Different Crystal Forms (different + crystal_form)

Distribution by Scientific Domains

Selected Abstracts

Polymorphism of racemic felodipine and the unusual series of solid solutions in the binary system of its enantiomers

Judith M. Rollinger
Abstract The aim of this study was to investigate the binary phase diagram and the polymorphism and pseudopolymorphism of racemic and enantiomeric felodipine, including their spectroscopic and thermodynamic properties. Different crystal forms were obtained by crystallization from solvents or from the annealed melt and investigated by thermal analysis (hot stage microscopy, differential scanning calorimetry, thermogravimetric analysis), spectroscopic methods (Fourier transform infrared,and Fourier transform,Raman spectroscopy), and X-ray powder diffractometry. The binary melting phase diagram was constructed based on thermoanalytical investigations of quantitative mixtures of (+)- and ()-felodipine. Two polymorphic forms of racemic felodipine, mod. I (mp, ,145C) and mod. II (mp, ,135C), as well as an acetone solvate (SAc,) were characterized. Melting equilibria of felodipine crystal forms decrease due to thermal decomposition. Enantiomeric felodipine was found to be dimorphic (En-mod. I: mp, ,144C; En-mod. II: mp, ,133C). Evaluation of the binary system of (+)- and (,)-felodipine results in the formation of a continuous series of mixed crystals between the thermodynamically stable and higher melting modifications, mod. I and En-mod. I. Their unusual curve course, termed as Roozeboom Type 2 b, passes through a maximum in the racemic mixture and is flanked by a minimum at 20% and at 80% (+)-felodipine. From the thermodynamic parameters, racemic mod. I and II are monotropically related. In contrast to SAc, the thermodynamically unstable mod. II shows a considerable kinetic stability. Because its crystallization is badly reproducible, the use of mod. II is not advisable for processing. However, desolvation of SAc leads to a profitable crystal shape of mod. I, representing a pseudoracemate by definition. 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:949,959, 2001 [source]

Promoting crystallization of antibody,antigen complexes via microseed matrix screening

Galina Obmolova
The application of microseed matrix screening to the crystallization of antibody,antigen complexes is described for a set of antibodies that include mouse anti-IL-13 antibody C836, its humanized version H2L6 and an affinity-matured variant of H2L6, M1295. The Fab fragments of these antibodies were crystallized in complex with the antigen human IL-13. The initial crystallization screening for each of the three complexes included 192 conditions. Only one hit was observed for H2L6 and none were observed for the other two complexes. Matrix self-microseeding using these microcrystals yielded multiple hits under various conditions that were further optimized to grow diffraction-quality H2L6 crystals. The same H2L6 seeds were also successfully used to promote crystallization of the other two complexes. The M1295 crystals appeared to be isomorphous to those of H2L6, whereas the C836 crystals were in a different crystal form. These results are consistent with the concept that the conditions that are best for crystal growth may be different from those that favor nucleation. Microseed matrix screening using either a self-seeding or cross-seeding approach proved to be a fast, robust and reliable method not only for the refinement of crystallization conditions but also to promote crystal nucleation and increase the hit rate. [source]

Analysis of multiple crystal forms of Bacillus subtilis BacB suggests a role for a metal ion as a nucleant for crystallization

M. Rajavel
Bacillus subtilis BacB is an oxidase that is involved in the production of the antibiotic bacilysin. This protein contains two double-stranded ,-helix (cupin) domains fused in a compact arrangement. BacB crystallizes in three crystal forms under similar crystallization conditions. An interesting observation was that a slight perturbation of the crystallization droplet resulted in the nucleation of a different crystal form. An X-ray absorption scan of BacB suggested the presence of cobalt and iron in the crystal. Here, a comparative analysis of the different crystal forms of BacB is presented in an effort to identify the basis for the different lattices. It is noted that metal ions mediating interactions across the asymmetric unit dominate the different packing arrangements. Furthermore, a normalized B -factor analysis of all the crystal structures suggests that the solvent-exposed metal ions decrease the flexibility of a loop segment, perhaps influencing the choice of crystal form. The residues coordinating the surface metal ion are similar in the triclinic and monoclinic crystal forms. The coordinating ligands for the corresponding metal ion in the tetragonal crystal form are different, leading to a tighter packing arrangement. Although BacB is a monomer in solution, a dimer of BacB serves as a template on which higher order symmetrical arrangements are formed. The different crystal forms of BacB thus provide experimental evidence for metal-ion-mediated lattice formation and crystal packing. [source]

