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: 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: 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: A new density modification technique - histogram matching - is being developed with encouraging results. Its application to the known structure of pig 2Zn insulin refines the 6500 1.9 Å MIR phases from a mean error of 60° to one of 46°. With these refined phases as a starting point for phase extension to 1.5 Å, the mean error for the final 13 000 phases is 46°. The original 1.9 Å phases continue to refine during the phase extension to a final mean error of 40°. A comparison is made with similar calculations already published.

Notes: To solve the problems related to the phases of the structure factors in surface structure analysis by transmission electron diffraction, the dynamical diffraction in the bulk crystal underneath the surface layer is considered by the many-beam theory. In the case of normal beam incidence, the intensities of superlattice reflections from surfaces and/or adsorbed layers are used to discriminate whether the surface structure is substitutional or displacive in type. In the case where only two beams are strongly excited in the bulk crystal, the phases of the structure factors of the surface layer are determined by the rocking curve of the superlattice reflections.

Notes: Homometric structures are non-congruent structures having identical X-ray intensity distributions. It has so far been assumed that such structures, while theoretically interesting, would not be realized in practice. Homometrism in close-packed structures is shown to be a realistic possibility. Some general rules applicable to homometric pairs are presented; it is shown that an infinite number of them can be derived from one-dimensional homometric pairs. An exhaustive search of close-packed structures with periods of up to 26 reveals that the smallest period of a homometric pair is 15 and that their number increases rapidly with the period. Homometrism in polytypic structures is further discussed.

Notes: Kato's statistical theory of diffraction [Kato (1980). Acta Cryst. A36, 763-769, 770-778] is reformulated in a self-consistent manner. The local displacement field u(r) occurs through the phase factor φ(r) = exp [2πih . u(r)]. The present paper is concerned with the limiting case where (φ(̃r)) = E = 0: this corresponds to the situation where only secondary extinction is present. There are two correlation lengths in the problem, the first one τ for the phase factor φ, the second one Γ for the wave-field amplitudes. Kato assumed Γ » τ. It is shown in the present paper that Γ ∼ τ, a property which has important consequences for the general theory, where E ≠ 0, to be discussed in the second paper of this series.

Notes: There is an unfortunate error in the caption to the figure on page 585 of the article by Kamminga [Acta Cryst. (1989). A45, 581-601]. The photograph was taken at the University of Leeds during the symposium held on 18-19 July 1946 and not 1948 as stated in the article.

Notes: The best X-ray atomic scattering factors for copper have been examined carefully to see which are most appropriate for charge density studies. The most consistent values were then used to generate a deformation charge density map, and it would appear that bonding in copper arises from electron charge build up between nearest-neighbour (n.n.) atoms, next-n.n. atoms etc. This is in agreement with conclusions obtained from γ-ray diffraction experiments and the best band-structure calculations, but in marked disagreement with the charge density obtained from earlier band-structure form factors.

Notes: With the use of modulated plane waves, a new method for n-beam dynamical calculations has been established on the basis of a paper by Watanabe, Kikuchi, Hiratsuka & Yamaguchi [Phys. Status Solidi A (1988), 109, 119-126]. The computing time is reduced to about one-sixth of what it originally was and a large reduction of memory is achieved, n-beam dynamical calculations of aluminium, copper and gold at several accelerating voltages and orientations were carried out in a completely parallel manner by the present method, the multi-slice method and Bethe's eigen-value method [Fujiwara (1959). J. Phys. Soc. Jpn 14, 1513-1524]. The present method turned out to be competitive with respect to accuracy and speed in comparison with the latter two methods. The new method makes n-beam dynamical calculations of complex systems and defects possible.

Notes: The symmetry conditions for flat points of minimal surfaces have been studied in relation to the order β of points on such surfaces. Using symmetry aspects, a set of rules for the derivation of fiat points have been developed. By means of these rules the flat points for the 45 families of minimal balance surfaces known so far have been determined. As a check for completeness the relation between the genus of a minimal surface and the orders of its flat points has been used.

Notes: There are two errors in the short communication by Velmurugan & Hauptman [Acta Cryst. (1989). A45, 656]. On line 9 in the Abstract, Φ2h′2k′0 should read Φ2h2k0 and on line 3 in Summary of final results, Φ2h′2k′0 should read Φ2h,2k,0.

Notes: Monoclinic crystals of Bacillus stearothermophilus tryptophanyl-tRNA synthetase grown in the presence of substrate trytophan (space group P21) display evidence of a low-resolution trigonal space group (P321). The origin and averaging transformations for the local 32 point group of this unusually clear sixfold non-crystallographic symmetry may be inferred without prior estimation of the electron density. This local symmetry was exploited in conjunction with solvent density contrast variation to determine the shape of the molecular envelope. X-ray intensities measured from crystals equilibrated in mother liquors of three different electron densities were used to estimate three parameters for each reflection: the modulus of the envelope transform, |Gh]; and components, Xh and Yh, relative to Gh, of the structure-factor vector for the transform of intramolecular density fluctuations. The moduli {|Gh|} behave somewhat like structure-factor amplitudes from small-molecule crystals, and estimation of their unknown phases was successfully carried out by statistical direct methods. Reflections to 18 Å resolution, which obey rather well the symmetry of space group P321, were merged to produce an asymmetric unit in that space group. |Gh| values for the 34 strongest of these were phased using the small-molecule direct-methods package MITHRIL [Gilmore (1984). J. Appl Cryst. 17, 42-46]. The best phase set was expanded back to the P21 lattice and negative density was truncated to generate initial phases for all reflections to 18 Å resolution. Phase refinement by iterative imposition of the local 32 symmetry produced an envelope with convincing features consistent with known properties of the enzyme. The envelope implies that the tryptophanyl-tRNA synthetase dimer is an elongated structure with an axial ratio of about 4: 1, in which the monomers have two distinct domains of unequal size. The smaller of these occurs at the dimer interface, and resembles the nucleotide binding portion of the tyrosyl-tRNA synthetase. It may therefore contain the amino-terminal one hundred or so residues, including all three cysteines, previously suggested to comprise a nucleotide-binding domain in the tryptophanyl enzyme. A purely crystallographic test of the overall features of this envelope was carried out by transporting it to a tetragonal crystal form of the same protein in which the asymmetric unit is a monomer. The small domain fits snugly inside three mercury and one gold heavy-atom binding sites for this crystal form; and symmetry-related molecules provide excellent, but very different, lattice contacts in nearly all directions.

