ALBERT

Notes: A knowledge-based approach to crystal structure determination is presented. The approach integrates direct-methods and artificial-intelligence strategies to rephrase the structure determination process as an exercise in scene analysis. A general joint probability distribution framework, which allows the incorporation of isomorphous replacement, anomalous scattering and a priori structural information, forms the basis of the direct-methods strategies. The accumulated knowledge on crystal and molecular structures is exploited through the use of artificial-intelligence strategies, which include techniques of knowledge representation, search and machine learning.

Notes: The OP-G2 monoclonal antibody binds to the platelet integrin, gpIIb/IIIa, in a mode that mimics fibrinogen binding. The specificity of this antibody is mediated by the third complementarity-determining region (CDR3) loop of the immunoglobulin heavy chain which contains a sequence (RYD) related to the RGD recognition sequence of fibrinogen. The OP-G2 Fab fragment has been crystallized by vapor diffusion from solutions containing polyethylene glycol and imidazole malate (pH 5.6). The crystals belong to space group P21212 with a = 93.1, b = 83.8 and c = 53.7 Å. One Fab molecule is present in the asymmetric unit. A complete data set to 2.0 Å resolution has been collected.

Notes: The entropy-dynamics method seeks maxima for the entropy of the electron density for N atoms in a crystal cell, when the Fourier amplitudes are fixed, but their phases are unknown. By analogy with molecular dynamics, the effective potential energy is the negative entropy V = −NS. The kinetic energy is proportional to the squared velocities of the electron densities at grid points in the map. It reduces to a sum of Fourier-mode rotor energies. Each rotor angle experiences a couple equal to the phase gradient of S, and local dynamical equilibrium yields a Boltzmann distribution of S. Discrete phase angles (e.g. signs) are treated as quantized rotor modes. The distributions depend on a popularity function of the entropy histogram. Trial calculations have been made of phase averages and correlations in a centrosymmetric projection of the membrane protein bacteriorhodopsin. The maximum-entropy solution and the correct solution do not always coincide.

Notes: The structure of aldose reductase, a monomeric enzyme of 314 amino acids which crystallizes in space group P1 with four monomers per asymmetric unit, has been solved using a combination of single isomorphous replacement (SIR), solvent flattening and local symmetry averaging. The self rotation showed evidence of 222 local symmetry. The map calculated from the original single isomorphous replacement phases showed a clear solvent envelope but was uninterpretable. A first averaging attempt failed because the molecular envelope obtained from the SIR map weighted with monomer correlation was too small and the averaging was biased by low-resolution truncation. A second attempt with an enlarged envelope and including low-resolution reflections succeeded in refining phases at 3.5 Å resolution but failed to extend them correctly. Rigid-body refinement of a partial model based on the 3.5 Å map calculated from refined phases showed significant departures from the 222 symmetry. A third averaging attempt using the improved symmetry succeeded in producing a clear map with phases extended to 3.07 Å resolution. This map revealed a (β/α)8 fold, not previously found in NADPH-dependent enzymes. This work shows the importance of mask definition and local symmetry elements accuracy for averaging, and describes a method for improving these parameters.

Notes: Ferredoxin I from Azotobacter vinelandii (AvFdI) is an iron–sulfur protein composed of 106 amino acids, seven Fe atoms and eight inorganic S* atoms. A crystallographic redetermination of its structure showed the originally reported structure to be incorrect. We report here the crystal structure of AvFdI at pH 6.5. Extensive refinement has led to a final R value of 0.170 for all 6986 non-extinct reflections in the range 10–2.3 Å using a solvent model which includes 98 discrete solvent atoms with occupancies between 0.3 and 1.0 and an average B value of 22.5 Å2. The first half of the peptide chain closely resembles that of the 55-residue ferredoxin from Peptococcus aerogenes (PaFd), while the remainder consists of three turns of helix and a series of loops which form a cap over part of the molecular core. Despite the similarities in structure and surroundings, the corresponding 4Fe4S* clusters in PaFd and AvFdI have strikingly different redox potentials; a possible explanation has been sought in the differing hydration models for the two molecules.

Notes: The molecular structure of an iron-containing 18 kDa fragment of duck ovotransferrin, obtained by proteolysis of the intact protein, has been elucidated by protein crystallographic techniques at 2.3 Å resolution. This structure supports a mechanism of iron uptake in the intact protein whereby the binding of the synergistic (bi)carbonate anion is followed by binding of the metal with the lobe in the open configuration. These stages are then followed by domain closure in which the aspartic acid residue plays a further key role, by forming an interdomain hydrogen-bond interaction in addition to serving as a ligand to the iron. This essential dual role is highlighted by model building studies on the C-terminal lobe of a known human variant. In this variant a mutation of a glycine by an arginine residue enables the aspartic acid to form an ion pair and reduce its effectiveness for both metal binding and domain closure. The X-ray structure of the 18 kDa fragment strongly suggests that the histidine residue present at the iron binding site of the intact protein and arising from the second interdomain connecting strand has been removed during the preparative proteolysis.

Notes: The crystal structure of the DNA hexamer d(TGATCA) complexed with the anthracycline antibiotic idarubicin has been determined at 1.6 Å resolution. The asymmetric unit consists of a single hexamer oligonucleotide strand, one drug molecule and 35 water molecules. The complex crystallizes in the tetragonal space group P41212, Z = 8 with lattice dimensions of a = b = 28.19 (3), c = 52.77 (4) Å, V = 41 935 A3. The structure is isomorphous with a series of hexamer–anthracycline complexes and was solved by molecular replacement. Restrained least-squares methods interspersed with computer-graphics map inspection and model manipulation were used to refine the structure. The R factor is 0.22 for 2032 reflections with F ≥ 3σ(F) in the resolution range 8.0–1.6 Å. The self-complementary DNA forms a distorted B-DNA double helix with two idarubicin molecules intercalated in the d(TpG) steps of the duplex. The duplex is formed by utilization of a crystallographic twofold axis of symmetry. The idarubicin chromophore is oriented at right angles to the long axis of the DNA base pairs with the anthracycline amino-sugar moiety positioned in the minor groove. Our structure determination allows for comparison with a d(CGATCG)–idarubicin complex recently reported. Despite the sequence alteration at the intercalation step, the structures are very similar. The geometry of the intercalation and the nature of the interactions are conserved irrespective of the DNA sequence involved in the binding.

Notes: The three-dimensional crystal structure of recombinant bovine interferon-γ was determined using the multiple isomorphous replacement method at 3.0 Å and refined to an R factor of 19.2%. This protein crystallizes in space group P212121 with unit-cell parameters of a = 42.8, b = 79.9 and c = 85.4 Å. There is one functional dimer in the asymmetric unit. The two polypeptide chains are related by a non-crystallographic twofold symmetry axis. The secondary structure is predominantly α-helical with extensive interdigitation of the α-helical segments of the polypeptide chains that make up the dimer. The secondary structure, tertiary structure and topology of this molecule are identical to the previously reported structures of recombinant rabbit interferon-γ and recombinant human interferon-γ. The molecular topology is also similar to that of murine interferon-β. These structural similarities strongly indicate the presence of a unique topological feature (fold) among γ-interferons from different species, and also among the different classes of interferons.

Notes: A practical generally applicable procedure for exponential modeling to maximum likelihood of macromolecular data sets constrained by a moderately large basis set of reliable phases and a molecular envelope is described, based on the computer program MICE [Bricogne & Gilmore (1990). Acta Cryst. A46, 284–297]. Procedures were first tested with simulated data sets. Exact and randomly perturbed amplitudes and phases were generated, together with a known envelope for solvent-free protein and for protein in an electron-dense crystal mother liquor typical of many real protein crystals. These experiments established useful guidelines and values for various parameters. Tests with basis sets chosen from the largest amplitudes indicate that exponential models with considerable correct extrapolated phase and amplitude information can be constructed from as few as 16% of the total number of reflections, with mean phase errors of about 30°, at resolution limits of either 5 or 3 Å. When the shape of the solvent channels in macromolecular crystals is known, it offers an important additional source of information. MICE was, therefore, adapted to average the density outside the molecular boundary defined by an input envelope. This flattening process imposes a uniform density distribution in solvent-filled channels as an additional constraint on the exponential model and is analogous to the treatment of solvent in conventional solvent flattening. Experimental data for cytidine deaminase, a structure recently solved by making extensive use of conventional solvent flattening, provides an example of the performance of maximum-entropy methods in a real situation and a compelling comparison of this method to standard procedures. Exponential models of the electron density constrained by the most reliable phases obtained by multiple isomorphous replacement with anomalous scattering (MIRAS) (figure of merit 〉 0.7, representing 34% of the total number of reflections) and by the envelope give rise to centroid electron-density maps which are quantitatively superior by numerous statistical criteria to conventionally solvent-flattened density. Similarity of these maps to the 2Fobs − Fcalc map calculated with phases obtained after crystallographic refinement of the model implies that maximum-entropy extrapolation provides better phases for the remaining 66% of the reflections than the original centroid MIRAS distributions. Importantly, the solvent-flattened electron density, although it did permit interpretation of the map which was not readily accomplished with the MIRAS map, contains substantial errors. It is proposed that errors of this sort may account for previously noted deficiencies of the solvent-flattening method [Fenderson, Herriott & Adman (1990). J. Appl. Cryst. 23, 115–131] and for the occasional tendency of incorrect interpretations to be `locked in' by crystallographic refinement [Brändén & Jones (1990). Nature (London), 343, 687–689, and references cited therein]. Solvent flattening with combined maximization of entropy and likelihood represents a phase-refinement path independent of atomic models, using the experimental amplitudes and the most reliable phases. It should, therefore, become a valuable and generally useful procedure in macromolecular crystal structure determination.