Heterogeneity and dynamics in villin headpiece crystal structures

Jianmin Meng
The villin headpiece domain (HP67) is the C-terminal F-actin-binding motif that confers F-actin-bundling activity to villin, a component of the actin bundles that support the brush-border microvilli. It has been investigated extensively by both experimental and theoretical measurements. Our laboratory, for example, has determined both its NMR and its crystal structures. This study presents the structures of HP67 and its pH-stabilized mutant (H41Y) in a different crystal form and space group. For both constructs, two molecules are found in each asymmetric unit in the new space group P61. While one of the two structures (Mol A) is structurally similar to our previously determined structure (Mol X), the other (Mol B) has significant deviations, especially in the N-terminal subdomain, where lattice contacts do not appear to contribute to the difference. In addition, the structurally most different crystal structure, Mol B, is actually closer to the averaged NMR structure. Harmonic motions, as suggested by the B -factor profiles, differ between these crystal structures; crystal structures from the same space group share a similar pattern. Thus, heterogeneity and dynamics are observed in different crystal structures of the same protein even for a protein as small as villin headpiece. [source]

Crystallization of the 43,kDa ATPase domain of Thermus thermophilus gyrase B in complex with novobiocin

V. Lamour
The 43,kDa ATPase domain of Thermus thermophilus gyrase B was overproduced in Escherichia coli and a three-step purification protocol yielded large quantities of highly purified enzyme which remained stable for weeks. Crystals of the 43,kDa domain in complex with novobiocin, one of the most potent inhibitors of bacterial topoisomerases, were obtained. Crystals obtained in the presence of PEG 8000 do not diffract, but a different crystal form was obtained using sodium formate as a precipitating agent. The plate-shaped crystals, which were less than 10,m in thickness, could be cryocooled directly from the mother liquor and a full diffraction data set was collected to 2.3, allowing the determination of the first structure of a gyrase B 43K domain in complex with a coumarin. [source]

Concanavalin A in a dimeric crystal form: revisiting structural accuracy and molecular flexibility

Katherine A. Kantardjieff
A structure of native concanavalin A (ConA), a hardy perennial of structural biology, has been determined in a dimeric crystal form at a resolution of 1.56, (space group C2221; unit-cell parameters a = 118.70, b = 101.38, c = 111.97,; two molecules in the asymmetric unit). The structure has been refined to an Rfree of 0.206 (R = 0.178) after iterative model building and phase-bias removal using Shake&wARP. Correspondence between calculated water,tyrosine interactions and experimentally observed structures near the saccharide-binding site suggests that the observed interactions between Tyr12 and water in various crystal forms are to be expected and are not unique to the presence of an active site. The present structure differs from previously reported atomic resolution structures of ConA in several regions and extends insight into the conformational flexibility of this molecule. Furthermore, this third, low-temperature, structure of ConA in a different crystal form, independently refined using powerful model-bias removal techniques, affords the opportunity to revisit assessment of accuracy and precision in high- or atomic resolution protein structures. It is illustrated that several precise structures of the same molecule can differ substantially in local detail and users of crystallographic models are reminded to consider the potential impact when interpreting structures. Suggestions on how to effectively represent ensembles of crystallographic models of a given molecule are provided. [source]

Structure of a new crystal form of tetraubiquitin

Cynthia L. Phillips
Polyubiquitin chains, in which the C-terminus and a lysine side chain of successive ubiquitin molecules are linked by an isopeptide bond, function to target substrate proteins for degradation by the 26S proteasome. Chains of at least four ubiquitin moieties appear to be required for efficient recognition by the 26S proteasome, although the conformations of the polyubiquitin chains recognized by the proteasome or by other enzymes involved in ubiquitin metabolism are currently unknown. A new crystal form of tetraubiquitin, which has two possible chain connectivities that are indistinguishable in the crystal, is reported. In one possible connectivity, the tetraubiquitin chain is extended and packs closely against the antiparallel neighbor chain in the crystal to conceal a hydrophobic surface implicated in 26S proteasome recognition. In the second possibility, the tetraubiqutitin forms a closed compact structure, in which that same hydrophobic surface is buried. Both of these conformations are quite unlike the structure of tetraubiquitin that was previously determined in a different crystal form [Cook et al. (1994), J. Mol. Biol.236, 601,609]. The new structure suggests that polyubiquitin chains may possess a substantially greater degree of conformational flexibility than has previously been appreciated. [source]