Notes: A Monte Carlo simulation of an ordering phase transition in the surface region of a f.c.c.-type A3B binary alloy is reported. The main emphasis of this simulation is the evaluation of short and long-range-order correlations near the surface which are used for calculating X-ray intensities under grazing-incident-angle conditions. These calculations suggest effective ways of conducting surface diffraction experiments on order-disorder phase transitions. The simulation results are also compared with available experimental data.

Notes: The previously reported distorted zeroth-order fringes in a bright-field Tanaka pattern from a dislocated region in silicon have been computer simulated and the experimental and the many-beam calculated patterns agree well. Calculations are carried out for nine distinct cases of edge, screw and 60° dislocations in a silicon crystal. The general usefulness of the distortion of the ZOLZ pattern in determining geometrical properties of a dislocation is discussed.

Notes: The group-theoretical method established for obtaining the non-vanishing independent number of constants required to describe a magnetic/physical property in respect of the 18 polychromatic crystal classes [Rama Mohana Rao (1987). J. Phys. A, 20, 47-57] has been explored to enumerate the second- order piezomagnetic coefficients (ni′) for the same classes. The advantage of Jahn's method [Jahn (1949). Acta Cryst. 2, 30-33] is appreciated in obtaining these ni′ through the reduction of a representation. The different group-theoretical methods are illustrated with the help of the point group 4. The results obtained for all 18 classes are tabulated and briefly discussed.

Notes: Measurements are reported of the mass attenuation coefficient of carbon taken by laboratories participating in the International Union of Crystallography X-ray Attenuation Project. Data resulting from a similar study using silicon were published earlier [Creagh & Hubbell (1987). Acta Cryst. A43, 102-112]. The data are self consistent, for the most part, to 0.5% for the energy range 6 to 60 keV, and accords well with earlier experimental data. These data are about 3% less than the theoretically calculated data [Saloman & Hubbell (1986). X-ray Attenuation Coefficients (Total Cross Sections). Comparison of the Experimental Data Base with the Recommended Values of Henke and the Theoretical Values of Scofield from 0.1 to 100 keV. Report NBSIR 86-3431. US Department of Commerce, NBS, Gaithersburg, MD, USA; Berger & Hubbell (1987). XCOM. Photon Cross Sections on a Personal Computer. Report NBSIR 87-3597. US Department of Commerce, Gaithersburg, MD, USA] over the energy range for which measurements were made. Hence carbon appears to be an example in which the renormalization, always a decrease, of Scofield's photo-effect calculations [Scofield (1973). Report UCRL-51236. Lawrence Livermore Laboratory, Livermore, California, USA], as was implemented by Hubbell [Int. J. Appl. Radiat. Isot. (1982), 33, 1269-1290], would improve agreement with measured data.

Notes: Entropy maximization has proven effective in treating certain aspects of the phase problem of X-ray diffraction. Much of its development has been expressed in probabilistic language, although image enhancement has been somewhat more physical or geometric in description. Here phasing and entropy maximization are embedded in the quantum mechanical problem of reconstructing an electronic one-matrix under experimental constraints. Entropy on an N-representable one-particle density matrix is well defined. The entropy is the expected form, and it is a simple function of the one-matrix eigenvalues which all must be non-negative. Certain other properties are pertinent to phasing which is implicit in one-matrix reconstruction governed by entropy maximization. Throughout this work reference is made to informational entropy, not the entropy of thermodynamics.

Notes: We describe the further development of phase refinement by iterative skeletonization (PRISM), a recently introduced phase-refinement strategy [Wilson & Agard (1993). Acta Cryst. A49, 97–104] which makes use of the information that proteins consist of connected linear chains of atoms. An initial electron-density map is generated with inaccurate phases derived from a partial structure or from isomorphous replacement. A linear connected skeleton is then constructed from the map using a modified version of Greer's algorithm [Greer (1985). Methods Enzymol. 115, 206–226] and a new map is created from the skeleton. This `skeletonized' map is Fourier transformed to obtained new phases, which are combined with any starting-phase information and the experimental structure-factor amplitudes to produce a new map. The procedure is iterated until convergence is reached. In this paper significant improvements to the method are described as is a challenging molecular-replacement test case in which initial phases are calculated from a model containing only one third of the atoms of the intact protein. Application of the skeletonization procedure yields an easily interpretable map. In contrast, application of solvent flattening does not significantly improve the starting map. The iterative skeletonization procedure performs well in the presence of random noise and missing data, but requires Fourier data to at least 3.0 Å. The constraints of linearity and connectedness prove strong enough to restore not only missing phase information, but also missing amplitudes. This enables the use of a powerful statistical test, analogous to the `free R factor' of conventional refinement [Brünger (1992). Nature (London), 355, 472–474], for optimizing the performance of the skeletonization procedure. In the accompanying paper, we describe the application of the method to the solution of the structure of the protease inhibitor ecotin bound to trypsin and to a single isomorphous replacement problem.

Notes: The previous paper described a phase-refinement strategy for protein crystallography which exploited the information that proteins consist of connected linear chains of atoms. Here the method is applied to a molecular-replacement problem, the structure of the protease inhibitor ecotin bound to trypsin, and a single isomorphous replacement problem, the structure of the N-terminal domain of apolipoprotein E. The starting phases for the ecotin-trypsin complex were based on a partial model (trypsin) containing 61% of the atoms in the complex. Iterative skeletonization gave better results than either solvent flattening or twofold non-crystallographic symmetry averaging as measured by the reduction in the free R factor [Brünger (1992). Nature (London), 355, 472–474]. Protection of the trypsin density during the course of the refinement greatly improved the performance of both skeletonizing and solvent flattening. In the case of apolipoprotein E, previous attempts using solvent flattening had failed to improve the SIR phases to the point of obtaining an interpretable map. The combination of iterative skeletonization and solvent flattening decreased the phase error with respect to the final refined structure, significantly more than solvent flattening alone. The final maps generated by the skeletonization procedure for both the ecotin–trypsin complex and apolipoprotein E were readily interpretable.