Notes: The crystal structure of a complex of ribonuclease from Streptomyces aureofaciens (RNase Sa) with guanosine-2′-monophosphate (2′-GMP) has been refined against synchrotron data recorded from a single crystal using radiation from beamline X31 at EMBL, Hamburg, and an imaging plate scanner. The crystals are in space group P212121 with cell dimensions a = 64.7, b = 78.8 and c = 39.1 Å. The structure has two enzyme molecules in the asymmetric unit, complexed with 2′-GMP inhibitor with occupancies of 1 and {2 \over 3} (different to the 3′-GMP complex crystal structure where only one of the two independent RNase Sa molecules binds nucleotide), 492 associated water molecules and one sulfate ion, and was refined using all data between 10.0 and 1.7 Å to a final crystallographic R factor of 13.25%. Binding of the base to the enzyme confirms the basis for the guanine specificity but the structural results still do not provide direct evidence of the identity and role of the particular residues involved in the catalytic process. New native RNase Sa data to 1.8 Å were recorded to provide a reference set measured under comparable experimental conditions. The crystals are in the same space group and have the same lattice as those of the 2′-GMP complex. The native structure with 423 water molecules was refined in a similar manner to the complex to a final R factor of 13.87%. 1.77 Å resolution data were independently measured on a 2′-GMP complex crystal at UCLA using an R-AXIS II image plate scanner mounted on a conventional source. The cell dimensions were essentially the same as above. 2′-GMP was bound more fully to molecule A than to molecule B of the RNase Sa. The structure was refined to an R factor of 14.64% with 388 water molecules. This work follows on from the structure determination of native RNase Sa and its complex with 3′-GMP [Sevcik, Dodson & Dodson (1991). Acta Cryst. B47, 240–253].

Notes: The hexagonal crystal form of the octamer d(GTGTACAC), grown in the presence of spermine, has unit-cell dimensions a = b = 32.18 and c = 78.51 Å, space group P6122, with one DNA strand in the asymmetric unit. The structure has been refined starting with the earlier lower resolution model and using high-resolution 1.4 Å data collected on a Siemens-Xentronics area detector at 258 K. There were 4365 unique reflections greater than 2σ(F) in the resolution range 5–1.4 Å. The model was refitted into 3Fo − 2Fc. Sim-weighted omit maps and difference maps were used to locate water molecules. The final model with 161 DNA atoms and 37 water molecules gave an R factor of 19.8%. Crystals of the same octamer were also grown in the presence of spermidine instead of spermine, and refinement using nominal 1.45 Å resolution data, 3292 unique reflections, final R = 19.1%, gave virtually identical DNA parameters. No bound spermine or spermidine was detected in either of these structure analyses. The electron density was clear for the DNA and showed holes in the center of the six-membered rings of bases, and also in the center of some of the sugar rings. The high-resolution structure has provided more precise DNA parameters and confirmed the features observed in the earlier 2 Å study including the packing-induced distortion in the A7 (A15) sugar pucker from C(3′)-endo and C(2′)-endo. This change causes the end base pairs to bend away from the helix axis while the rest of the duplex is nearly linear. The hydration patterns in the deep and shallow grooves have been characterized. Chains of water molecules were found, but no rings. The familiar intermolecular contact region between the end base pair and the minor groove of a symmetry-related duplex, involving four residues on one strand and two on the other, has been analyzed. One of these interactions is a hydrogen bond.

Notes: The white-beam Laue-diffraction method is a useful tool for rapid measurement of crystallographic intensities with synchrotron radiation. Considerations of the signal-to-noise ratio to be expected from scattering of X-rays within a limited wavelength range suggest that it will pay to limit that range to something like an octave. This rule-of-thumb has the added advantage that there will be significantly fewer diffraction spots that are overlapping harmonics of one another. To maximize the number of reflections recorded in a single stationary-crystal exposure, one should choose this octave of wavelengths in a region where the curvature of the Ewald sphere is greatest, that is at the longest wavelength allowable after other considerations are taken into account.

Notes: The development of high-intensity X-ray sources and the use of insertion devices will make it possible to collect data routinely from protein crystals at very short wavelengths (λ ≤ 0.5 Å). Possible benefits of using shorter wavelengths can be inferred from the improvement in the quality of the data when using a wavelength λ ∼ 0.9 Å instead of one close to the Cu Kα emission edge. In addition to fewer absorption errors, two factors might contribute to this improvement. These are an increase in the lifetime of the protein crystal and a better signal-to-background ratio. In this paper we address the second of these. In order to compare the quality of the data and the relative background level in the diffraction patterns at different wavelengths two data sets have been collected at λ = 0.92 and 0.55 Å. The results obtained from data processing and careful measurement of the background in the raw images suggest that, in the absence of absorption errors and radiation damage, data collection at very short wavelengths does not provide higher quality data. There is no improvement in the signal-to-background ratio in the short-wavelength data.

Notes: The crystal structure of metmyoglobin from yellowfin tuna (Thunnus albacares) has been determined by molecular replacement methods and refined to a conventional R factor of 0.177 for all observed reflections in the range of 6.0–1.70 Å resolution. Like other myoglobins for which a high-resolution structure is available, the polypeptide chain is organized into several helices that cooperate to form a hydrophobic pocket into which the heme prosthetic group is non-covalently bound; however, the D helix observed in other myoglobins is absent in myoglobin from yellowfin tuna and has been replaced with a random coil. As well, the A helix has a pronounced kink due to the presence of Pro16. The differences in structure between this and sperm whale myoglobin can be correlated with their reported dioxygen affinity and dissociation. The structure is in agreement with reported fluorescence data which show an increased Trp14...heme distance in yellowfin tuna compared to sperm whale myoglobin.

Notes: High-resolution single crystals of a catalytic RNA molecule derived from the sequence of the satellite RNA of tobacco ringspot virus have been obtained. The unit-cell volumes of the RNA crystals vary depending on the crystallization conditions and temperature. The best crystal form, when flash frozen, has space group P1 with unit-cell dimensions a = 53.08, b = 71.81, c = 28.03 Å, α = 98.43, β = 104.32 and γ = 74.54°. This form diffracts to a resolution of 2.4 Å. A heavy-atom derivative search is in progress.

Notes: The automated microbatch technique developed at Imperial College has been used to establish a phase diagram for crystallization. The concentrations of the protein (carboxypeptidase G2) and precipitant (PEG 4000) were varied, while pH and temperature were kept constant. The diagram consists of an undersaturation and a supersaturation zone, the latter being subdivided into the metastable, nucleation and precipitation zones. In the metastable zone, crystals may grow but nucleation of crystals does not occur. It is the best zone for growth of X-ray diffraction quality crystals because of the slower growth rate and the avoidance of uncontrolled nucleation, which uses up protein in the formation of tiny crystals. Nevertheless, in practice, it is rarely well defined or used because nuclei must be introduced artificially into the system. The new method used here consists of setting crystallization droplets at nucleation conditions and later diluting them to conditions where nucleation has not been observed. Single diffracting crystals of typical dimensions 0.3 × 0.3 × 0.2 mm were routinely obtained in the metastable zone, equivalent to the best (very rarely) obtained crystals in the nucleation zone.

Notes: The crystallization of a variant of Bacillus lentus subtilisin and the native enzyme was achieved using identical conditions. The variant B. lentus was found to grow in two crystal forms, form 1 and form 2, whereas the native B. lentus subtilisin enzyme crystallized in only one, form 1. Form 2 crystals, once obtained, were found to grow much more rapidly than form 1 crystals. The lattice contacts and structural changes giving both crystal forms have been examined. The results show that crystal form 2 has a more complex network of interactions. There is also a small surface conformational change in the form 2 structure relative to the native and variant form 1 crystals and at least two solvent molecules bound to the enzyme in crystal form 1 are displaced in crystal form 2. In addition, a site specific substitution in the variant at position 27 induces a `short' lattice contact which does not exist in the native B. lentus or the form 2 variant B. lentus. These results suggest that in some circumstances engineered variants could be designed to crystallize more rapidly than the native enzyme.

Notes: X-ray or neutron diffraction studies have shown, at the atomic level, that water molecules occupy well determined sites inside or at the surface of biological macromolecules. These water molecules are constitutive of biomolecules and play a crucial role in their structural and functional properties. Upon crystallization some water molecules are either desolvated or involved in crystal packing. The role of water in determining crystal packing has been experimentally confirmed by several X-ray analyses.

Notes: Large single crystals of the dodecylmaltoside (DDM) complex of a polytopic integral membrane transport protein, the Neurospora plasma membrane H+-ATPase, have been obtained using an approach that attempts to take into account the possibly radically different physicochemical properties of the protein surfaces and the detergent micellar collar. The overall goal of the crystallization strategy employed was to identify conditions in which the protein surfaces of the DDM–ATPase complex are moderately insoluble and in which the DDM micellar collar is also near its solubility limit. The first step was to screen a variety of commonly used protein precipitants for those that were able to induce the aggregation of pure DDM micelles. The concentration at which any precipitant induced DDM micellar aggregation was hoped to be close to the concentration at which it might induce insolubility of the detergent micellar collar of the DDM–ATPase complex. Of the nine precipitants tried, seven, all polyethylene glycols (PEGs), were able to induce DDM micelle insolubility. The seven PEGs were then tested for their effect on the solubility of the DDM–ATPase complex at a concentration slightly below that necessary to induce DDM micellar aggregation. Three of the PEGs caused extensive precipitation of the ATPase at this concentration and were, therefore, shelved. The other four PEGs did not induce precipitation at the concentration employed and were subsequently used at this concentration for crystallization trials in which the protein concentration was varied. Encouragingly, crystalline plates of the ATPase were obtained for each of the four PEGs tried, indicating that the overall approach may be valid. Unfortunately, the crystals obtained were visibly flawed, suggesting that the correct balance of protein surface and DDM micelle insolubility had not yet been reached. The ionic strength of the crystallization trials was then raised, which was known from other experiments to render the protein surfaces of the ATPase less soluble while having no effect on the DDM micellar aggregation point. For one of the PEGs, PEG 4000, this brought on a new, well formed hexagonal crystal habit. Subsequent optimization of the initial conditions has yielded large single hexagonal crystals of the H+-ATPase roughly 0.4 × 0.4 × 0.15 mm in size, holding promise for exploration of the structure of the ATPase by X-ray diffraction analysis.