Structural analysis of photosystem II in far-red-light-adapted thylakoid membranes

FEBS JOURNAL, Issue 1 2000
New crystal forms provide evidence for a dynamic reorganization of light-harvesting antennae subunits
We studied two-dimensional crystals of the major pigment,protein complex, photosystem II, in far-red-light-adapted thylakoid membranes of the viridis-zb63 mutant of barley. Significantly larger grana membranes were produced with an increased synthesis of the entire photosystem II complex. These red-light-adapted membranes also contained two-dimensional crystals with a high frequency. Three different crystal forms of photosystem II were observed, providing the following data which further our understanding of the architecture of the native complex. (a) The oligomeric form of photosystem II in the membrane was monomeric in all crystal forms, but with a clear non-crystallographic pseudo-twofold symmetry. This was more apparent on the lumenal face of the complex. (b) The variability of unit cell contacts in different crystal forms implied that the peripheral light-harvesting antenna complex and the core of the complex were loosely connected. These peripheral subunits were predicted to rearrange so that they can either encircle the core complex or associate in parallel channels separated by lines of core complexes. (c) Grana membranes were found to retain a double-layered inside-out character, with a stromal face-to-stromal face packing. However, the presence of a crystal in one membrane did not necessarily impose crystallinity on its pair. [source]

High-order simulation of polymorphic crystallization using weighted essentially nonoscillatory methods

AICHE JOURNAL, Issue 1 2009
Martin Wijaya Hermanto
Abstract Most pharmaceutical manufacturing processes include a series of crystallization processes to increase purity with the last crystallization used to produce crystals of desired size, shape, and crystal form. The fact that different crystal forms (known as polymorphs) can have vastly different characteristics has motivated efforts to understand, simulate, and control polymorphic crystallization processes. This article proposes the use of weighted essentially nonoscillatory (WENO) methods for the numerical simulation of population balance models (PBMs) for crystallization processes, which provide much higher order accuracy than previously considered methods for simulating PBMs, and also excellent accuracy for sharp or discontinuous distributions. Three different WENO methods are shown to provide substantial reductions in numerical diffusion or dispersion compared with the other finite difference and finite volume methods described in the literature for solving PBMs, in an application to the polymorphic crystallization of L -glutamic acid. 2008 American Institute of Chemical Engineers AIChE J, 2009 [source]

On the Lamellar Crystal Structure of Nylons 6,8 and 8,10: A Study Combining Electron Microscopy and Energy Analysis

Marta Bermdez
Abstract Lamellar crystals of nylon 6,8 and nylon 8,10 with chains in a fully extended conformation were obtained by crystallization in solution, and examined by electron and X-ray diffraction. The same crystal structure appeared to be shared by both nylons crystallized in glycerin. This structure consisted of a mixture of ,- and ,-forms with features similar to that previously described for lamellar crystals of nylon 4,6 grown in the same solvent. Crystallization of nylon 8,10 in hexane-2,5-diol afforded lamellar crystals with sheets stacked in the ,-form. A comparative energy evaluation of the different models that are feasible for the crystal structure of these nylons with fully extended chains indicated very small energy differences among them. The presence of different chain arrangements in the same lamellar crystal, as well as the crystallization of the same nylon in different crystal forms upon slight modification of the crystallization conditions, are fully consistent, therefore, with the results obtained in the energy analysis. Nylon 8,10 crystals grown in hexane-2,5-diol at 150,C. Inset: The respective electron diffraction patterns. [source]