Notes: The Pseudomonas aeruginosa azurin mutant Asn47Asp has been isolated, its spectroscopic and kinetic properties characterized, and the X-ray crystal structure of its zinc derivative determined. While the optical and electron paramagnetic resonance spectra as well as the electron-transfer activity of the mutant are very similar to the wild-type values, the Asn47Asp reduction potential is slightly increased by 20 mV. The mutant crystallized in the orthorhombic space group P212121 with cell dimensions a = 57.8, b = 81.5 and c = 112.6 Å. There are four molecules in the asymmetric unit, packed as a tetramer which consists of two independent dimers. The zinc site of this mutant structure is similar to the wild-type zinc azurin and, in particular, the metal-binding site is almost identical to the site found in the wild-type zinc-azurin structure [Nar, Huber, Messerschmidt, Filippou, Barth, Jaquinod, Kamp & Canters (1992). Eur. J. Biochem. 205, 1123–1129]. The Asp47 side chain at that mutation site takes on a very similar orientation to Asn47 in the wild-type structure preserving the two hydrogen bonds with the neighbouring Thr113 NH and OγH. Therefore, the increased reduction potential of the mutant is probably a result of an altered charge distribution close to the metal site.

Notes: The structures of two hexanucleotide–anthracycline complexes d(CGGCCG)/daunomycin and d(TGGCCA)/adriamycin have been determined using single-crystal X-ray diffraction techniques. In both cases the anthracycline molecule is bound to non-preferred d(YGG) base-pair triplet sites. For both complexes the crystals are tetragonal and belong to the space group P41212. Unit-cell dimensions are a = 28.07 (2), c = 53.35 (1) and a = 28.01 (1), c = 52.99 (1) Å, respectively, and the asymmetric unit of both structures consists of one strand of DNA, one drug molecule and approximately 50 water molecules. For the d(CGGCCG) complex the refinement converged with an R factor of 0.21 for 1108 reflections with F ≥ 2σ(F) in the resolution range 7.0–1.9 Å. For the complex involving d(TGGCCA) the final R value was 0.22 for 1475 reflections in the range 7.0–1.7 Å with the same criterion for observed data. Both complexes are essentially isomorphous with related structures but differ in terms of the number of favourable van der Waals interactions of the amino sugars of the drug molecules with the DNA duplexes and the formation in the minor groove of heterodromic pentagonal arrangements of hydrogen bonds involving water molecules which link the amino sugars to the DNA. These differences in structure are used to rationalize the lack of affinity of daunomycin-type anthracyclines for d(YGG) and d(YGC) sites.

Notes: In an earlier study [Heinemann & Hahn (1992). J. Biol. Chem. 267, 7332–7341], the crystal structure of the double-stranded B-DNA decamer d(CCAGGCm5CTGG) was refined with NUCLSQ to R = 17.4% against 3799 2σ structure amplitudes in the resolution range 8–1.7 Å. This structure has now been re-refined against the same diffraction data using either TNT or X-PLOR in order to determine to what extent the resulting DNA conformations would differ and to examine the suitability of these programs for the refinement of oligonucleotide structures. The R value from the NUCLSQ refinement could not be reached with either TNT or X-PLOR, although both programs yield reasonably refined DNA models showing root-mean-square deviations against the NUCLSQ model of the decamer duplex of 0.25 and 0.32 Å, respectively. Some derived local structure parameters differ depending on the refinement procedure used. This holds true for several exocyclic torsion angles of the sugar-phosphate backbone, whereas sugar puckers as well as helical and base-pair stacking parameters are only weakly influenced. A subset of 15 solvent sites with low temperature factors is conserved in all three models.

Notes: Canavalin, the major reserve protein of the jack bean, was obtained in four different crystal forms. From the structure determined by multiple isomorphous replacement in a hexagonal unit cell, the structures of three other crystals were determined by molecular replacement. In two cases, the rhombohedral and cubic crystals, placement was facilitated by coincidence of threefold molecular symmetry with crystallographic operators. In the orthorhombic crystal the canavalin trimer was the asymmetric unit. The rhombohedral, orthorhombic and cubic crystal structures were subsequently refined using a combination of several approaches with resulting R factors of 0.194, 0.185 and 0.211 at resolutions of 2.6, 2.6 and 2.3 Å, respectively. Variation in the conformation of the molecule from crystal to crystal was small with an r.m.s. deviation in Cα positions of 0.89 Å. Packing is quite different among crystal forms but lattice interactions appear to play little role in the conformation of the molecule. Greatest variations in mean position are for those residues that also exhibit the greatest thermal motion. Crystal contacts in all crystals are mediated almost exclusively by hydrophilic side chains, and three to six intermolecular salt bridges per protein subunit are present in each case.

Notes: Aldehyde reductase from porcine kidney has been crystallized from buffered ammonium sulfate solutions. Two crystal forms are monoclinic, space group P21, with a = 56.2, b = 98.1, c = 73.2 Å, β = 112.5° and a = 92.4, b = 62.1, c = 59.0 Å, β = 94.6°. A third crystal form is hexagonal with a = b = 166.0, c = 66.0 Å, α = β = 90.0° and γ = 120.0°. Molecular-replacement structure solutions have been successfully obtained for the two monoclinic crystal forms. The crystallographic R factor at 8–2.8 Å resolution for the two monoclinic crystal forms is currently 0.23 and 0.25, respectively. There are two molecules per asymmetric unit related by a non-crystallographic twofold axis. The aldehyde reductase models are supported by the arrangement of the molecules in their respective unit cells and by electron densities corresponding to amino-acid side chains not included in the search structures.

Notes: Monoclinic crystals of turkey egg lysozyme (TEL, E.C. 3.2.1.17) were obtained from 2.2 M ammonium sulfate solution at pH 4.2. They belong to space group P21 with unit-cell dimensions a = 38.07, b = 33.20, c = 46.12 Å and β = 110.1°, and contain one molecule in the asymmetric unit (Vm = 1.91 Å3 Da−1). The three-dimensional structure of TEL was solved by the method of multiple isomorphous replacement with anomalous scattering. Area detector data to 1.5 Å resolution from native and heavy-atom derivatives were used for the structure determination. The structure was refined by the simulated-annealing method with diffraction data of 10–1.30 Å resolution. The conventional R factor was 0.189. The root-mean-square deviations from ideal bond distances and angles were 0.016 Å and 2.9°, respectively. The backbone structure of TEL is very similar to that of hen egg lysozyme (HEL) and the difference in seven amino-acid residues does not affect the basic folding of the polypeptide chain. Except for the region from Gly101 to Gly104, the geometry of the active-site cleft is conserved between TEL and HEL. The Gly101 residue is located at the end of the sugar-binding site and the structural change in this region between TEL and HEL is considered to be responsible for the difference in their enzymatic properties.