Notes: Protein crystal growth often depends on the combination of many different factors. Some affect protein solubility directly; others may act indirectly by causing conformational changes. Systematic characterization of these factors can be important for generating good crystals. It can also provide useful insight into the biochemical behavior of the protein being crystallized. Here we focus on statistical methods to achieve these two objectives. (1) Characterization of a protein system by analyzing patterns of crystal polymorphism under different levels of biochemical parameters, such as ligands and pH. Tests of the reproducibility of crystal growth experiments indicate that quantitative scales of crystal quality can be statistically significant. Analysis of variance for a replicated, full-factorial design in which four factors were tested at two levels has been used to demonstrate highly significant, biochemically relevant, two-factor interactions strongly implicating pH and ligand-dependent conformational changes. (2) Optimization of crystal growth via response-surface methods. `Minimum predicted variance' designs provide for efficient response-surface experiments aimed at constructing quadratic models in several dimensions. We have used such models to improve crystal size and quality significantly for three forms of Bacillus stearothermophilus tryptophanyl-tRNA synthetase. In one case we can now avoid having to increase the size by repeated seeding, a difficult procedure that also produces unwanted growth of satellite crystals. Graphs of two-dimensional level surfaces reveal a number of ridges, where the same result is obtained for many combinations of the factors usually varied when trying to improve crystals. An important inference is that it may be better to sample simultaneously for the effects of protein concentration and supersaturation. For a system involving only one crystallizing agent, supersaturation can be approximated as the product of protein and precipitant concentrations. Use of this search direction significantly improves the performance of response-surface experiments. Advantages of growing crystals at stationary points of their response surfaces include better crystals and higher reproducibility, since crystal growth at stationary points is insulated from the deleterious effects of experimental fluctuations. This arises because the derivatives of the response are by definition zero with respect to the experimental variables. Quantitative analysis of appropriately designed crystal growth experiments can thus be a powerful way to characterize complex and interacting biochemical dependencies in macromolecular systems and optimize parameters important to the crystallography.

Notes: The Escherichia coli molecular chaperone cpn60 oligomer, [cpn60]14, also called GroEL, has been crystallized and examined by X-ray crystallography and self-rotation function calculations. The crystals show unit-cell dimensions a = 143.3, b = 154.6 and c = 265 Å, with α = 82, β = 95 and γ = 107°. The space group is P1 and crystals diffract to 7 Å. X-ray analysis shows that the oligomer has one sevenfold symmetry axis and seven twofold axes that are all perpendicular to the sevenfold. The symmetry suggests that [cpn60]24 consists of two heptamers, [cpn60]7, stacked on top of each other. The orientations of the symmetry axes of the two independent [cpn60]14 oligomers in the triclinic unit cell have been determined relative to the crystallographic axes. The two oligomers in the unit cell are arranged side-by- side, but the second oligomer is rotated 26° around the sevenfold axis relative to the first oligomer.

Notes: The crystals of most proteins are poorly ordered and diffract to lower resolutions than other crystals of simple and inorganic compounds. The use of two novel methods for gel protein crystal growth, utilizing liquid diffusion and vapor diffusion, are described for the growth of lysozyme and canavalin. Crystallization using gels has been demonstrated to improve crystal quality by reducing convective flow, sedimentation, nucleation and twinning. Preliminary X-ray diffraction data are also presented.

Notes: A procedure which allows an investigator to supply a crystal with fresh mother material without inducing significant growth defects is described. This technique requires that the crystal is grown in a gelled hanging or sitting drop. An example concerning a model macromolecule, hen egg-white lysozyme, is given. Extension of this procedure to other macromolecules is discussed.

Notes: Two populations of aggregates are generally indentified in supersaturated solutions of biological macromolecules: small aggregates of a size which is less than 5 nm and large aggregates, the largest of which are at least one order of magnitude bigger. In order to understand the role played by the microporous network of a gel in the growth and behaviour of these different species in the prenucleation period, an in situ observation of nucleation has been carried out using either free solutions or solutions trapped in agarose gels. In a previous study, free solutions were investigated by small-angle neutron scattering (SANS) to identify the small aggregates. Optical observations, made under the same conditions, revealed the formation of an amorphous precipitate which disappeared at the end of the experiment. The sedimentation of this phase, which occurs in free solution but never occurs in gelled solution, depletes the solution bulk and this could explain why the nucleation density is higher in agarose gel than in free solution. The case of silica gel, the behaviour of which is completely different with respect to nucleation, will be discussed.

Notes: Crystals of the binary complex of alcohol dehydrogenase from Sulfolobus solfataricus with NADH were shown to be twinned and not suitable for automated data collection. Several crystallization trials, performed with the aim of eliminating twinning, are described. Interestingly, crystals grown from agarose gel have been demonstrated to have a unique reciprocal lattice. These crystals are monoclinic, space group C2, with cell dimensions a = 134.47 (9), b = 85.26 (5), c = 71.76 (8) Å, β = 97.53 (4)°, and showed significant diffraction beyond 3.0 Å resolution.

Notes: The parameters affecting the crystal quality of complexes between p21H-ras and caged GTP have been investigated. The use of pure diastereomers of caged GTP complexed to the more stable p21(G12P)′ mutant of p21 and the addition of n-octyl-β-D-glucopyranoside improved the reproducibility and decreased the mosaicity of the crystals significantly. Furthermore, the crystallization technique was changed from the batch method to the sitting-drop technique. With the availability of a larger yield of well ordered crystals, it was possible to extend the time-resolved crystallographic investigations on p21H-ras. A structure of p21(G12P)′:GTP could be obtained 2 min after photolytic removal of the cage group and led to the identification of a previously unidentified conformation for the so-called catalytically active loop L4. The refinement of five data sets collected within 2 min at different times (2–4, 11–13, 20–22, 30–32 and 90–92 min) after the initiation of the intrinsic GTPase reaction of the protein indicates that the synchrotron Laue method can be used to detect small structural changes and alternative conformations, but is presently limited in the analysis of larger rearrangements since these produce diffuse and broken electron density.

Notes: The tetrameric flavoenzyme 2,4-pentadienoyl-CoA reductase has been crystallized from solutions containing polyethylene glycol as precipitant. The crystals grow in the monoclinic space group C2 with unit-cell dimensions a = 160.2, b = 120.2, c = 95.3 Å, β = 99.0°. The packing parameter VM is 2.3 Å3 Da−1 (Matthews parameter) for four monomers per asymmetric unit. Complete data sets to about 2.9 Å resolution have been collected.

Notes: Ribosome-inactivating protein from barley seeds has been crystallized using polyethylene glycol as precipitant. The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 88.36, b = 62.59, c = 53.18 Å and β = 108.62°. The asymmetric unit contains one molecule of ribosome-inactivating protein with a corresponding crystal volume per protein mass (Vm) of 2.32 Å3 Da−1 and a solvent content of 47% by volume. The crystal diffracts to about 2.3 Å with X-rays from a rotating-anode source and is very stable in the X-ray beam. X-ray data (nearly complete to 2.4 Å Bragg spacing) have been collected from a native crystal.

Notes: A platinum chromophore, chloro(2,2′:6′,2′′-terpyridine)platinum(II) chloride, previously used in labelling active-site histidines of serine proteases, proves to be a useful reagent in heavy-atom derivatization of protein crystals for X-ray crystallographic phase determination.

Notes: Galactose-1-phosphate uridylyltransferase catalyzes the formation of UDP-galactose during normal cellular metabolism, making it an essential enzyme in all cells. The enzyme from Escherichia coli has been crystallized at pH 5.9 in the presence of phenyl-UDP (P1-5′-uridyl-P2-phenyl diphosphate), a substrate analog, using PEG 10 000 in combination with Li2SO4 and NaCl. Crystals belong to space group P21212 with unit-cell dimensions a = 58.6, b = 217.6 and c = 69.6 Å. There is one dimer or two subunits in the asymmetric unit. Crystals are relatively insensitive to X-ray radiation and diffract beyond 2.5 Å resolution. A low-resolution native data set has been recorded.

Notes: The three-dimensional structure of human dicupric monooxalate lactoferrin, Cu2oxLf, has been determined to 2.0 Å resolution, using X-ray diffraction data collected by diffractometry to 2.5 Å resolution, and oscillation photography on a synchrotron source to 2.0 Å resolution. Difference electron-density maps calculated between Cu2oxLf and both dicupric lactoferrin, Cu2Lf, and diferric lactoferrin, Fe2Lf, showed that the oxalate had replaced a carbonate in the C-terminal binding site, and that, relative to Cu2Lf, there were no significant differences in the N-terminal site. The structure was then refined crystallographically by restrained least-squares methods. The final model, in which the r.m.s. deviation in bond distances is 0.017 Å, contains 5314 protein atoms (691 residues), two Cu2+ ions, one bicarbonate ion, one oxalate ion, 325 solvent molecules and one sugar residue. The crystallographic R factor of 0.193 is for 46 134 reflections in the range 8.0 to 2.0 Å resolution. The oxalate ion is coordinated to copper in a 1,2-bidentate fashion, and the added bulk of the anion results in the rearrangement of the side chains of nearby arginine and tyrosine residues. No other major alterations in the molecule can be observed, the overall protein structure being the same as that for Cu2Lf and Fe2Lf.

Notes: It is often found in the crystallization of enzyme–inhibitor complexes that an inhibitor causes crystal packing which is different to that of native protein. This is the case for crystals of human non-pancreatic secreted phospholipase A2 (124 residues) containing six molecules in the asymmetric unit when the protein is complexed with a potential acylamino analogue of a phospholid. The hexameric structure was determined by molecular replacement using the structure of monomeric native protein as a probe. As an extension to the experiment, it was tested whether a backbone polypeptide composed of 17% of a known monomeric structure could find its correct position on a target molecule in molecular replacement. A probe model composed of the backbone atoms of the N-terminal 77 residues of lysine-, arginine-, ornithine-binding protein (LAO, a total of 238 residues) liganded with lysine correctly finds its position on LAO liganded with histidine which crystallizes as a monomer in the asymmetric unit. The results indicate that as little as 17% of total diffracting matter can be used in molecular replacement to solve crystal structures or to obtain phase information which can be combined with phases obtained by the isomorphous-replacement method.