Polymorphism of microcrystalline urate oxidase from Aspergillus flavus

Ines Collings
Different polymorphs of rasburicase, a recombinant urate oxidase enzyme (Uox) from Aspergillus flavus, were obtained as a series of polycrystalline precipitates. Different crystallization protocols were followed in which the salt type, pH and polyethylene glycol 8000 (PEG 8000) concentration were varied. The related crystalline phases were characterized by means of high-resolution synchrotron X-ray powder diffraction. In all cases, Uox complexed with the inhibitor 8-azaxanthine (AZA) was not altered from its robust orthorhombic I222 phase by variation of any of the factors listed above. However, in the absence of AZA during crystallization ligand-free Uox was significantly affected by the type of salt, resulting in different crystal forms for the four salts tested: sodium chloride, potassium chloride, ammonium chloride and ammonium sulfate. Remarkable alterations of some of these phases were observed upon gradual increase of the exposure time of the sample to the synchrotron beam in addition to variation of the PEG 8000 concentration. When Uox was crystallized in Tris buffer or pure water in the absence of salt, a distinct polymorph of orthorhombic symmetry (P21212) was obtained that was associated with significantly altered lattice dimensions in comparison to a previously reported isosymmetrical structure. The latter form of Uox exhibits enhanced stability to variation of pH and PEG 8000 concentration accompanied by minor modifications of the unit-cell dimensions in the ranges under study. Accurate lattice parameters were extracted for all crystalline phases. This study reveals the rich phase diagram of Uox, a protein of high pharmaceutical importance, which is associated with an enhanced degree of polymorphism. The outcome of our analysis verifies previously reported results as well as demonstrating polymorphs that have altered unit-cell dimensions with respect to known structural models. [source]

Structure of d -tyrosyl-tRNATyr deacylase using home-source Cu,K, and moderate-quality iodide-SAD data: structural polymorphism and HEPES-bound enzyme states

Manickam Yogavel
d -Tyrosyl-tRNATyr deacylase (DTD) is an editing enzyme that removes d -amino acids from mischarged tRNAs. The crystal structure of Plasmodium falciparum DTD (PfDTD) was determined using the iodide-SAD phasing method. Iodide-derivatized PfDTD crystals were obtained using the quick cryo-soaking procedure in which native crystals were soaked for a short period of 10,30,s in cryoprotectant solution containing 0.2,1,M NaI. Iodide-SAD data sets were collected to 3.3 and 2.74, resolution from PfDTD crystals that belonged to two different space groups, P43 and P1, using an in-house X-ray copper-anode source. This is the first report to detail structure solution using low iodide anomalous signal, modest resolution and redundancy and average solvent content for SAD phasing of 984 and 1312 amino acids in the triclinic P1 and tetragonal P43 space groups, respectively. A total of 85% and 56% of the residues were automatically built into the iodide-phased electron-density maps using PHENIX AutoBuild. The structure of HEPES-bound PfDTD was subsequently determined by molecular replacement and refined to 2.83, resolution. The crystals obtained from various batches of crystallization trials of PfDTD exhibited polymorphism in terms of belonging to different crystal forms and space groups. Even within a given crystal system the unit-cell parameters showed high non-isomorphism. These packing variations were exploited in order to conduct a systematic study of conformational changes in PfDTD. It is shown that the disposition of a ten-residue insertion loop affects packing within the PfDTD crystals and seems to determine the non-isomorphism in unit-cell parameters. By tracking the changes in PfDTD unit cells, it was possible to map conformational differences within PfDTD that may be of significance for enzyme activity. [source]

Analysis of multiple crystal forms of Bacillus subtilis BacB suggests a role for a metal ion as a nucleant for crystallization

M. Rajavel
Bacillus subtilis BacB is an oxidase that is involved in the production of the antibiotic bacilysin. This protein contains two double-stranded ,-helix (cupin) domains fused in a compact arrangement. BacB crystallizes in three crystal forms under similar crystallization conditions. An interesting observation was that a slight perturbation of the crystallization droplet resulted in the nucleation of a different crystal form. An X-ray absorption scan of BacB suggested the presence of cobalt and iron in the crystal. Here, a comparative analysis of the different crystal forms of BacB is presented in an effort to identify the basis for the different lattices. It is noted that metal ions mediating interactions across the asymmetric unit dominate the different packing arrangements. Furthermore, a normalized B -factor analysis of all the crystal structures suggests that the solvent-exposed metal ions decrease the flexibility of a loop segment, perhaps influencing the choice of crystal form. The residues coordinating the surface metal ion are similar in the triclinic and monoclinic crystal forms. The coordinating ligands for the corresponding metal ion in the tetragonal crystal form are different, leading to a tighter packing arrangement. Although BacB is a monomer in solution, a dimer of BacB serves as a template on which higher order symmetrical arrangements are formed. The different crystal forms of BacB thus provide experimental evidence for metal-ion-mediated lattice formation and crystal packing. [source]