Notes: A survey of 129 protein crystal structures with more than one molecule per asymmetric unit shows that local (non-crystallographic) symmetry axes are not randomly oriented. When compared to the crystal cell edges, face diagonals, body diagonal and reciprocal cell edges, 65% of the local symmetry axes are found to be parallel to one of the reference directions to within 15°; another 18% are orthogonal to within 3°; only 17% are in general orientations. In monoclinic, trigonal and hexagonal crystals, a majority of the local symmetry axes are orthogonal to the unique axis, while preferred orientations are parallel to the cell edges in orthorhombic crystals.

Notes: Chloromuconate cycloisomerase (E.C. 5.5.1.7) is an enzyme involved in the 2,4-dichlorophenoxyacetate degradation pathway of Alcaligenes eutrophus JMP134 (pJP4). The crystal structure of this protein was determined at 3 Å resolution by molecular-replacement techniques using atomic coordinates from the reported crystal structure of the homologous muconate cycloisomerase (E.C. 5.5.1.1) from Pseudomonas putida as the search model (42% identical positions in the sequences). Structure refinement by simulated-annealing and restrained least-squares techniques converged at R = 0.195. In the crystals studied, space group I4, the protein is present as two octamers per unit cell with two subunits per asymmetric unit. Each subunit consists of two globular domains, one of which forms an α/β-barrel. Comparison of this structure with that of muconate cycloisomerase reveals the reasons for the altered substrate specificity of chloromuconate cycloisomerase. Marked differences are observed in polarity, accessibility and hydrogen-bonding potential in the channel leading into the active site as well as in the active center itself.

Notes: Transforming growth factor-β is a multifunctional cell-growth regulator and is a member of the TGF-β superfamily of cytokines. Each monomer is 112 amino acids long and the mature active form is a 25 kDa homodimer. Recently, the crystal structure of TGF-β2 has been determined independently in two laboratories [Daopin, Piez, Ogawa & Davies (1992). Science, 257, 369–373; Schlunegger & Grütter (1992). Nature (London), 358, 430–434] and subsequently refined to higher resolutions [Daopin, Li & Davies (1993). Proteins Struct. Funct. Genet. In the press; Schlunegger & Grütter (1993). J. Mol. Biol. In the press]. A detailed structural comparison shows that the two structures are nearly identical with the differences mostly located on the mobile regions of the molecule. The r.m.s. differences between the two structures are 0.10 Å for 104 pairs of Cα atoms, 0.15 Å for 434 pairs of main-chain atoms, 0.33 Å for 860 out of 890 pairs of protein atoms and a correlation of 90% between the temperature B factors of all protein atoms. Based on a comparison of the water molecules, a B value of 60.0 Å2 is recommended as the cut off for modeling new waters. The structural identity is striking because in one case the material was expressed in vivo in CHO cells whereas in the other case it was expressed in E. coli and had to be refolded in vitro. The overall coordinate errors are estimated to be 0.21 Å from the Luzzati plot, 0.18 Å from the σA plot, 0.24 Å with Cruickshank's equations and 0.25 Å using the empirical method of Perry & Stroud. These estimates are comparable to the r.m.s. structure superposition. The r.m.s. differences correlate very well with the crystallographic B values and the relation is best described with the Cruickshank formula. In addition to the estimation of an overall error, a new application of the Cruickshank formula is presented here to estimate the local errors.

Notes: The X-ray structures of native carboxypeptidase A and of the enzyme–inhibitor complex with L-phenyl lactate have been refined at 1.54 and 1.45 Å resolution to R factors of 0.151 and 0.161, respectively. Crystals of the complex were isomorphous with the native crystals (space group P21, a = 51.60, b = 60.27, c = 47.25 Å, β = 97.27°). The high-resolution electron density allowed correction of many side-chain positions in the classical carboxypeptidase A model. This reflects the advantages of the high-quality complete synchrotron data collected with an imaging plate detector. The conformational changes in the active centre of the enzyme upon binding of the inhibitor are restricted to only two residues, Tyr248 and Arg145. L-Phenyl lactate is bound in the S1′ pocket and forms hydrogen bonds to Arg145, Glu270 and to the zinc-bound water molecule. The present structure provides an explanation for the higher stability of the complexes with the products of esterolysis in comparison with those of amidolysis. This is consistent with the finding that product release is rate limiting for esters but not for peptides.

Notes: The X-ray diffraction pattern of the crystals of the non-selfcomplementary hexadeoxyribonucleotide d(CGCACG)·d(CGTGCG) can be indexed in four different space groups: (i) P65 and P21, with cell parameters a = 17.75 (1), b = 17.76 (1), c = 42.77 (3) Å, α = 90, β = 90, γ = 120°, and (ii) P212121 and C2, with cell parameters a = 17.75 (1), b = 30.74 (2), c = 42.77 (3) Å, α = 90, β = 90, γ = 90°. While the Rmerge for the equivalent reflections in the different space groups indicates that P21 is the correct choice in the present case, it is demonstrated that the near degeneracy of the space groups arises out of the fact that the DNA molecule is nearly cylindrical. A perfect cylinder would show perfect degeneracy.

Notes: We report the crystallization of samples of a recombinant preparation of human interleukin-1 receptor antagonist protein (IRAP) and solution of the crystal structure by isomorphous replacement methods. Crystals were obtained by the hanging-drop vapor-diffusion method at 277 K from solutions of PEG 4000 containing sodium chloride, dithiothreitol and PIPES [sodium piperazione-N,N′-bis(2-ethanesulfonate)] buffer at pH 7.0. Crystals appear within about a week and grow as truncated tetragonal bipyramids to 0.3–0.6 mm on an edge. X-ray diffraction data from these crystals specify space group P43212 and unit-cell dimensions of a = b = 72.35(26), c = 114.7(8) Å and Z = 16 (two molecules per asymmetric unit). Fresh crystals diffract to about 2.3 Å resolution. The search for heavy-atom derivatives has produced two, potassium gold cyanide and trimethyl lead chloride, as same-site, single-site derivatives. Inspection of an electron-density map at 4 Å resolution calculated with these derivatives confirms that the IRAP molecule is a member of the interleukin-1 structural family.