Notes: This paper gives an overview of the science of crystals of biological macromolecules. The historical background of the field is outlined and the main achievements and open problems are discussed from both biological and physical–chemical viewpoints. Selected results, including data from the authors, illustrate this overview. The perspectives of crystallogenesis for structural biology, but also more general trends, are presented.

Notes: A dilute solution parameter obtained from static light-scattering measurements is proposed as a predictor for protein crystallization experiments. The osmotic second virial coefficients, B22, have been measured for a variety of proteins in solvents that are known to promote crystallization and the values for B22 were found to lie within a fairly narrow range which we refer to as a crystallization slot. Solution conditions which were known not to favor crystallization of the proteins resulted in B22 values well outside the crystallization slot.

Notes: The early stages of the crystallization process of porcine pancreatic α-amylase were investigated by quasi-elastic light scattering. It is shown that at 288 and 293 K the diffusion coefficient does not monotonically change with increasing protein concentration but passes through a maximum at 10 mg ml−1. In supersaturated solutions, prior to nucleation, the protein is strictly monodisperse. Nucleation induces the formation of aggregates and a polydispersity of, for example, 18% for an initial supersaturation C/Ce = 5.8. Monodispersity is restored after the nuclei have grown and partially consumed the solute. On the other hand, polydispersity increases up to 20% at 298 K if the protein concentration decreases to 3–4 mg ml−1, values at which the solutions are under-saturated. When the protein concentration exceeds 5–6 mg ml−1 the protein becomes monodisperse again. These results, confirmed by those of another system we are studying (bovine pancreatic trypsin inhibitor), are at variance with the statements that supersaturation is always at the origin of aggregation and polydispersity, and that in undersaturated solutions the diffusion coefficient should remain constant for obtaining crystals once the solutions are supersaturated.

Notes: The NAD(P)-dependent glutamate dehydrogenase from Pyrococcus furiosus has been crystallized by the hanging-drop method of vapour diffusion using lithium sulfate as the precipitant. The crystals belong to the tetragonal system and are in space group P42212 with unit-cell dimensions of a = b = 167.2, c = 172.9 Å. Consideration of the values of Vm and possible packing of the molecules within the cell suggest that the asymmetric unit contains a trimer. P. furiosus belongs to the family of Archaea and is one of the most thermostable organisms known, having an optimal growth temperature of 376 K. The glutamate dehydrogenase isolated from this organism has a half-life of 12 h at 373 K and, therefore, the determination of the structure of this enzyme will be important in advancing our understanding of how proteins are adapted to enable them to survive at such extreme temperatures.

Notes: The formation of protein single crystals grown with the shape controlled by the geometry of the capillary used as a growth cell is presented. The shaped crystals show strong birefringence under crossed nicols and diffract as single crystals up to 1.74 Å.

Notes: For a quarter of a century X-ray diffraction in single crystals was unique in its ability to solve three-dimensional structures of proteins and nucleic acids at atomic resolution. The situation changed in 1984 with the completion of a protein structure determination by nuclear magnetic resonance (NMR) spectroscopy in solution, and today NMR is a second widely used method for biomacromolecular structure determination. This review describes the method of NMR structure determination of biological macromolecules, and attempts to place NMR structure determination in perspective with X-ray crystallography. NMR is most powerful for studies of relatively small systems with molecular weights up to about 30000, but these structures can be obtained in near-physiological milieus. The two techniques have widely different time scales which afford different insights into internal molecular mobility as well as different views of protein or nucleic acid molecular surfaces and hydration. Generally, in addition to information on the average three-dimensional structure, NMR provides information on a wide array of short-lived transient conformational states. Combining information from the two methods can yield a more detailed insight into the structural basis of protein and nucleic acid functions, and thus provide a more reliable platform for rational drug design and the engineering of novel protein functions.

Notes: The crystal structures of the B-DNA dodecamer d(CGCGTTAACGCG) duplex (T2A2), with the inverted tetranucleotide core from the duplex d(CGCGAATTCGCG) [A2T2, Dickerson & Drew (1981). J. Mol. Biol. 149, 761–768], and its netropsin complex (T2A2–N) have been determined at 2.3 Å resolution. The crystals are orthorhombic, space group P212121, unit-cell dimensions of a = 25.7, b = 40.5 and c = 67.0 Å, for T2A2 and a = 25.49, b = 40.87, c = 67.02 Å for T2A2–N and are isomorphous with A2T2. The native T2A2 structure, with 70 water molecules had a final R value of 0.15 for 1522 reflections (F 〉 2σ), while for the netropsin complex, with 87 water molecules, the R value was 0.16 for 2420 reflections. In T2A2, a discontinuous string of zigzagging water molecules hydrate the narrow A·T minor groove. In T2A2–N, netropsin binds in one orientation in the minor groove, covering the TTAA central region, by displacing the string of waters, forming the majority of hydrogen bonds with DNA atoms in one strand, and causing very little perturbation of the native structure. The helical twist angle in T2A2 is largest at the duplex center, corresponding to the cleavage site by the restriction enzymes HpaI and HincII. The sequence inversion AATT→TTAA of the tetranucleotide at the center of the molecule results in a different path for the local helix axis in T2A2 and A2T2 but the overall bending is similar in both cases.

Notes: Quasi-elastic light scattering (QELS) was used to investigate quantitatively the mechanisms of nucleation, postnucleation growth, and dissolution in ensembles of both crystalline and amorphous aggregates of satellite tobacco mosaic virus (STMV), ferritin, apoferritin and pumpkin seed globulin. At low supersaturation conditions, as described previously for small molecule crystallization, the metastable region was obtained. Under these conditions aggregation took place, but crystallization did not proceed and critical nuclei did not form over a long period of time. The critical solution supersaturation necessary to obtain crystals, σ = ln(c/s) where c and s are concentration and solubility of protein, varied from ∼0.1 for pumpkin seed globulin to ∼0.9 for STMV. For higher supersaturation conditions when aggregation processes leading to formation of crystals are not established immediately but after a certain induction period, the supersaturation-dependent critical nuclear size, Rc, for different macromolecular systems was estimated from time-dependent size-distribution analyses to be in the range of ∼103 for proteins such as pumpkin globulin to approximately 10 for virus particles. From the same data, the molar interfacial free energy was deduced to be 3.3–9.2 kJ mol−1. These are believed to be among the first estimates for macromolecular crystals. Under conditions of moderate supersaturation where induction periods preceded the appearance of critical nuclei, the potential barriers for formation were estimated to be in the range 8.3–50 kJ mol−1. Growth and dissolution kinetics for pumpkin seed globulin were investigated. These experiments allowed determination of protein solubility versus solution temperature, protein and precipitant concentrations. Aggregation patterns which lead to crystal formation are distinctly different to those which produce an amorphous precipitate. The results provide additional evidence that QELS can be used to find general criteria that allow one to discriminate between conditions for a given protein system leading to crystalline or amorphous states at early stages of the aggregation process.

Notes: This laboratory has explored the potential of a combination of three analytical techniques to study the nucleation of chicken egg-white lysozyme. Collisional quenching of the fluorescent molecule SPQ [6-methoxy-N-(3-sulfopropyl)quinolinium] by chloride ions was used to determine the binding of the crystallizing agent to the protein at equilibrium and kinetically. Calorimetric measurements show that this binding generates an exothermic peak larger than the energy released during the early stages of nucleation. Light scattering intensity measurements were used to follow the aggregation kinetics.

Notes: The structure of cytochrome c′ from two bacterial species, Alcaligenes sp and Alcaligenes denitrificans, have been determined from X-ray diffraction data to 3.0 Å resolution using the anomalous scattering of the single Fe atom in each to identify and refine a weak molecular-replacement solution. Molecular-replacement studies, with the program AMORE, used two isomorphous data sets (from the two species), two independent search models (the cytochromes c′ from Rhodospirillum molischianum and Rhodospirillum rubrum), both with and without side chains, and two different resolution ranges (10.0–4.0 and 15.0–3.5Å) to generate a large number of potential solutions. No single solution stood out and none appeared consistently. The Fe-atom position in each structure was then determined from its anomalous-scattering contribution and all molecular- replacement solutions were discarded which did not (i) place the Fe atom correctly and (ii) orient the molecule such that a crystallographic twofold axis generated a dimer like those of the two search models. Finally, electron-density maps phased solely by the Fe-atom anomalous scattering were calculated. As these were combined and subjected to solvent flattening and histogram matching (with the program SQUASH), correlation with the remaining molecular-replacement solutions identified one as correct and enabled it to be improved and subjected to preliminary refinement. The correctness of the solution is confirmed by parallel isomorphous-replacement studies.

Notes: The crystallization of homogeneous or highly purified macromolecules depends on many variables such as precipitant, pH, choice of buffer, protein concentration, temperature, the participation of different mono- and divalent ions, as well as the presence of minute amounts of detergent and organic molecules. Finding the best combination among these many parameters is a multi-variable optimization problem. This kind of problem can be treated mathematically by sampling techniques. We have used this technique for protein crystallization. The iterative procedure starts with random sampling, followed by quantitative evaluation and cycles with weighted sampling. A simple procedure, derived from this concept and called MON48, has been successfully applied to many protein crystallization problems.

