Showing papers in "Acta Crystallographica Section D-biological Crystallography in 2000"

Maximum-likelihood density modification

Abstract: A likelihood-based approach to density modification is developed that can be applied to a wide variety of cases where some information about the electron density at various points in the unit cell is available. The key to the approach consists of developing likelihood functions that represent the probability that a particular value of electron density is consistent with prior expectations for the electron density at that point in the unit cell. These likelihood functions are then combined with likelihood functions based on experimental observations and with others containing any prior knowledge about structure factors to form a combined likelihood function for each structure factor. A simple and general approach to maximizing the combined likelihood function is developed. It is found that this likelihood-based approach yields greater phase improvement in model and real test cases than either conventional solvent flattening and histogram matching or a recent reciprocal-space solvent-flattening procedure [Terwilliger (1999), Acta Cryst. D55, 1863–1871].

An approach to multi-copy search in molecular replacement.

Abstract: The molecular-replacement method has been extended to a simultaneous search for multiple copies of the macromolecule in the unit cell. The central point of this approach is the construction of a multi-copy search model from the properly oriented monomers using a special translation function. The multi-copy search method has been implemented in the program MOLREP and successfully tested using experimental data.

Structures of recombinant native and E202Q mutant human acetylcholinesterase complexed with the snake-venom toxin fasciculin-II

Abstract: Structures of recombinant wild-type human acetylcholinesterase and of its E202Q mutant as complexes with fasciculin-II, a `three-finger' polypeptide toxin purified from the venom of the eastern green mamba (Dendroaspis angusticeps), are reported. The structure of the complex of the wild-type enzyme was solved to 2.8 A resolution by molecular replacement starting from the structure of the complex of Torpedo californica acetylcholinesterase with fasciculin-II and verified by starting from a similar complex with mouse acetylcholinesterase. The overall structure is surprisingly similar to that of the T. californica enzyme with fasciculin-II and, as expected, to that of the mouse acetylcholinesterase complex. The structure of the E202Q mutant complex was refined starting from the corresponding wild-type human acetylcholinesterase structure, using the 2.7 A resolution data set collected. Comparison of the two structures shows that removal of the charged group from the protein core and its substitution by a neutral isosteric moiety does not disrupt the functional architecture of the active centre. One of the elements of this architecture is thought to be a hydrogen-bond network including residues Glu202, Glu450, Tyr133 and two bridging molecules of water, which is conserved in other vertebrate acetylcholinesterases as well as in the human enzyme. The present findings are consistent with the notion that the main role of this network is the proper positioning of the Glu202 carboxylate relative to the catalytic triad, thus defining its functional role in the interaction of acetylcholinesterase with substrates and inhibitors.

Structural changes in a cryo-cooled protein crystal owing to radiation damage

Abstract: The high intensity of third-generation X-ray sources, along with the development of cryo-cooling of protein crystals at temperatures around 100 K, have made it possible to extend the diffraction limit of crystals and to reduce their size. However, even with cryo-cooled crystals, radiation damage becomes a limiting factor. So far, the radiation damage has manifested itself in the form of a loss of overall diffracted intensity and an increase in the temperature factor. The structure of a protein (myrosinase) after exposure to different doses of X-rays in the region of 20 × 1015 photons mm−2 has been studied. The changes in the structure owing to radiation damage were analysed using Fourier difference maps and occupancy refinement for the first time. Damage was obvious in the form of breakage of disulfide bonds, decarboxylation of aspartate and glutamate residues, a loss of hydroxyl groups from tyrosine and of the methylthio group of methionine. The susceptibility to radiation damage of individual groups of the same kind varies within the protein. The quality of the model resulting from structure determination might be compromised owing to the presence of radiolysis in the crystal after an excessive radiation dose. Radiation-induced structural changes may interfere with the interpretation of ligand-binding studies or MAD data. The experiments reported here suggest that there is an intrinsic limit to the amount of data which can be extracted from a sample of a given size.

Novel approach to phasing proteins: derivatization by short cryo‐soaking with halides

Abstract: A quick (less than 1 min) soak of protein crystals in a cryo-solution containing bromide or iodide anions leads to incorporation of these anomalous scatterers into the ordered solvent region around the protein molecules. These halide anions provide a convenient way of phasing through their anomalous scattering signal: bromides using multiwavelength anomalous dispersion (MAD) and bromides and/or iodides using single-wavelength anomalous dispersion (SAD) or single isomorphous replacement with anomalous scattering (SIRAS) methods. This approach has been tested successfully on four different proteins and has been used to solve the structure of a new protein of molecular weight 30 kDa.