Notes: The structure of the ADP complex of the enzyme 3-phosphoglycerate kinase (PGK, E.C. 2.7.2.3) from Bacillus stearothermophilus NCA-1503 has been determined by the method of molecular replacement. The structure has been refined to an R factor of 0.16 for all data between 10.0 and 1.65 Å resolution, using data collected on the Hendrix–Lentfer imaging plate at the EMBL outstation in Hamburg. The r.m.s. deviations from stereochemical ideality are 0.010 and 0.011 Å for bonds and planes, respectively. Although crystallized in the presence of the nucleotide product MgATP, the high-resolution structure reveals the bound nucleotide to be MgADP reflecting the low intrinsic ATPase activity of PGK. Although the two domains of this enzyme are found to be some 4.5° closer together than is found in the yeast and horse-muscle apo-enzyme structures, this structure represents the `open' rather than the `closed', catalytically competent form, of the enzyme.

Notes: Yeast initiator tRNA crystals exhibit strong X-ray diffuse scattering. This scattering can be used to extract information about lattice-coupled and intramolecular motions in the crystals. The amplitudes and correlation distances of these motions can be estimated by calculating the diffuse scattering and comparing the results with the observed scattering. Results indicate that both anisotropic, lattice-coupled motions as well as short-range correlated local disorder in the anticodon arm contribute to the overall disorder in the crystals. These types of motions can be correlated with aspects of tRNA function. This additional information complements the results from analysis of crystallographic data and provides a more detailed picture of the structure and dynamics of the molecule. The degree to which the methodology presented here can account for the observed diffuse scattering from tRNA represents a significant step forward in the ability to use this conventionally discarded information, and encourages the ultimate extension of these ideas to a wide variety of macromolecular systems.

Notes: Two new crystal forms of isoenzyme 3-3 of rat liver glutathione S-transferase (GST 3-3) have been obtained. They were grown under essentially the same crystallization conditions as those reported for the C2 crystal form [Fu, Rose, Chung, Tam & Wang (1991). Acta Cryst. B47, 813–814]. The new crystals belong to space group P21 with one form having cell dimensions a = 101.6, b = 69.5, c = 81.4 Å, and β = 113.6°, and the other form having cell parameters a = 97.4, b = 81.1, c = 69.4 Å and β = 109.2°. These new crystals diffract to at least 2.5 Å, resolution. The molecular packing arrangements in these P21 crystals have been found by molecular replacement studies.

Notes: Lipase from Chromobacterium viscosum has been purified to homogeneity and crystallized in a form suitable for X-ray diffraction analysis from 10-14% polyethylene glycol 4000 and 10-14% 2-methyl-2,4-pentane diol at pH 6.4 in the presence of 0.25%(w/v) n-octyl-β-D-glucopyranoside. These crystals belong to space group P21212 with refined lattice constants a = 41.1 Å, b= 156.8, c = 43.6 Å, indicating a cell content of one monomer per asymmetric unit of the crystal. The crystals diffract to a resolution of 2.2 Å.

Notes: Xylose isomerase from Bacillus coagulans has been crystallized in two different crystal forms. One crystal form is in space group P21212, cell dimensions a = 462, b = 165, c = 82 Å. The other is in space group I422, cell dimensions a = b = 113, c = 153 Å.

Notes: The packing of glutathione reductase from Escherichia coli in crystal form T showed a place where two molecules are at a distance of only 6 Å between the closest atoms, i.e. where a contact is almost made. In order to form this contact with hydrogen bonds, two amino-acid residues were exchanged. This mutation had no effect on molecular packing or the resolution limit of the X-ray diffraction, but facilitated crystal nucleation dramatically and possibly increased the crystal growth rate and shortened the crystallization time.

Notes: Crystals of the tetra-heme cytochrome c3 (Mr = 13 kDa, 107 residues, four heme groups) from sulfate- and nitrate-reducing Desulfovibrio desulfuricans ATCC 27774 have been obtained and crystallographically characterized. They belong to space group P6122 with cell dimensions a = b = 61.84 (4) and c = 109.7 (2) Å, and Z = 12. Intensity data were initially collected on a FAST system with a rotating-anode X-ray source leading to a total of 22 592 observations, from which only 4930 were unique, in the resolution range 20.0–2.4 Å with an Rmerge(I) of 7.0%. Higher resolution data were measured on a FAST system at station 9.6 of the SRS (Daresbury, England), leading to 19 328 intensities, of which 11 179 were unique, in the resolution range 20.0–1.75 Å and an Rmerge(I) of 5.5%. Cross-rotation and translation functions were performed with ALMN and TFSGEN programs from the CCP4 suite. The packing of the molecules in the unit cell was checked with TOM/FRODO. Rigid-body refinement of the model and subsequent refinement using molecular dynamics were performed with X-PLOR, leading to a current R factor of 25.9%, for data up to 2.3 Å.

Notes: An error in the paper by Naismith, Habash, Harrop, Helliwell, Hunter, Wan, Weisgerber, Kalb & Yariv [Acta Cryst. (1993), D49, 561–571] is corrected. The first sentence of the caption for Fig. 6 on p. 568 should read: The S1 (Cd2+) and S2 (Ca2+) metal sites.

Notes: The speed of electron-density fitting during X-ray structure solution and refinement, and the quality of the protein model resulting, can both be enhanced by the use of databases of main- and side-chain conformations. Three structures are compared in this report, one refined at high resolution (1.7 Å), and two at lower resolutions using either the database method (2.4 Å resolution) or more traditional empirical electron-density fitting (1.9 Å resolution). An analysis of peptide orientation was used as an aid in finding unusual portions of main-chain structure. The fit of side chains to known rotamer conformations was used to help determine the accuracy of these atomic positions. In addition, the use of an objective measure of the fit of structures to electron-density maps was evaluated, both alone and in combination with side-chain conformational information.

Notes: Ferredoxin I (Fd I) from Equisetum arvense is an iron–sulfur protein composed of 95 amino-acid residues and one [2Fe–2S] cluster. It crystallized in the space group P21, a = 30.4, b = 57.4, c = 47.5 Å and β = 78.7° with two molecules per asymmetric unit. X-ray diffraction data up to 1.8 Å resolution were collected by using a Rigaku four-circle diffractometer. The initial model of Fd I, which was derived by the molecular replacement method using a structure of the Fd I from the blue–green alga Aphanothece sacrum, was refined by molecular dynamics simulation and a least-squares minimization with stereochemical restraints. Positional parameters and isotropic temperature factors for 1420 non-H protein atoms and 183 water molecules were refined on 13 838 observed structure factors (Fo 〉 σFo) between 10.0 and 1.8 Å resolution. The final Rfactor was 17.0%, and the standard deviation of atomic position estimated by Luzzati plot [Luzzati (1952). Acta Cryst. 5, 802–810] was 0.2 Å. The electron-density map was well defined for the two independent molecules except for the N-terminal residue and the three C-terminal residues. Equivalent Cα atoms of two independent molecules in the asymmetric unit were superposed by the least-squares method with root-mean-square deviations of 0.26 Å. Reasonable structural differences were observed at a polypeptide segment having few intramolecular interactions. Highly flexible regions of the molecule were assigned from the structural differences between the two independent molecules in the crystal and the distribution of temperature factors along the polypeptide chain.