Notes: Studies on the low-humidity (88%) forms of tetragonal and monoclinic lysozyme, resulting from water-mediated transformations, have provided a wealth of information on the variability in protein hydration, its structural consequences and the water structure associated with proteins, in addition to facilitating the delineation of the rigid and the flexible regions in the protein molecule and the invariant features in its hydration shell. Surprisingly, monoclinic lysozyme continues to diffract even when the environmental humidity is drastically reduced, thus permitting the structural study of the enzyme at different levels of hydration. As part of a study in this direction, three very low humidity forms, two of them occurring at a nominal relative humidity of 38% and the other at 5% relative humidity, have been characterized. These have unprecedented low solvent contents of 16.9, 17.6 and 9.4%, respectively, as determined by the Matthews method.

Notes: Anionic salmon trypsin in a second crystal form (ST-IIB) has been refined at 1.83 Å, resolution. The crystals are orthorhombic and belong to space group P21212 with lattice parameters a = 77.09, b = 82.33 and c = 31.16 Å. The present structure has been compared to salmon trypsin as it appears in a previously reported crystal form (ST-IIA) with cell dimensions a = 61.95, b = 84.33 and c = 39.11 Å [Smalås & Hordvik (1993). Acta Cryst. D49, 318–330]. The presence of a sulfate group involved in several hydrogen bonds to active-site residues, and the location of an additional benzamidine site in the crystal lattice, are the most striking differences between the present and the previous structure. Superposition of main-chain atoms in the two structures give an overall r.m.s. difference of 0.26 Å, with the main differences located to areas with different molecular packing. The overall coordinate error is estimated to be between 0.20 and 0.25 Å, by the method of Luzzati.

Notes: Most rotation functions try to achieve maximal correlation between two Patterson functions by systematically rotating one and computing the overlap with the other. In contrast, the direct rotation function rotates a search model relative to the crystal unit cell and evaluates the linear correlation coefficient (Patterson correlation, PC) between squared normalized structure-factor amplitudes of the observed and calculated diffraction data. Structure factors are calculated from the rotated search model in a P1 unit cell identical to that of the target crystal. PC makes use of all self-Patterson vectors of the search model. A comparison of the direct rotation function, a real-space rotation function, and a fast rotation function suggests that the direct rotation function provides a considerable enhancement of the signal-to-noise ratio compared to other two. Combined with PC refinement, the direct rotation function was successful in solving multidomain macromolecular crystal structures.

Notes: Electron-density averaging, fast Fourier synthesis and fast Fourier analysis programs have been adapted for parallel-computing systems. These have been linked to perform iterative phase improvement and extension utilizing non-crystallographic symmetry and solvent flattening. Various strategies for parallel algorithms have been tested on a variety of computers as a function of the number of computer nodes. Some experimental timing results are discussed.

Notes: The X-ray structure of a dimeric, cyanomet-liganded hemoglobin D-chain (Hb-D) from Caudina arenicola has been determined by the molecular-replacement method. The search model was a concatenated model of three hemoglobin structures superimposed on the backbone of monomeric, hemichrome hemoglobin C-chain (Hb-C) from the same organism. Hb-D crystallizes in space group P41212 with cell constants a = b = 77.0 and c =61.5 Å with one subunit in the asymmetric unit. The dimer twofold axis corresponds to a crystallographic twofold along one of the body diagonals of the unit cell. Rotation and translation searches as well as model refinement were carried out in X-PLOR with the final model having an R value of 0.19 using the data from 5.0 to 2.9 Å resolution (R = 0.26 for 10.0 to 2.9 Å resolution). The homodimeric structure of Caudina Hb-D features close heme–heme contacts with an Fe—Fe distance of 19.0 Å. The subunit-subunit interface involves both the E and F helices from each subunit with the E helices oriented antiparallel at 50° with respect to one another, similar to the quaternary structure observed for the homodimeric hemoglobin from Scapharca inaequivalvis.

Notes: Microisopiestic measurements of the concentrations of polyethylene glycol (PEG 8000) paired with the salts sodium chloride, ammonium sulfate and magnesium sulfate heptahydrate have been made in a sitting-drop arrangement with PEG in the droplet and salt in the reservoir. Resulting graphs of the concentrations of PEG and salt that are equivalent with respect to the vapor pressure of water are non-linear, do not intersect their origins, and demonstrate that relatively low (mM) salt concentrations are equivalent to relatively high PEG concentrations. The consequences of each of these observations for macromolecular crystallization by the vapor-diffusion technique when PEG is employed as the crystallizing agent are discussed.

Notes: The X-ray structure of bovine ribonuclease A cocrystallized with the dinucleotide deoxycytidylyl-3′,5′-guanosine has been determined at 1.9 Å resolution and refined by restrained least squares to R = 0.218 for 7807 reflections. The structure established that the recently observed retrobound mode of attachment of substrate analogues cytidylyl-2′,5′-guanosine and deoxycytidylyl-3′,5′-guanosine found in soaked RNase A crystals is also present in the cocrystallized complex. Retrobinding is thus unlikely to be the result of restrictions imposed by the crystalline environment as the ligands soak into the lattice but rather a phenomenon specific to small nucleotides containing guanine.

Notes: The crystal structure of native salmon pancreatic elastase (SPE) has been solved by molecular-replacement methods, and refined by conventional conjugate-gradient methods and simulated-annealing techniques. The final R value is 17.2% for 21 389 reflections between 8.0 and 1.61 Å, and the corresponding free R value is 23.9%. The overall tertiary structure of SPE is remarkably similar to that of porcine pancreatic elastase I (PPE), to which it shows about 67% sequence identity. The primary structure of SPE is determined from the electron-density maps, and only about 15 side chains are somewhat uncertain. Interesting differences between SPE and PPE, are one sequence deletion assigned to position 186, the residue 192 at the entrance of the specificity pocket is substituted from a Gln in PPE to Asn in SPE, and one of the calcium ligands is different. Furthermore, electron density is missing in SPE for the last three residues of the C-terminal helix. A comparison of the present amino-acid sequence of SPE with other sequences available indicates that SPE belongs to the class 1 pancreatic elastases.

Notes: Liver alcohol dehydrogenase (LADH) is a ZnII-dependent dimeric enzyme. LADH with the active-site ZnII substituted by CuII resembles blue (type I) copper proteins by its spectroscopic characteristics. In this work we present the X-ray structure of the active site CuII-substituted LADH complex with NADH and dimethyl sulfoxide (DMSO). The structure was solved by molecular replacement. The space group is P21 with cell dimensions a = 44.4, b = 180.6, c = 50.8 Å and β = 108°. There is one dimer of the enzyme in the asymmetric unit. The refinement was carried out to a crystallographic R-factor of 16.1% for 41 119 unique reflections in the resolution range 12.0 to 2.1 Å. The coordination geometry of CuII in LADH is compared with the active-site metal coordination in the Zn–LADH–NADH–DMSO complex and blue-copper proteins. The distances from the metal to the protein ligands (Cys46, His67 and Cys174) are similar for the ZnII and CuII ions. The distances of the O atom of the inhibitor DMSO to the CuII ion in the two subunits of the dimer are 3.19 and 3.45 Å. These are considerably longer than the corresponding distances for the ZnII enzyme, 2.19 and 2.15 Å. The CuII ion is positioned nearly in the plane of the three protein ligands (NS2) with a geometry similar to the trigonal arrangement of the three strongly bound ligands (N2S) in blue-copper proteins. This coordination probably accounts for the similarity of the spectral characteristics of CuII–LADH and type I copper proteins.

Notes: Synchrotron sources provide a continuously tunable X-ray beam which makes it possible to optimize the anomalous contribution to phase determination using heavy-atom replacement. This method was used to solve two protein structures, those of Dictyostelium discoideum nucleoside diphosphate kinase and of lobster enolase. The first had 17 kDa of protein in the asymmetric unit, the second, 47 kDa. In both cases, a single mercury derivative yielded single isomorphous replacement with anomalous-scattering phases from which an interpretable electron-density map was derived by solvent flattening. The efficient solution of the X-ray structure was largely due to the large anomalous scattering of mercury at a wavelength shorter than the LIII absorption edge.

Notes: The initial structural analysis of the ternary complex of procarboxypeptidase A from hemihedrally twinned crystals diffracting up to 2.8 Å is described. Detection of twinning by different techniques is presented, including biochemical and intensity statistics approaches. The structure was initially solved using Patterson-search techniques, and the three positioned search models were used to effectively deconvolute the twinned data.

Notes: The filamentous bacteriophage Pf1 is structurally similar to the well known Ff (fd, fl, M13) strains, but it gives much better X-ray diffraction patterns, enabling a more detailed analysis of the molecular structure. The 46-residue protein subunit can be closely approximated by a single gently curved stretch of α-helix. The axes of the subunits are at a small angle to the virion axis, and several thousand subunits form an overlapping inter-digitated helical array surrounding a DNA core. We have derived a detailed model of the virion based on X-ray data and stereochemical constraints. We have considered potential sources of error in the diffraction data, and used the improved data to study regions where the protein subunit of Pf1 may deviate from a continuous α-helix. We use simulated annealing to escape from local minima, and various kinds of electron-density maps to guide the model building. Refinement of the model shows that the first few residues at the N terminus are non-helical, and there is a slight discontinuity in the α-helix near the middle of the sequence. The model is consistent both with general structural principles derived from high-resolution analysis of other proteins, and with specific chemical and spectroscopic data about Pf1. We apply the same refinement techniques to an alternative model with a non-helical surface loop between residues 13 and 19. Comparative analysis of models with and without a loop shows that the loop model is not supported by 3.3 Å resolution X-ray diffraction data.

Notes: The extracellular form of glutathione peroxidase from human plasma has been crystallized by the sitting-drop vapour-diffusion method. The crystals belong to tetragonal space group I41 with cell dimensions of a = b = 83.1 and c = 131.0 Å. They diffract beyond 2.9 Å resolution and have one dimer in the asymmetric unit. A self-rotation function analysis shows the possible (222) symmetry for the tetramers of this enzyme.