Diffraction data analysis in the presence of radiation damage

Dominika Borek
In macromolecular crystallography, the acquisition of a complete set of diffraction intensities typically involves a high cumulative dose of X-ray radiation. In the process of data acquisition, the irradiated crystal lattice undergoes a broad range of chemical and physical changes. These result in the gradual decay of diffraction intensities, accompanied by changes in the macroscopic organization of crystal lattice order and by localized changes in electron density that, owing to complex radiation chemistry, are specific for a particular macromolecule. The decay of diffraction intensities is a well defined physical process that is fully correctable during scaling and merging analysis and therefore, while limiting the amount of diffraction, it has no other impact on phasing procedures. Specific chemical changes, which are variable even between different crystal forms of the same macromolecule, are more difficult to predict, describe and correct in data. Appearing during the process of data collection, they result in gradual changes in structure factors and therefore have profound consequences in phasing procedures. Examples of various combinations of radiation-induced changes are presented and various considerations pertinent to the determination of the best strategies for handling diffraction data analysis in representative situations are discussed. [source]

Type II dehydroquinase: molecular replacement with many copies

Kirsty Anne Stewart
Type II dehydroquinase is a small (150-amino-acid) protein which in solution packs together to form a dodecamer with 23 cubic symmetry. In crystals of this protein the symmetry of the biological unit can be coincident with the crystallographic symmetry, giving rise to cubic crystal forms with a single monomer in the asymmetric unit. In crystals where this is not the case, multiple copies of the monomer are present, giving rise to significant and often confusing noncrystallographic symmetry in low-symmetry crystal systems. These different crystal forms pose a variety of challenges for solution by molecular replacement. Three examples of structure solutions, including a highly unusual triclinic crystal form with 16 dodecamers (192 monomers) in the unit cell, are described. Four commonly used molecular-replacement packages are assessed against two of these examples, one of high symmetry and the other of low symmetry; this study highlights how program performance can vary significantly depending on the given problem. In addition, the final refined structure of the 16-dodecamer triclinic crystal form is analysed and shown not to be a superlattice structure, but rather an F -centred cubic crystal with frustrated crystallographic symmetry. [source]

On the routine use of soft X-rays in macromolecular crystallography.

Part IV.
23 different crystal forms of 19 different biological macromolecules were examined with respect to their anomalously scattering substructures using diffraction data collected at a wavelength of 2.0, (6.2,keV). In more than 90% of the cases the substructure was found to contain more than just the protein S atoms. The data presented suggest that chloride, sulfate, phosphate or metal ions from the buffer or even from the purification protocol are frequently bound to the protein molecule and that these ions are often overlooked, especially if they are not bound at full occupancy. Thus, in order to fully describe the macromolecule under study, it seems desirable that any structure determination be complemented with a long-wavelength data set. [source]

Extraction of functional motion in trypsin crystal structures

Andrea Schmidt
The analysis of anisotropic atomic displacement parameters for the direct extraction of functionally relevant motion from X-ray crystal structures of Fusarium oxysporum trypsin is presented. Several atomic resolution structures complexed with inhibitors or substrates and determined at different pH values and temperatures were investigated. The analysis revealed a breathing-like molecular motion conserved across trypsin structures from two organisms and three different crystal forms. Directional motion was observed suggesting a change of the width of the substrate-binding cleft and a change in the length of the specificity pocket. The differences in direction of motion across the structures are dependent on the mode of substrate or inhibitor binding and the chemical environment around the active-site residues. Together with the occurrence of multiple-residue conformers, they reflect spatial rearrangement throughout the deacylation pathway. [source]

Urate oxidase from Aspergillus flavus: new crystal-packing contacts in relation to the content of the active site