Notes: A computer program, VOIDOO, is described which can be employed in the study of cavities such as they occur in macromolecular structures (in particular, in proteins). The program can be used to detect unknown cavities or to delineate known cavities, either of which may be connected to the outside of the molecule or molecular assembly under study. Optionally, output files can be requested that contain a description of the shape of the cavity which can be displayed by the crystallographic modelling program O. Additionally, VOIDOO can be used to calculate the volume of a molecule and to create a file containing data pertaining to the surface of the molecule which can also be displayed using O. Examples of the use of VOIDOO are given for P2 myelin protein, cellular retinol-binding protein and cellobiohydrolase II. Finally, operational definitions to discern different types of cavity are introduced and guidelines for assessing the accuracy and improving the comparability of cavity calculations are given.

Notes: An automatic molecular-replacement procedure has been applied to solve the crystal structure of cytochrome c2 from Rhodopseudomonas viridis. The structure was solved on the basis of the structure of tuna cytochrome c as a search model using an automatic processing program system, AUTOMR. The refinements by molecular dynamics and restrained least-squares methods result in a current crystallographic R factor of 0.219 for diffraction data at 3 Å resolution.

Notes: Crystals of cadmium-substituted azurin have been prepared by diffusing CdII into crystals of apo-azurin grown previously and their structure has been determined at high resolution by X-ray crystallography. Data to 1.8 Å resolution were collected by Weissenberg photography (with image plates) using synchrotron radiation. These data were combined with a 2.2 Å diffractometer data set to give 90% coverage to 1.8 Å. An initial model was derived from the isomorphous CuII-azurin structure, and the cadmium and ligand positions added from `omit' maps. Refinement was by restrained least squares (program PROLSQ), to a final R value of 0.168 for all data in the range 10.0–1.8 Å (23 349 reflections). The final model of 1954 protein atoms, two CdII ions (occupancy 0.75), four SO{_4^{2-}} ions and 239 water molecules has r.m.s. deviations of 0.015, 0.045 and 0.013 Å from standard bond lengths, angle distances and planar groups. The protein structure is essentially the same as that of CuII-azurin, with an r.m.s. deviation of 0.18 Å for 97% of main-chain atoms after superposition of the two structures. The Cd atom is within 0.2 Å of the equivalent copper position, displaced slightly away from the axial Met ligand towards the carbonyl O atom of Gly45. The latter has also moved slightly towards the metal, by a rotation of the peptide unit, to give a Cd—O bond of 2.76 Å. The Cd—S(Cys) bond is lengthened to 2.39 Å. The coordination geometry is slightly more tetrahedral than for CuII, and the cadmium–oxygen interaction is consistent with the presence of an oxygen ligand in the coordination sphere of stellacyanin.

Notes: . Sulfur atoms, an integral part of many proteins, are possible candidates for anomalous scattering in phase determination by multiple-wavelength methods. The main difficulty encountered is that a wavelength of about 5 Å is required to obtain a large anomalous signal from these atoms, leading to very large absorption effects. Initial experiments have been carried out using a synchrotron X-ray source, evacuated beam tubes, a diffractometer inside a vacuum chamber, a special sample holder and a suitable scattering geometry. The results are encouraging, showing that Bragg reflections can be measured, and that changes in their intensities around the absorption edge are observable.

Notes: The structure of the serine protease trypsin from the North Atlantic salmon (Salmo salar) has been solved by molecular replacement and refined by restrained least-squares methods to a conventional R factor of 16.4% using diffractometer data in the 6.0–1.82 Å resolution range (14 443 reflections greater than 3σ). The model comprises 1793 protein atoms and 180 solvent molecules which were given unit occupancies, and the average temperature factors for protein atoms and solvent oxygen atoms are 15.2 and 36.8 Å2, respectively. The estimated error in atomic positions is about 0.2 Å. The structure of salmon trypsin was solved and refined with only a small part of the amino-acid sequence known. However, a gene sequence of salmon trypsin has later become available. Some discrepancies between this sequence and the sequence obtained from the present X-ray crystal study indicate that the mentioned sequences may correspond to isoenzymes. The structure of salmon trypsin is similar to other trypsins of known structure.

Notes: The crystal structure of prolyl-glutaminyl-valyl-statyl-alanyl-leucine (Pro-Gln-Val-Sta-Ala-Leu, C32H57N709.5H20, Mr = 683.9 + 90.1), a putative HTLV-1 protease inhibitor based on one of the consensus retroviral protease cleavage sequences, and containing the statine residue [(4S,3S)-4-amino-3-hydroxy-6-methylheptanoic acid], has been determined by X-ray diffraction. The same molecule has been modelled in the active site of the HTLV-1 protease and both conformations have been compared. The peptide crystallizes as a pentahydrate in space group P21 with a = 10.874(2), b = 9.501(2), c = 21.062(5) Å, β = 103.68 (1)°, Z = 2, V= 2114.3 Å3, Dx = 1.21 g cm−3, μ = 8.02 cm−1, T= 293 K, λ(Cu Kα) = 1.5418 Å. The structure has been refined to an R value of 0.070 for 2152 observed reflections. The peptide main chain can be described as extended and adopts the usual zigzag conformation from the prolyl to the statyl residue. The main difference in conformation between the individual observed and modelled molecules is located on the Sta, Ala and Leu residues with the main chain of the modelled molecule rotated by about 180° as compared to the observed conformation in the crystal state.

Notes: Monoclonal antibody 4B7 is a neutralizing antibody that binds the protein Pfs25 in the sexual stages of the malaria parasite Plasmodium falciparum and completely blocks transmission of the parasite from human serum to the mosquito host. Here we report the identification of the epitope on Pfs25 recognized by 4B7 and the crystallization of the intact murine monoclonal antibody with peptides corresponding to that epitope. This study highlights the importance of ligands in the crystallization of proteins. In this case peptides have been used to modulate the solubility of the peptide–IgG complex and may have provided different or additional crystal contacts to create or enhance a crystalline reticulum. Multiple crystal forms characterize this crystallization and the various peptides, differing both in length and sequence, have been used to investigate how such changes affect nucleation and crystal growth.