Notes: Carbamoyl Phosphate synthetase catalyzes the formation of carbamoyl phosphate, a high-energy intermediate used in several biosynthetic pathways. The enzyme from Escherichia coli has been crystallized at pH 8 in the presence of L-ornithine, MnCl2 and ADP, using PEG 8000 in combination with NEt4Cl and KCl. The crystals (apparently) belong to the orthorhombic space group P212121 with unit-cell dimensions of a = 154.4, b = 166.5 and c = 338.7 Å. The crystals are relatively sensitive to radiation damage, but show diffraction to beyond 2.8 Å resolution. A low-resolution (3.5 Å) native data set has been recorded and conditions for flash cooling the crystal have been established.

Notes: We have obtained two additional crystal forms of the metal-dependent class II fructose-1,6-bisphosphate aldolase from Escherichia coli. Crystals in the shape of elongated plates have unit-cell dimensions a = 73.4, b = 120.0, c = 190.1 Å, orthorhombic space group P212121. Monoclinic prisms have unit-cell dimensions a = 67.7, b = 104.3, c = 52.8 Å, β = 105°, space group P21. Diffraction to slightly better than 3.0 Å, has been observed for both forms using in-house and synchrotron facilities. These crystal forms may aid the structure solution of this enzyme by presenting additional forms for heavy-atom derivatization. These forms have multiple copies of the enzyme in the asymmetric unit and averaging methods might also be useful in the analysis.

Notes: A glutamic acid specific proteinase from Bacillus licheniformis has been crystallized as a complex with the inhibitor Z-Leu-Glu-CH2Cl. Crystals were grown by the vapor-diffusion method using sodium formate as a precipitant. The crystals diffracted to about 2.0 Å resolution and belonged to the trigonal space group P3121 (P3221) with unit-cell parameters a = b = 134.3, c = 109.7 Å. A total of 26 964 independent reflections were obtained up to 2.2 Å resolution, the merging R-factor being 0.05 for 42 614 measurements.

Notes: FK506 (tacrolimus) is a natural product now approved in the US and Japan for organ transplantation. FK506, in complex with its 12 kDa cytosolic receptor (FKBP12), is a potent agonist of immunosuppression through the inhibition of the phosphatase activity of calcineurin. Rapamycin (sirolimus), which is itself an immunosuppressant by a different mechanism, completes with FK506 for binding to FKBP12 and thereby acts as an antagonist of calcineurin inhibition. We have solved the X-ray structure of unliganded FKBP12 and of FKBP12 in complex with FK506 and with rapamycin; these structures show localized differences in conformation and mobility in those regions of the protein that are known, by site-directed mutagenesis, to be involved in calcineurin inhibition. A comparison of 16 additional X-ray structures of FKBP12 in complex with FKBP12-binding ligands, where those structures were determined from different crystal forms with distinct packing arrangements, lends significance to the observed structural variability and suggests that it represents an intrinsic functional characteristic of the protein. Similar differences have been observed for FKBP12 before, but were considered artifacts of crystal-packing interactions. We suggest that immunosuppressive ligands express their differential effects in part by modulating the conformation of FKBP12, in agreement with mutagenesis experiments on the protein, and not simply through differences in the ligand structures themselves.

Notes: Crystals of human recombinant N-acetylgalactosamine-4-sulfatase have been grown using vapour diffusion. The protein contains approximately 13%(w/w) carbohydrate. The crystals belong to the tetragonal space group P41212 or its enantiomorph P43212 with a = b = 108.0 and c = 145.5 Å. The crystals diffract to 2.7 Å resolution.

Notes: Crystals of the fructose-l,6-bisphosphate-dependent pyruvate kinase from Escherichia coli have been grown using the hanging-drop vapour-diffusion technique. The space group was found to be C2221 with cell dimensions a = 76.8, b = 247.5, c = 132.6 Å. Diffraction data to 3.0 Å resolution have been recorded and the enzyme molecular symmetry analysed through inspection of the self-rotation function. The crystallized protein is in the allosterically inactive T state.

Notes: With a size of 372 kDa, the F1 ATPase particle is the largest asymmetric structure solved to date. lsomorphous differences arising from reacting the crystals with methyl-mercury nitrate at two concentrations allowed the structure determination. Careful data collection and data processing were essential in this process as well as a new form of electron-density modification, `solvent flipping'. The most important feature of this new procedure is that the electron density in the solvent region is inverted rather than set to a constant value, as in conventional solvent flattening. All non-standard techniques and variations on new techniques which were employed in the structure determination are described.

Notes: The structures of two mimetic inhibitor complexes of human α-thrombin have been determined by X-ray crystallography. One mimics a β-turn with a bicyclic ring system; the other mimics two different active-site binding modes. The β-turn mimetic is used to approximate a turn found in the conformation of fibrinopeptide A, which is catalytically released by thrombin in the activation of fibrinogen to fibrin. The binding of the second mimetic is a hybrid between normal substrate and the abnormal binding of the potent natural leech inhibitor hirudin. The binding of the β-turn mimetic is tenuous, because it is like a substrate, while that of the substrate–hirudin hybrid is that of a tenacious inhibitor (which it is). Structurally retrospect modifications for rational design and improvement of both mimetic inhibitors are proposed.

Notes: Several empirical modeling protocols are evaluated allowing a quantification of the interaction between an enzyme and a series of inhibitors. The evaluation and optimization of the modeling protocols used a database of 35 non-covalently bound inhibitors of human thrombin. Intermolecular interaction energies were calculated with CHARMm after energy minimization of the modeled complexes using various dielectric functions and constraining strategies. These calculated binding energies were correlated with the experimentally obtained binding constants of the inhibitors. The best protocols resulted in linear correlations with correlation coefficients 〉 0.80. In the best protocols the enzyme was fully constrained, the ligand was allowed to move freely and electrostatics were scaled down drastically or fully neglected during the energy minimization. For the interaction energy evaluation step, a distance-dependent dielectric function ε = R proved to be optimal. This simple empirical protocol, that neglects solvation or entropy effects, can be implemented readily in other force field packages and may be applied on various enzyme–inhibitor complexes, providing a tool for the evaluation and rank-ordering of newly designed inhibitors.

Notes: Starting from the known three-dimensional structure of the class I major histocompatibility complex-encoded HLA-B*2705 protein, three non-natural nonapeptides were designed to fit optimally the HLA-B*2705-binding groove. The optimization was performed using structure-based drug design methods and the fact that all the possible interactions of the secondary anchor residue (position 3) with its human leukocyte antigen-binding pocket (pocket D) in nature are not entirely utilized. 150 ps molecular-dynamics (MD) simulation in water was employed to study the stability of the bimolecular complexes with three non-natural peptides (P3 = homophenylalanine, β-naphthylalanine, α-naphthylalanine) as well as with the two natural homologues (P3 = Gly, Leu). Various structural and dynamical properties (atomic fluctuations, solvent-accessible surface areas, peptide Cα-atom positions) of the simulated bimolecular complexes were used to compare the three non-natural with the two natural ligands. Since the various molecular properties have been shown previously to be related to the binding affinity of nonapeptide ligands to the major histocompatibility complex (MHC) HLA-B*2705 protein, the MD data predict a rather higher stability of MHC–ligand complexes with the three non-natural peptides, suggesting that the unnatural peptides studied show an enhanced binding affinity to the HLA-B*2705 protein. These results are in agreement with the experimental values of a semi-quantitative in vitro assembly assay, performed on the five nonapeptides (P3 = Gly, Leu, homophenylalanine, β-naphthylalanine, α-naphthylalanine), which shows their ability to stabilize the native conformation of the HLA-B*2705 heavy chain and also shows that the three non-natural ligands bind with higher affinity (0.5 µM) to the MHC protein than the two natural homologues (40 µM). Thus, this study demonstrates that structural information combined with rational design and molecular-dynamics simulations can illustrate and predict MHC binding and potential T-cell epitope properties as well as contribute to the design of new non-peptidic MHC inhibitors that may be useful for the selective immunotherapy of autoimmune diseases to which HLA alleles are directly associated.

Notes: A loop-deleted mutant form of 3-isopropylmalate dehydrogenase from Thermus thermophilus was constructed to investigate the relationship between the flexibility of the structure and the thermostability of the enzyme. The structure of the mutant enzyme was determined by X-ray crystallography and was found to be almost the same as that of the native enzyme with a reduced temperature factor. Although the mutant protein had lost the flexible loop, its function and thermostability had remained unchanged. This phenomenon can be explained by an internal reprieve tolerance mechanism.

Notes: The canine parvovirus structure (CPV) [Tsao, Chapman, Agbandje, Keller, Smith, Wu, Luo, Smith, Rossmann, Compans & Parrish (1991). Science, 251, 1456–1464] has been refined by a real-space refinement procedure [Chapman (1994). Acta Cryst. A51, 69–801. The fit of an atomic model to electron density was optimized while taking into account the resolution limit of the data and the stereochemistry of the structure. The refined model had a reasonable free R factor [Brtinger (1992). Nature (London), 355, 472–4751 of 0.29. The method is particularly fast and convenient when only a small fraction of the crystallographic asymmetric unit needs to be refined, as is the case when there is high non-crystallographic redundancy. Cycles of refinement for virus capsids were completed in about 1/50th of the time required for equivalent reciprocal-space procedures.

Notes: The structure of a tetragonal crystal of bovine pancreatic RNase B complexed with d(pA)4 was determined by molecular replacement and difference Fourier methods. This crystal belongs to space group P41212 and has unit-cell dimensions a = b = 44.5, c = 156.5 Å. The model consists of the enzyme and a tetranucleotide with fractional occupancies, suggesting multiple modes of oligonucleotide binding. It does not include any polysaccharide residues or solvent molecules. After refinement at 2.7 Å, the R value was 0.163 with acceptable stereochemistry. The model illustrates a set of well defined interactions for substrate binding, particularly between the central dinucleotide and the enzyme.

Notes: Recombinant transaldolase from Escherichia coli, an enzyme of the pentose phosphate pathway has been crystallized by the vapor-diffusion method using polyethylene glycol 6000 as precipitant. The crystals are orthorhombic, space group P212121 with cell dimensions a = 68.9, b = 91.3 and c = 130.5 Å and diffract to 2 Å resolution on a conventional X-ray source. The asymmetric unit very likely contains two subunits, corresponding to a packing density of 2.9 Å3 Da−1.

