies on the B800-850 antenna complex from Rhodopseudomonas acidophila strain 10050.

Large single crystals (up to 1 mm in each dimension) of the B800-850 antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown in the presence of beta-octyl-glucoside. These crystals have the space group R32 and unit cell dimensions of a = b = 119.9 A and c = 297.0 A. Recently we have improved our crystallization procedures so that all crystals now diffract reliably to beyond 3.5 A, with some diffracting to below 3 A. A range of isomorphous heavy atom derivatives have been prepared and we are now engaged in locating the heavy atom sites within the unit cell.

Trimethyllead acetate: A first-choice heavy atom derivative for protein crystallography

The three-dimensional conformation of a protein provides a wealth of biochemical information and with the advent of cloning techniques that allow the preparation of proteins almost at will, a renewed interest has arisen in the crystallographic determination of protein structures. As in any research technique, however, there are often many difficulties encountered in an X-ray crystallographic investigation. One of these is the “phase problem”. Although in recent years there has been considerable progress in the development of techniques for phase determination, including the use of molecular replacement and multiple wavelength measurements, the multiple isomorphous replacement method is still the most successful method for obtaining a three-dimensional structure. Here we report the use of trimethyllead acetate as a heavy atom compound of first choice in the preparation of an isomorphous heavy atom derivative.

Crystallization and structure determination of 2.5-.ANG. resolution of the oxidized iron-sulfur [2Fe-2S] ferredoxin isolated from Anabaena 7120

The molecular structure of the oxidized form of the [2Fe-2S] ferredoxin isolated from the cyanobacterium Anabaena species strain PCC 7120 has been determined by X-ray diffraction analysis to a nominal resolution of 2.5 A and refined to a crystallographic R factor of 18.7%. Crystals used in this investigation belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 37.42 A, b = 38.12 A, and c = 147.12 A and two molecules in the asymmetric unit. The three-dimensional structure of this ferredoxin was solved by a method that combined X-ray data from one isomorphous heavy-atom derivative with noncrystallographic symmetry averaging and solvent flattening. As in other plant-type [2Fe-2S] ferredoxins, the iron-sulfur cluster is located toward the outer edge of the molecule, and the irons are tetrahedrally coordinated by both inorganic sulfurs and sulfurs provided by protein cysteine residues. The main secondary structural elements include four strands of beta-pleated sheet and three alpha-helical regions.

Crystal structure of the antigen-binding fragment of the murine anti-arsonate monoclonal antibody 36-71 at 2.9-A resolution.

The structure of the antigen-binding fragment (Fab) of an anti-phenylarsonate monoclonal antibody (36-71) bearing a major crossreacting idiotype of A/J mice has been solved and refined to an R factor of 19.3% at a resolution of 2.9 A. An initial electron density map was obtained with phase information from a total of six isomorphous heavy-atom derivatives (from two different compounds) and a molecular replacement solution using the HED10 Fab crystal structure as a model. The structure of the McPC603 Fab was used to provide an initial set of atomic coordinates. The electron density maps are clear and easily interpretable for the entire sequence except for sections from two of the heavy-chain complementarity-determining regions totaling 21 residues. These residues have been left out of the refinement and are not represented in our current model. The antigen-combining site was located by means of a difference Fourier synthesis with one of the heavy-atom derivatives, which contained arsanilic acid. It lies in a small pocket formed by residues from the hypervariable regions of both the heavy and the light chains. Interactions with the hapten from framework residues are also possible.

Preparation of heavy-atom derivatives using site-directed mutagenesis: Introduction of cysteine residues into γδ resolvase

The ability to determine protein structures by X-ray crystallography is often thwarted by the difficulty of finding isomorphous heavy-atom derivatives. The crystal structure of the site-specific recombinase, resolvase, has been difficult to determine for this reason. We have overcome this problem by introducing 13 single cysteine substitutions into the resolvase catalytic domain using oligonucleotide mutagenesis. The mutant proteins were screened for their ability to crystallize into the orthorhombic form and bind mercury ions isomorphously. Two mutant proteins provided excellent heavy-atom derivatives. This approach should be of general use and particularly helpful in cases where traditional methods have failed to produce a derivative.

Preliminary x-ray data on crystals of mandelate racemase.

The recent development of a high-yield expression system and purification scheme for mandelate racemase has enabled us to produce sufficiently large quantities of pure enzyme to pursue x-ray crystallographic study. Large, single crystals of mandelate racemase have been grown from buffered polyethylene glycol (pH 8.0) in the presence of 10 mM magnesium chloride. The crystals grow in several habits, and we have identified two distinct tetragonal space groups in preliminary x-ray diffraction analysis. Crystals shaped as rectangular plates demonstrate 4/mmm Laue symmetry and systematic absences consistent with the space group I422. They have cell dimensions of a = b = 153 A and c = 181 A. Octahedrally shaped crystals of mandelate racemase display 4/m Laue symmetry and systematic absences consistent with the space group 14. Cell dimensions for these crystals are a = b = 113 A and c = 124 A. Based on estimates of Vm and on the measured density of the 1422 form, we suggest that two subunits of mandelate racemase (38,570 daltons/subunit) occupy the asymmetric unit in both crystal forms. Crystals of both forms diffract to beyond 3.0-A resolution. We are currently screening for isomorphous heavy-atom derivatives.