Notes: The diffraction resolution of crystals of the guanine nucleotide-exchange factor complex, EF–Tu–Ts, has been extended from 5.0 to 2.5 Å by lowering the solvent content in the crystals as well as the temperature of data collection. The common form of EF–Tu–Ts crystal belongs to space group P212121 with a = 81.1, b = 109.9, c = 207.5 Å and has a solvent content of 61%. The crystals diffract to a resolution of 5.0 Å at 293 K and 4.0 Å at 273 K. When cryoprotective agents are slowly diffused into the crystals, the cell constants shrink to a = 74.4, b = 109.9, c = 198.7 Å and the solvent content falls to 55%. After the cryoprotective agent has been added, the crystals diffract to 2.7 Å resolution at 293 or 273 K and 2.5 Å at 250 K. X-ray diffraction data, collected before and after the transformation of individual EF–Tu–Ts crystals, demonstrate that a large percentage of the improvement in diffraction resolution is due solely to the addition of cryoprotective agents. The transfer procedures for the successful introduction of cryoprotective agents into EF–Tu–Ts crystals as well as the general applicability to other crystal systems will be discussed.

Notes: A computer-simulation method is proposed for studying the hydrodynamic interactions of rigid protein molecules. It is a combination of Stokes dynamics and continuum hydrodynamics. The Stokes equations of motion for the protein molecules, the creeping-flow equation for the solvent together with the no-slip boundary conditions give a complete representation of the system. The resulting three-dimensional boundary-value problem can be rewritten in a two-dimensional form (without any loss of information) considering the surfaces of the particles only. Then, by solving the equations on discrete surface elements, the so-called mobility matrix is determined in which all hydrodynamic interactions are included. Finally, after calculation of the conservative forces and the stochastic force, the new velocities of the protein molecules can be determined. The simulation method can be applied to arbitrary particle shapes. It can also handle arbitrary flow fields, and the effects of applying a flow field to the system can be studied. From analysis of the trajectories, information can be gained on the kinetics and thermodynamics in the early stages of the crystallization process.

Notes: All hitherto solved crystal structures of the catalytic (C) subunit of cAMP-dependent protein kinase can be classified into two groups, those with a closed and those with an open conformation of the ATP-binding lobe. The molecules with the closed conformation are all related by a crystallographic 21 axis that connects them into an infinite-chain motif. The motif has only one large contact region that involves many residues, several of them in the ATP-binding lobe, embedded in an extensive network of water molecules. The dominant feature of this region is the hydrophobic interaction between Trp196 and Arg133, Arg134. This motif has been found so far in three different crystal forms, two correspond to ternary enzyme–inhibitor–ATP complexes with mammalian and recombinant C, and one to a binary enzyme–inhibitor complex with recombinant C. The open conformation has been found in two closely related crystal structures, both of cubic symmetry, of the apoenzyme and a binary complex of the mammalian catalytic subunit. In this cubic structure of the binary complex, the hydrogen-bonded intramolecular contacts between Arg18 of the inhibitor and the ATP-binding lobe of the binary and ternary complexes of the recombinant enzyme are missing due to a strong hydrophobic intermolecular contact involving the diiodinated Tyr7. In solution, no crystal contacts prevent these hydrogen bonds involving Arg18 from forming so that it is likely that the binary complex with Tyr7 of the peptide inhibitor iodinated or not, can assume the closed conformation in solution. While the closed structure very likely represents a stable conformation in solution, there is no evidence to suggest that the open conformation represents a unique stable conformational state of the enzyme in solution.

Notes: A novel procedure has been developed for locating heavy-atom positions in crystals of macromolecules. This method used genetic algorithms (GA's) to search for heavy-atom sites that are consistent with an observed difference Patterson function. The procedure is straightforward to apply, space-group independent, and particularly powerful for cases involving non-crystallographic symmetry of multiple heavy atoms in the asymmetric unit. In this paper, we introduce how GA's are used for determining the heavy-atom positions and show how this method is more efficient than a sequential search.

Notes: Ferricytochromes c were crystallized at low ionic strength by macroseeding techniques. Large crystals were grown by seed-induced self-nucleation which occurred anywhere in the drop, regardless of the location of the seed crystal. This unusual crystal-seeding method worked reproducibly in our hands, and X-ray quality crystals have been prepared of several ferricytochromes c: horse, rat (recombinant wild type), and two site-directed mutants of the latter, tyrosine 67 to phenylalanine (Y67F) and asparagine 52 to isoleucine (N52I). Crystals of any one of these four proteins could be used as seeds for the crystallization of any one of the others. All the crystals are of the same crystal form, with space group P212121. There are two protein molecules per asymmetric unit. The crystals are stable in the X-ray beam and diffract to at least 2.0 Å, resolution. Full crystallographic data sets have been collected from single crystals of all four proteins.

Notes: Crystals of macromolecules often have two or more molecules per asymmetric unit, or contain domains of a macromolecule or a macromolecular complex that are structurally independent. In such cases the conventional molecular-replacement method attempts to determine the position of each structural unit independently. Typically, some parts of the structure can be determined more easily or more reliably than other parts. Methods are proposed whereby information from a part of a crystal structure that has been determined can be used to help determine the structure of the remainder. Two different strategies are discussed, `subtraction' and `addition'. With `subtraction' strategy the Patterson function of the known part of the structure is subtracted from the `observed' Patterson. This approach is found to be most effective in the context of the rotation function in that it eliminates peaks that are irrelevant to the desired solution. With `addition' strategy the structure factors of the known component are added to those of the search model. This procedure is most effective in the context of the translation function because it brings the structure factors calculated from the search model closer to those observed. Methods of applying the fast Fourier transform to facilitate these calculations are described. A number of examples are provided including structures of mutants of T4 lysozyme that might not have been solved without recourse to the proposed methods. A method of including information from a heavy-atom derivative in a translation function is also developed and shown to be superior in some situations to the conventional translation function.

Notes: Core tracing is a threshold-independent method of determining connectivity (long chains of high-density values) in electron-density maps. It gives visually sparse pictures of large volumes which are useful for initial fitting and for molecular-boundary determination. New methods for visual presentation of the traces are suggested by the way that the connectivity is parameterized in terms of local connections between maxima and the saddle (lowest) points along the connecting paths. The algorithm also partitions the density into small compact volumes containing the maxima. These volumes are useful for localization and statistical analysis.