Notes: Xenopus laevis Cu,Zn superoxide dismutase (recombinant isoenzyme b) has been crystallized and the structure determined at 1.49 Å resolution. The crystals belong to space group P212121, with cell constants a = 73.33, b = 68.86, c = 59.73 Å, and contain one dimeric molecule of Mr 32 000 per asymmetric unit. The structure was solved by molecular-replacement techniques using the semisynthetic Cu,Co bovine enzyme as search model, and refined by molecular dynamics with a crystallographic pseudo-energy term. During the final steps, positional and anisotropic thermal parameters of the atoms were refined. The R factor for the 49 209 unique reflections in the 10.0–1.49 Å resolution range is 0.104, for a model comprising 2023 protein atoms, two Cu2+, two Zn2+, and 353 water molecules. The overall temperature factor for the model, including solvent, is 20.3 Å2, while the calculated r.m.s. coordinate error for the refined model is 0.036 Å. As suggested by the primary structure homology to any other known intracellular eukaryotic superoxide dismutase (〉 50%), the typical structural scaffolding of flattened antiparallel eight-stranded (β-barrel is well conserved in X. laevis Cu,Zn superoxide dismutase b, together with the coordination geometry of the metal centers in the active site. The higher thermal stability of the bb X. laevis superoxide dismutase homodimer, with respect to dimers involving the a-type isoenzyme subunit(s), can be related, on the basis of the high-resolution structure, to side-chain and solvent interactions centered on residue Tyr149, in both b-type subunits. The analysis of the overall solvent structure reveals a number of equivalent water molecule sites in the two subunits, and in homologous superoxide dismutase models. Their locations are discussed in detail and classified on the basis of their structural role.

Notes: Haemorrhagin I from the snake venom of Agkistrodon acutus (AaHI) has been crystallized using the hanging-drop vapour diffusion method. The crystals belong to space group P41212 or P43212 with unit-cell dimensions a = b = 63.61 and c = 95.69 Å. There is one molecule in the asymmetric unit. Data to 2.35 Å resolution have been collected using a single-crystal.

Notes: Crystals of 7α-hydroxysteroid dehydrogenase from E. coli, which is a tetramer in its active form, have been obtained by a hanging-drop vapor-diffusion method in the presence of the coenzyme NAD+. Crystals as large as 0.25 × 0.25 × 0.75 mm could be grown within a month at pH 8.5 with polyethylene glycol as precipitating agent. Preliminary X-ray crystallographic analysis revealed that they belong to one of the enantiomorphic space groups P41212 or P43212 with dimensions a = b = 81.66 and c = 214.6 Å, having two subunits in an asymmetric unit. The crystals diffract to at least 2.3 Å resolution.

Notes: Overexpressed dimeric E. coli aspartyl-tRNA synthetase (AspRS) has been crystallized in its free state and complexed with yeast tRNAAsp. Triclinic crystals of the enzyme alone (a = 104.4, b = 107.4, c = 135.0 Å, α = 102.9, β = 101.0, γ = 106.3°), have been grown using ammonium sulfate as the precipitant and monoclinic crystals (a = 127.1, b = 163.6, c = 140.1 Å, β = 111.7°), space group C2, have been grown using polyethylene glycol 6000. They diffract to 2.8 and 3.0 Å, respectively. Crystals of the heterologous complex between E. coli AspRS and yeast tRNA have been obtained using ammonium sulfate as the precipitant and 2-propanol as the nucleation agent. They belong to the monoclinic space group P21 (a = 76.2, b = 227.3, c = 82.3 Å, β = 111.7°) and diffract to 2.7 Å.

Notes: T4 deoxynucleotide kinase catalyzes the phosphorylation of 5-hydroxymethyldeoxycytidylate, dTMP and dGMP while excluding dCMP and dAMP. In order to understand the mechanism of this remarkable specificity, the enzyme was over-expressed in Escherichia coli, purified and crystallized for X-ray diffraction analysis. The crystals belong to the monoclinic space group C2 with cell dimensions a = 155.2, b = 58.5, c = 75.7 Å, β = 108.1°. There are two protein monomers in the asymmetric unit related by a twofold axis. Diffraction data to 2.0 Å resolution have been collected.

Notes: Collagenase from the fly larvae Hypoderma lineatum cleaves triple-helical collagen in a single region. It was crystallized at neutral pH in the absence of inhibitor and 1.8 Å data were collected using synchrotron radiation and a Mark II prototype detector. The structure was solved by combining multiple isomorphous replacement methods and rotation translation function in real space. Refinement between 7 and 1.8 Å using the program X-PLOR led to a final R factor of 16.9%. The overall fold is similar to that of other trypsin-like enzymes but the structure differs mainly by the presence of a β-sheet at position 31–44. The two embedded molecules of the asymmetric unit are related by a pseudo twofold axis. The β-sheet 31–44 of one molecule is involved in hydrogen bonds with binding-pocket residues of the other molecule. It thus completely prevents access to the active site. The specificity of this enzyme probably results from the position of Phe192 and Tyr99 at the entrance of the active site.

Notes: Two new crystal forms of calmodulin from Gallus gallus are reported. Crystals in space group P1 (cell dimensions a = 59.7, b = 53.1, c = 24.6 Å, α = 93.2, β = 96.7, γ = 89.2 and Z = 2), grow as long thin needles. Water content on density considerations is \sim50%. They diffract to \sim2.0 Å but give wide multiply peaked spot profiles. Crystals in space group P212121 (cell dimensions a = 32.2, b = 56.0, c = 67.3 Å and Z = 4), grow as clusters of thin tablets and contain \sim30% water by volume. These small crystals (\sim0.4 × 0.15 × 0.1 mm) diffracted well to \sim 1.4 Å and some appreciable intensities were observed at resolutions better than 1.2 Å.

Notes: Genetic algorithms have been investigated as computational tools for the de novo phasing of low-resolution X-ray diffraction data from crystals of icosahedral viruses. Without advance knowledge of the shape of the virus and only approximate knowledge of its size, the virus can be modeled as the symmetry expansion of a short list of nearly tetrahedrally arranged lattice points which coarsely, but uniformly, sample the icosahedrally unique volume. The number of lattice points depends on an estimate of the non-redundant information content at the working resolution limit. This parameterization permits a simple matrix formulation of the model evaluation calculation, resulting in a highly efficient survey of the space of possible models. Initially, one bit per parameter is sufficient, since the assignment of ones and zeros to the lattice points yields a physically reasonable low-resolution image of the virus. The best candidate solutions identified by the survey are refined to relax the constraints imposed by the coarseness of the modeling, and then trials whose intensity-based statistics are comparatively good in all resolution ranges are chosen. This yields an acceptable starting point for symmetry-based direct phase extension about half the time. Improving efficiency by incorporating the selection criterion directly into the genetic algorithm's fitness function is discussed.

Notes: Chitinase from barley seeds is a monomeric enzyme with 243 amino-acid residues and it plays a role as a defense protein. Its structure, previously determined at 2.8 Å resolution by multiple isomorphous replacement method, is mainly α-helical [Hart, Monzingo, Ready, Ernst & Robertus, (1993). J. Mol. Biol. 229, 189–193]. The crystallization and preliminary X-ray data of the same enzyme in a different crystal form has been reported independently [Song, Hwang, Kim & Suh, (1993). Proteins, 17, 107–109}, the asymmetric unit of which contains two chitinase molecules. As a step toward understanding the general principles of catalysis, reported here is the structure of chitinase from barley seeds in this crystal form, as determined by molecular replacement and subsequently refined at 2.0 Å resolution, with incorporation of partial data to 1.9 Å (R factor of 18.9% for 31 038 unique reflections with Fo〉 2σF in the range 8.0–1.9 Å). The r.m.s. deviations from ideal stereochemistry are 0.013 Å for bond lengths and 1.32° for bond angles. A superposition of the two independent molecules in the asymmetric unit gives an r.m.s. difference of 0.55 Å for all protein atoms (0.43 and 0.74 Å for main-chain and side-chain atoms, respectively). When the refined model of each chitinase molecule in the asymmetric unit is superposed with the starting model, the r.m.s. difference for all shared protein atoms is 0.99 Å for molecule 1 and 0.85 Å for molecule 2, respectively. Through a sequence comparison with homologous plant chitinases as well as a structural comparison with the active sites of other glycosidases, key catalytic residues have been identified and the active site has been located in the three-dimensional structure of the barley chitinase. The present structure, refined at an effective resolution of 2.0 Å with incorporation of partial data to 1.9 Å, represents a significant improvement in resolution compared to the previously reported model. The improved resolution has enabled the location of solvent atoms, including water molecules near the catalytic residues, in addition to the positioning of protein atoms with greater accuracy.

Notes: In 1961 Rossmann & Blow published a simple procedure for analytically combining the phase probabilities derived from various isomorphous derivatives or other phase-determining procedures [Rossmann & Blow (1961). Acta Cryst. 14, 641–647]. However, they found it necessary to make an approximation in obtaining the expression for the lack of closure (ε) of the phase triangle. In 1970 Hendrickson & Lattman [Hendrickson & Lattman (1970). Acta Cryst. B26, 136–143] suggested an alternative method of defining the lack of closure of the phase triangle which did not require any approximation in deriving the same analytical expression for the phase-probability function. It is now shown that it is possible to avoid the Rossmann-Blow approximation and thereby maintain the original meaning of the lack of closure as defined by Blow & Crick [(1959). Acta Cryst. 12, 794–802] and Dickerson, Kendrew & Strandberg [(1961). Acta Cryst. 14, 1188–1195].