Pascal Retailleau
Urate oxidase from Aspergillus flavus (uricase or Uox; EC is a 135,kDa homotetramer with a subunit consisting of 301 amino acids. It catalyses the first step of the degradation of uric acid into allantoin. The structure of the extracted enzyme complexed with a purine-type inhibitor (8-azaxanthin) had been solved from high-resolution X-ray diffraction of I222 crystals. Expression of the recombinant enzyme in Saccharomyces cerevisiae followed by a new purification procedure allowed the crystallization of both unliganded and liganded enzymes utilizing the same conditions but in various crystal forms. Here, four different crystal forms of Uox are analyzed. The diversity of the Uox crystal forms appears to depend strongly on the chemicals used as inhibitors. In the presence of uracil and 5,6-diaminouracil crystals usually belong to the trigonal space group P3121, the asymmetric unit (AU) of which contains one tetramer of Uox (four subunits). Chemical oxidation of 5,6-diaminouracil within the protein may occur, leading to the canonical (I222) packing with one subunit per AU. Coexistence of two crystal forms, P21 with two tetramers per AU and I222, was found in the same crystallization drop containing another inhibitor, guanine. Finally, a fourth form, P21212 with one tetramer per AU, resulted fortuitously in the presence of cymelarsan, an additive. Of all the reported forms, the I222 crystal forms present by far the best X-ray diffraction resolution (,1.6, resolution compared with 2.3,3.2, for the other forms). The various structures and contacts in all crystalline lattices are compared. The backbones are essentially conserved except for the region near the active site. Its location at the dimer interface is thus likely to be at the origin of the crystal contact changes as a response to the various bound inhibitors. [source]

Purification, crystallization and preliminary X-ray analysis of Triatoma virus (TrV) from Triatoma infestans

Gabriela S. Rozas-Dennis
Triatoma virus (TrV) is a viral pathogen of the blood-sucking reduviid bug Triatoma infestans, the most important vector of American human trypanosomiasis (Chagas' disease). TrV has been putatively classified as a member of the Cripavirus genus (type cricket paralysis virus) in the Dicistroviridae family. This work describes the purification of TrV particles from infected T. infestans and their crystallization and preliminary crystallographic analyses. Two different crystal forms, rhombohedral and orthorhombic, were obtained at room temperature by the hanging-drop vapour-diffusion technique using polyethylene glycol and polyethylene glycol monomethylether as precipitants. The rhombohedral crystals have unit-cell parameters a = b = 306.6, c = 788.4, (hexagonal setting), diffract to 3.2, resolution and contain one-third of the viral particle per asymmetric unit. The orthorhombic crystals have cell parameters a = 336, b = 351, c = 332,, diffract to about 2.5, resolution, and contain one-half of a virus particle in the asymmetric unit. A complete diffraction data set has been collected to 3.2, resolution, using synchrotron radiation, from a single rhombohedral crystal under cryogenic conditions. [source]

Expression, purification, crystallization and preliminary crystallographic studies of the Enterococcus faecalis cytolysin repressor CylR2

Adelia Razeto
The expression of an exotoxin called cytolysin contributes to the virulence of Enterococcus faecalis, one of the organisms responsible for antibiotic resistant infections acquired in hospitals. The DNA-binding protein CylR2 is a transcriptional repressor of cytolysin. At a specific cell density, cytolysin triggers signaling events, which result in the dissociation of CylR2 from its DNA-binding site. CylR2 was overexpressed in Escherichia coli and purified and crystals diffracting to 1.9, were obtained in two different crystal forms. One crystal form belongs to space group P41, with unit-cell parameters a = 63.7, b = 63.7, c = 41.2,, , = , = , = 90, and the other belongs to space group P1, with unit-cell parameters a = 36.9, b = 45.0, c = 47.7,, , = 67, , = 90, , = 66. [source]

Crystallization of a carbamatase catalytic antibody Fab fragment and its complex with a transition-state analogue

Carbamatase catalytic antibody Fab fragment
Catalytic antibodies showing carbamatase activity have significant potential in antibody-directed prodrug therapy against tumours. The Fab fragment of an IgG1 mouse monoclonal carbamatase catalytic antibody JC1 raised against a transition-state analogue, ethyl N -(3,5-dicarboxyphenyl)- P -{N- [5,-(2,,,5,,-dioxo-1,,-pyrrolidinyl)oxy-1,,5,-dioxopentyl]-4-aminophenylmethyl}phosphonamidate, was obtained by digestion of the whole antibody with papain and was purified by two-step ion-exchange chromatography. Using hanging-drop vapour-diffusion crystallization techniques, three different crystal forms of the Fab fragment were obtained in the presence and absence of the transition-state analogue. All crystals diffract X-rays to between 3.5 and 3.2, resolution. The two crystal forms grown in the presence of the transition-state analogue contain up to four or eight copies of the Fab in the asymmetric unit and diffract to 3.5 and 3.2,, respectively. The crystal of the Fab alone is most likely to contain only two copies of the Fab in the asymmetric unit and diffracts to beyond 3.5,. Determination of the structure will provide insights into the active-site arrangement of this antibody and will help to increase our understanding of the molecular mechanisms by which the immune system can evolve catalytic function. [source]