Notes: The crystal structures of two anionic inhibitor complexes of human carbonic anhydrase I (HCAI), namely, HCAI–iodide and HCAI–Au(CN)2−, have been refined by the restrained least-squares method at 2.2 and 2 Å nominal resolution, respectively, with good stereochemistry for the final models. The R values have improved from 30.3 to 16.6% for HCAI–iodide and from 28.8 to 17.1% for HCAI–Au(CN)2−. The sites of inhibitor binding as elucidated are totally different in the two structures. The iodide anion replaces the zinc-bound H2O/OH− ligand and renders the enzyme inactive. This result confirms that the zinc-bound H2O/OH− is the activity-linked group in carbonic anhydrase enzymes. Au(CN)2− binds at a different and new site near the zinc ion, without liganding to the metal. The N atom of Au(CN)2− is within hydrogen-bonding distance of the zinc-bound H2O/OH− group which shifts by about 0.4 Å away from the zinc ion in relation to its position in the native HCAI. It is proposed that the presence of the inhibitor Au(CN)2− results in a conformational reorientation of the activity-linked group, due to hydrogen-bond formation with the inhibitor, which in turn sterically hinders the binding of the substrate CO2 molecule in the active site, leading to the inhibition of HCAI enzyme.

Notes: Ferritin, the iron-storage protein, binds porphyrins, metalloporphyrins and the fluorescent dyes ANS (8-anilino-1-naphthalenesulfonic acid) and TNS (2-p-toluidinyl-6-naphthalenesulfonic acid), similarly to apo-myoglobin. Octahedral crystals of horse-spleen apo-ferritin (HSF; 174 amino acids) complexes prepared by the addition of haem, hematoporphyrin or Sn-protoporphyrin IX to a solution of apo-ferritin crystallize in space group F432 with cell parameter a = 184.0 Å. X-ray crystallographic analysis of single crystals prepared from a mixture containing haem or Sn-protoporphyrin IX shows that the haem-binding sites in these crystals are occupied by protoporphyrin IX, which is free of metal, rather than by the original metalloporphyrin. The present paper describes the structure of horse-spleen apo-ferritin cocrystallized with Sn-protoporphyrin IX. The 6797 reflections up to 2.6 Å resolution used in the refinement were obtained from a data set recorded on a Nicolet/Xentronics area detector with Cu Kα radiation from a Rigaku RU 200 rotating anode. The final structure comprises 1613 non-H atoms, two Cd atoms and 170 solvent molecules. Four residues are described as disordered. The root-mean-square deviations from ideal bond lengths and angles are 0.013 A and 2.88°, respectively. Protoporphyrins are observed in special positions on the twofold axes of the ferritin molecule with a stoichiometry of 0.4 per subunit.

Notes: The structure of the copper protein plastocyanin from poplar leaves (Populus nigra var. italica) at 173 K has been subjected to two independent refinements, using a single set of synchrotron X-ray data at 1.6 A resolution. Energy-restrained refinement using the program EREF resulted in lower root-mean-square deviations from ideal geometry (e.g. 0.011 Å for bond lengths) but a higher residual R (0.153) than restrained least-squares refinement using the program PROLSQ (0.014 Å, 0.132). Electron-density difference maps in both refinements provided evidence for disorder at some side chains and solvent atoms, and the PROLSQ refinement made allowance for this disorder. The number of solvent sites identified at the 4σ(ρ) level was 171 in the EREF refinement and 189 in the PROLSQ refinement; 159 of the solvent sites are common to both refinements within 1 Å. The root-mean-square differences between the atomic positions produced by the two refinements are 0.08 Å for Cα atoms, 0.08 Å for backbone atoms and 0.12 Å for all non-H atoms (excluding six obvious outliers) of the protein molecule. The two sets of Cu–ligand bond lengths differ by up to 0.07 Å, and the ligand–Cu–ligand angles by up to 7°. At 173 K the volume of the unit cell is 4.2% smaller than at 295 K. Greater order in the solvent region is indicated by the location of 79 more solvent sites, the identification of extensive networks of hydrogen-bonded rings of solvent molecules, and a general decrease in the thermal parameters. Within the unit cell, the protein molecules are significantly translated and rotated from their positions at ambient temperature. An important structural change at low temperature is a 180° flip of the peptide group at Ser48-Gly49. Nearly all other significant differences between the structures of the protein at 173 and 295 K occur at exposed side chains. If the backbone atoms in the 173 and 295 K structures are superposed, excluding atoms involved in the peptide flip, the root-mean- square difference between the positions of 393 atoms is 0.25 Å. Two internal water molecules, not included in previous descriptions of poplar plastocyanin, have been located. The plastocyanin Cu-site geometry at 173 K is not significantly different from that at 295 K. If plastocyanin undergoes a change in Cu-site geometry at low temperature, as has been suggested on the basis of resonance Raman spectroscopic evidence, then the change is not detected within the limits of precision of the present results.

Notes: Crystals have been obtained of glyceraldehyde 3-phosphate dehydrogenase from the extreme thermophile, Thermus aquaticus. This enzyme is stable and active at 363 K, thus its three-dimensional structure should add insight into the structural basis of protein thermostability. Large high-quality crystals were grown using isopropanol and polyethylene glycol at pH 8.4. They crystallize in the orthorhombic space group P212121 with cell dimensions a = 144.77 (6), b = 148.77 (5), c = 149.50 (7) Å, and diffract to beyond 2.8 Å. The volume of the unit cell and the packing observed in other GAPDH structures suggest that there are two tetramers per asymmetric unit. With 300 kDa/asymmetric unit expected in this form, its solution represents a challenging molecular replacement problem. A low-resolution data set has been recorded and used to carry out self-rotation, cross-rotation and Patterson-correlation refinement calculations. We found that the Q molecular axes of both tetramers are approximately coincident with the crystallographic a axis, and the non-crystallographic symmetry relating the two tetramers is approximately a rotation of 90° about the a axis.

Notes: Diffraction data to 2.7 Å resolution were measured on crystals of the homotetramers of components II and III of the cytoplasmic hemoglobin of the symbiont-harboring clam Lucina pectinata. Even though the crystallization conditions are different and the sequence homology of the two hemoglobins is only 63%, the crystals are isomorphous to each other and to the heterotetramer Hb II/III, implying that the residues primarily involved in the intermolecular interactions and responsible for crystal cohesion may be invariant.