Notes: Thc crystal structure of an α-chymotrypsin inhibitor (P6122; a = 61.4, c = 210.9 Å) isolated from winged bean (Psophocarpus. tetragonolobus) seeds has been determined at 2.95 Å resolution by the molecular-replacement method using the 2.6 Å coordinates of Erythrina trypsin inhibitor (ETI) as the starting model (57% sequence homology). This protease inhibitor, WCI, belongs to the Kunitz (STI) family and is a single polypeptide chain with 183 amino-acid residues having a molecular weight of 20 244 Da. Structure refinement with RESTRAIN and X-PLOR has led to a crystallographic R factor of 19.1% for 3469 observed reflections (I 〉 2σ) in the resolution range 8–2.95 Å. A total of 56 water molecules have been incorporated in the refined model containing 181 amino-acid residues. In the refined structure the deviations of bond lengths and bond angles from ideal values are 0.015 Å and 2.2°, respectively. The inhibitor molecule is spherical and consists of 12 antiparallel β-strands with connecting loops arranged in a characteristic folding (a six-stranded β-barrel and a six-stranded lid on one hollow end of the barrel) common to other homologous serine protease inhibitors in the Kunitz (STI) family as well as to some non-homologous proteins like interleukin-lα and interleukin-lβ. In the structure the conformation of the protruding reactive-site loop is stabilized through hydrogen bonds mainly formed by the side chain of Asnl4, which intrudes inside the cavity of the reactive-site loop, with the side-chain and main-chain atoms of some residues in the loop region. A pseudo threefold axis exists parallel to the barrel axis of the structure. Each of the three subdomains comprises of four β-strands with connecting loops.

Notes: The amyloidogenic Leu55Pro variant of transthyretin has been expressed, purified and crystallized in space group C2. The cell constants are a = 149.99, b = 78.74, c = 98.95 Å, β = 100.5° and the crystals diffract to 2.7 Å resolution. There are eight monomers in the asymmetric unit giving a VM = 2.6 Å3 Da−1 and 53% solvent content. In the wild-type protein, the crystals are orthorhombic with two monomers in the asymmetric unit. The wild-type protein is a tetramer composed of four identical subunits [Blake, Geisow, Oatley, Rerat & Rerat (1978). J. Mol. Biol. 121, 339–356.] and a molecular-replacement solution for the Leu55Pro variant was obtained using one monomer of the wild-type protein as a model. Rigid-body refinement of the eight monomers in the asymmetric unit and subsequent refinement using molecular dynamics were performed with X-PLOR, leading to a current R factor of 20.3% for all the data. The crystallographic packing of the molecules is different from the one presented by the wild-type protein, opening new perspectives for understanding how this protein aggregates to form amyloid fibrils.

Notes: A new crystal form of the histone octamer, crystallized in 1.6 M KCl, 1.6 M phosphate, diffracts to appreciably better than 2.6 Å resolution. The crystals have space group P61 or P65 and lattice parameters a = b = 158.29, c = 103.27 Å, α = β = 90, γ = 120°, with one molecule per asymmetric unit. The new crystals promise to yield more detail of the histone basic domains and a higher resolution structure for the histone octamer.

Notes: The ParE subunit of Escherichia coli topoisomerase IV has been crystallized in the presence of the non-hydrolyzable ATP analogue, 5′-adenylyl-β,γ-imidodiphosphate (ADPNP). The crystals are of the orthorhombic space group, P212121, with unit-cell dimensions a = 92.6, b = 119.1, c = 135.3 Å. Data have been collected to 3.5 Å resolution from frozen native crystals. Self-rotation function analysis of these data indicate the position of a molecular twofold axis. Higher resolution native data are being collected and a derivative search is underway.

Notes: Spo0F, a member of a superfamily of bacterial response regulatory proteins, is crucial to the regulation of sporulation in Bacillus subtilis. As there were difficulties in reproducing crystals of wild-type Spo0F, we report here the crystallization and preliminary studies of a mutant, Y13S protein, which gave well diffracting reproducible crystals. The crystals of the mutant obtained by the hanging-drop method belong to the tetragonal space group P41212 (P43212) a = b = 105.1, c = 85.9 Å. Diffraction data were collected at 2.8 Å at the laboratory source and subsequently 2.05. Å data were collected upon flash freezing the crystal at the Stanford Synchrotron Radiation Laboratory. This mutant participates in the phosphorelay in a similar manner to the wild-type protein. The presence of divalent cations are essential for wild-type phosphorylation and the present mutant crystal form is obtained in the presence of calcium.

Notes: Crystals of the hydroxynitrile lyase from Hevea brasiliensis overexpressed in Pichia pastoris have been obtained by the hanging-drop technique at 294 K with ammonium sulfate and PEG 400 as precipitants. The crystals belong to the orthorhombic space group C2221 with cell dimensions of a = 47.6, b = 106.8 and c = 128.2 Å. The crystals diffract to about 2.5 Å resolution on a rotating-anode X-ray source.

Notes: Uridine 5′-diphospho-N-acetylenolpyruvylglucosamine reductase (MurB), the second enzyme in the peptidoglycan synthetic pathway of Escherichia coli, has been crystallized in two previously unreported forms, one orthorhombic and the other monoclinic. MurB (molecular mass 38 kDa) crystallizes in a range of conditions that utilize polyethylene glycol fractions as precipitants, and crystals can be grown with or without the enzyme's substrate, uridine 5′-diphospho-N-acetylenolpyruvylglucosamine. X-ray diffraction from crystals of the orthorhombic form extends to 2 Å resolution and shows the symmetry and systematic absences of space group P212121. These crystals show significant variations in cell dimensions at room temperature and at 100 K. A crystal used to collect a 2.0 Å resolution data set at a synchrotron source showed cell dimensions at ca 100 K of a = 51.0, b = 79.3 and c = 87.1 Å, indicating one molecule peroasymmetric unit. The monoclinic crystals scatter X-rays to 3.0 Å resolution consistent with space group P21, unit-cell dimensions (ca 100 K) a = 50.7, b = 92.4, c = 85.5 Å, and β = 104°, and two molecules per asymmetric unit. Mercury derivatives have been prepared with both orthorhombic and monoclinic forms, and efforts are underway to exploit these derivatives to determine the structure of this protein.

Notes: Peroxidase from the brown alga Ascophyllum nodosum, a non-heme vanadium-dependent haloperoxidase, has been purified to homogeneity and crystallized from ammonium sulfate solutions in a form suitable for X-ray diffraction analysis. The crystals have been grown by the vapour-diffusion technique using the sitting-drop method. X-ray diffraction studies show that the crystals belong to the tetragonal space group P41212 or P43212 with a = b = 114.3 and c = 276.0 Å. The crystals diffract to at least 2.4 Å resolution.

Notes: Human salivary cystatin, a thiol proteinase inhibitor, has been implicated in potential antimicrobial and antiviral functions of saliva. A variant of human salivary cystatin SN expressed and purified in an Escherichia coli expression system lacking residues 12–16 near the N-terminus (Δ12–16) has been crystallized by the vapor-diffusion technique. The crystals are of the hexagonal space group P622 and have cell constants of a = 85.41, b = 85.41, c = 131.6 Å, α = β = 90, γ = 120°, and contain two molecules of molecular weight 13 500 per asymmetric unit. The crystals diffract up to a resolution of 2.2 Å and are suitable for X-ray diffraction analysis.

Notes: Concanavalin A was co-crystallized in two crystal forms with 3,6-di-O-methyl- (α-D-mannopyranosyl) α- D-mannopyranoside, which is primarily responsible for the high-affinity binding of N-linked carbohydrates to concanavalin A. Both crystal forms have space group P21 and contain a complete concanavalin A tetramer in the asymmetric unit. Form A was crystallized using polyethylene glycol methyl ether as the precipitant and has unit-cell dimensions a = 59.83, b = 64.84 and c = 125.92 Å, β = 93.87°. Form B was obtained using phosphate as the precipitant and has unit-cell dimensions a = 81.94, b = 66.75 and c = 108.92 Å, β = 97.58°. Form B was stable in the X-ray beam for several days and diffracted to 3.15 Å resolution. Form A crystals could not withstand X-ray radiation at room temperature, but produced high-quality data under cryogenic conditions. The latter are suitable for a 2.3 Å resolution structure determination by molecular replacement.

Notes: Carboxypeptidase G2 is a zinc-dependent exopeptidase which has applications in cancer therapy. Crystallization of carboxypeptidase G2, first achieved more than a decade ago, yields large crystals; however, problems with non-isomorphism between native crystals as well as failure to obtain any useful heavy-atom derivatives have precluded structure solution. A modification of the crystallization protocol leading to a promising new crystal form which diffracts beyond 3.0 Å resolution on a rotating-anode source is now reported. These crystals are readily indexed on an apparent C-centred orthorhombic lattice with a = 81.35, b = 230.9 and c = 105.5 Å, but the correct crystal system is monoclinic. The crystals have space group P21, with a = 81.35, b = 105.5, c = 122.4 Å and β = 109.3°. There are two possible non-equivalent monoclinic indexings with these lattice constants. A partial native data set collected at the SRS, Daresbury, indicates that 1.9 Å diffraction is attainable. Structure determination using MIR methods is in progress.

Notes: The one-wavelength anomalous scattering (OAS) X-ray diffraction data of azurin II, a copper-containing protein from Alcaligenes xylosoxidans were collected at the Photon Factory, Japan at a `routine' wavelength of 0.97 Å. The structure had been originally solved by the molecular-replacement method [Dodd, Hasnain, Abraham, Eady & Smith (1995). Acta Cryst. D51, 1052–1064]. As a technique of ab initio structure determination, the direct method [Fan, Hao, Gu, Qian, Zheng & Ke (1990). Acta Cryst. A46, 935–939] was attempted to break the phase ambiguity intrinsic to OAS data. The phases were then improved using the solvent-flattening method. The final electron-density map clearly shows most Cα positions and many side chains and it is traceable without prior knowledge of the structure. It is concluded that the direct method is capable of phasing anomalous scattering data collected at one wavelength from moderate-sized native proteins (Mw ∼ 20 kDa) which contain copper or atoms with a similar scattering power.