A genetic algorithm for the identification of conformationally invariant regions in protein molecules

Thomas R. Schneider
Understanding macromolecular function often relies on the comparison of different structural models of a molecule. In such a comparative analysis, the identification of the part of the molecule that is conformationally invariant with respect to a set of conformers is a critical step, as the corresponding subset of atoms constitutes the reference for subsequent analysis for example by least-squares superposition. A method is presented that categorizes atoms in a molecule as either conformationally invariant or flexible by automatic analysis of an ensemble of conformers (e.g. crystal structures from different crystal forms or molecules related by non-crystallographic symmetry). Different levels of coordinate precision, both for different models and for individual atoms, are taken explicitly into account via a modified form of Cruickshank's DPI [Cruickshank (1999), Acta Cryst. D55, 583,601] and are propagated into error-scaled difference distance matrices [Schneider (2000), Acta Cryst. D56, 715,721]. All pairwise error-scaled difference distance matrices are then analysed simultaneously using a genetic algorithm. The algorithm has been tested on several well known examples and has been found to converge rapidly to reasonable results using a standard set of parameters. In addition to the description of the algorithm, a criterion is suggested for testing the identity of two three-dimensional models within experimental error without any explicit superposition. [source]

Crystallization and preliminary X-ray analysis of a thermoalkalophilic lipase from Bacillus stearothermophilus L1

Seong-Tae Jeong
A thermoalkalophilic lipase from Bacillus stearothermophilus L1 (L1 lipase) was crystallized in two different crystal forms using a low concentration of the enzyme and a calcium-exchange process. The first, needle-like, crystal form, which diffracts to about 3.5,, belongs to the orthorhombic space group P212121, with unit-cell parameters a = 67.84, b = 72.96, c = 104.41,. The second, monoclinic, crystal form, which behaves better than the first form for crystallographic analyses, belongs to the monoclinic space group C2 and has unit-cell parameters a = 119.62, b = 85.05, c = 98.36,, , = 99.73. From the monoclinic crystals, a native data set and a samarium-derivative data set were collected to 2.0 and 2.3, resolution, respectively. The difference Patterson map between the two data sets shows strong heavy-atom peaks, indicating that the crystals are suitable for a high-resolution structure determination. [source]

Structural effects of monovalent anions on polymorphic lysozyme crystals

M. C. Vaney
Understanding direct salt effects on protein crystal polymorphism is addressed by comparing different crystal forms (triclinic, monoclinic, tetragonal and orthorhombic) for hen, turkey, bob white quail and human lysozymes. Four new structures of hen egg-white lysozyme are reported: crystals grown in the presence of NapTS diffracted to 1.85,, of NaI to 1.6,, of NaNO3 to 1.45, and of KSCN to 1.63,. These new structures are compared with previously published structures in order to draw a mapping of the surface of different lysozymes interacting with monovalent anions, such as nitrate, chloride, iodide, bromide and thiocyanate. An analysis of the structural sites of these anions in the various lysozyme structures is presented. This study shows common anion sites whatever the crystal form and the chemical nature of anions, while others seem specific to a given geometry and a particular charge environment induced by the crystal packing. [source]

Structure of a new `aspzincin' metalloendopeptidase from Grifola frondosa: implications for the catalytic mechanism and substrate specificity based on several different crystal forms

Tetsuya Hori
Crystal structures of a peptidyl-Lys metalloendopeptidase (MEP) from the edible mushroom Grifola frondosa (GfMEP) were solved in four crystal forms. This represents the first structure of the new family `aspzincins' with a novel active-site architecture. The active site is composed of two helices and a loop region and includes the HExxH and GTxDxxYG motifs conserved among aspzincins. His117, His121 and Asp130 coordinate to the catalytic zinc ligands. An electrostatically negative region composed of Asp154 and Glu157 attracts a positively charged Lys side chain of a substrate in a specific manner. A Tyr133 side chain located on the S1, pocket had different configurations in two crystal forms and was not observed in the other crystal forms. The flexible Tyr133 plays two roles in the enzymatic function of GfMEP. The first is to provide a hydrophobic environment with Phe83 in order to accommodate the alkyl part of the Lys side chain of a substrate and the second is as a `proton donor' to the oxyanion of the tetrahedral transition state to stabilize the reaction transition state. [source]

Crystallization of agGST1-6, a recombinant glutathione S -transferase from a DDT-resistant strain of Anopheles gambiae

Pamela H. Roberts
Glutathione S -transferases (GSTs) belong to a family of detoxification enzymes that conjugate glutathione to various xenobiotics, thus facilitating their expulsion from the cell. GST activity is elevated in many insecticide-resistant insects, including the DDT-resistant malaria vector Anopheles gambiae. Crystals of the recombinant form of a GST from A. gambiae, agGST1-6, have been grown in at least five different crystal forms, with a broad range of diffraction resolution limits. A complete 2.0, data set has been collected on a C -centered orthorhombic crystal form with unit-cell parameters a = 99.0, b = 199.4, c = 89.6,. A search for heavy-atom derivatives has been initiated, along with phase-determination efforts by molecular replacement. [source]

Structural studies of the catalytic core of the primate foamy virus (PFV-1) integrase

Stphane Rty
Retroviral integrases are vital enzymes in the viral life cycle and thus are important targets for antiretroviral drugs. The structure of the catalytic core domain of the integrase from human foamy virus, which is related to HIV-1, has been solved. The structure of the protein is presented in two different crystal forms, each containing several molecules in the asymmetric unit, with and without the essential manganese or magnesium ion, and the structures are compared in detail. This allows regions of high structural variability to be pinpointed, as well as the effect of divalent cations on the conformation of the catalytic site. [source]

Crystallization and initial X-ray diffraction studies of the flavoenzyme NAD(P)H:(acceptor) oxidoreductase (FerB) from the soil bacterium Paracoccus denitrificans

The flavin-dependent enzyme FerB from Paracoccus denitrificans reduces a broad range of compounds, including ferric complexes, chromate and most notably quinones, at the expense of the reduced nicotinamide adenine dinucleotide cofactors NADH or NADPH. Recombinant unmodified and SeMet-substituted FerB were crystallized under similar conditions by the hanging-drop vapour-diffusion method with microseeding using PEG 4000 as the precipitant. FerB crystallized in several different crystal forms, some of which diffracted to approximately 1.8, resolution. The crystals of native FerB belonged to space group P21, with unit-cell parameters a = 61.6, b = 110.1, c = 65.2,, , = 118.2 and four protein molecules in the asymmetric unit, whilst the SeMet-substituted form crystallized in space group P21212, with unit-cell parameters a = 61.2, b = 89.2, c = 71.5, and two protein molecules in the asymmetric unit. Structure determination by the three-wavelength MAD/MRSAD method is now in progress. [source]

X-ray investigation of gene-engineered human insulin crystallized from a solution containing polysialic acid

V. I. Timofeev
Attempts to crystallize the noncovalent complex of recombinant human insulin with polysialic acid were carried out under normal and microgravity conditions. Both crystal types belonged to the same space group, I213, with unit-cell parameters a = b = c = 77.365,, , = , = , = 90.00. The reported space group and unit-cell parameters are almost identical to those of cubic insulin reported in the PDB. The results of X-ray studies confirmed that the crystals obtained were cubic insulin crystals and that they contained no polysialic acid or its fragments. Electron-density maps were calculated using X-ray diffraction sets from earth-grown and microgravity-grown crystals and the three-dimensional structure of the insulin molecule was determined and refined. The conformation and secondary-structural elements of the insulin molecule in different crystal forms were compared. [source]

Crystallization and preliminary crystallographic analysis of eukaryotic transcription and mRNA export factor Iws1 from Encephalitozoon cuniculi

Michael Koch
Transcription elongation by eukaryotic RNA polymerase II requires the coupling of mRNA synthesis and mRNA processing and export. The essential protein Iws1 is at the interface of these processes through its interaction with histone chaperone and elongation factor Spt6 as well as with complexes involved in mRNA processing and export. Upon crystallization of the evolutionarily conserved domain of Iws1 from Encephalitozoon cuniculi, four different crystal forms were obtained. Three of the crystal forms belonged to space group P21 and one belonged to space group P2221. Preliminary X-ray crystallographic analysis of one of the crystal forms allowed the collection of data to 2.5, resolution. [source]