Asymmetries in the nucleosome core particle at 2.5 A resolution.

Abstract: The 2.5 A X-ray crystal structure of the nucleosome core particle presented here provides significant additions to the understanding of the nucleosome, the fundamental unit of chromatin structure. Extensions are made to the structure of the N-terminal histone tails and details are provided on hydration and ion binding. The structure is composed of twofold symmetric molecules, native chicken histone octamer cores and the DNA palindrome, which were expected to form a perfectly twofold symmetric nucleosome core particle. In fact, the result is asymmetric owing to the binding of the DNA to the protein surface and to the packing of the particles in the crystal lattice. An analysis is made of the asymmetries by comparisons both within the nucleosome core particle and to the structure of the histone octamer core of the nucleosome.

Accurate calculation of the density of proteins.

Abstract: On the basis of theoretical calculations, Andersson & Hovmoller have recently suggested that the long-established value of 1.35 g cm−3 for the mean density of proteins should be revised to 1.22 g cm−3 [Andersson & Hovmoller (2000), Acta Cryst. D56, 789–790]. To substantiate their assertion, these authors used the Voronoi algorithm to calculate the mean atomic volume for 30 representative protein structures. The Voronoi procedure requires that atoms of interest be bounded on all sides by other atoms. Volume calculations for surface atoms that are not surrounded or are only sparsely surrounded by other atoms either are not possible or may be unreliable. In an attempt to circumvent this problem, Andersson & Hovmoller rejected atoms with calculated volumes that were indeterminate or were greater than 50 A3. In the present study, it is shown that this criterion is not sufficiently restrictive to ensure accurate volume determinations. When only strictly buried atoms are included in the volume calculations using the Voronoi algorithm, the mean density is found to be 1.47 ± 0.05 g cm−3. In addition, an alternate procedure based on the Connolly algorithm that permits all protein atoms to be included in volume calculations gives 1.43 ± 0.03 g cm−3 for the mean density of the same set of proteins. The latter two calculated values are mutually consistent and are in better agreement with the experimental value.

Fitting atomic models into electron-microscopy maps

Abstract: Combining X-ray crystallographically determined atomic structures of component domains or subunits with cryo-electron microscopic three-dimensional images at around 22 A resolution can produce structural information that is accurate to about 2.2 A resolution. In an initial step, it is necessary to determine accurately the absolute scale and absolute hand of the cryo-electron microscopy map, the former of which can be off by up to 5%. It is also necessary to determine the relative height of density by using a suitable scaling function. Difference maps can identify, for instance, sites of glycosylation, the position of which helps to fit the component structures into the EM density maps. Examples are given from the analysis of alphaviruses, rhinovirus–receptor interactions and poliovirus–receptor interactions.

Docking structures of domains into maps from cryo-electron microscopy using local correlation.

Abstract: Accurate maps of large macromolecular complexes can be calculated from cryo-electron micrographs of non-crystalline specimens to resolutions of about 10 A. A method to dock the atomic structures of domains solved by X-ray crystallography or nuclear magnetic resonance into cryo-EM maps is presented. Domains can be docked independently into large complexes without prior definition of the boundaries. No special symmetry is assumed and the procedure is suitable for general application to almost any system where a cryo-EM map (at 15 A resolution or better) of a complex has been obtained and the atomic structures of the components are available. This is achieved through use of a real-space density-matching procedure based on local correlation. A complete asymmetric unit search correlating a density object derived from the atomic coordinates and the density of the EM map is performed. The correlation coefficient is calculated locally in real space using only values of the search object and corresponding samples extracted from the EM map which are under the `footprint' of the positioned search object. The procedure has been demonstrated by docking the domains of GroEL from the crystal structure into a cryo-EM map Fourier filtered to 12 or 15 A resolution. The correct positions were found without applying any additional constraints. A model of the oligomer built from the docked domains compared favourably with the known crystal structure, confirming the validity of the approach. The procedure is designed to facilitate the incorporation of additional constraints on the docking solutions, which could help to dock using lower resolution maps.

Validation of protein crystal structures.

Abstract: Since the process of building and refining a model of a biomacromolecule based on crystallographic data is subjective, quality-control techniques are required to assess the validity of such models. During the 1990s, much experience was gained; the methods used and some of the lessons learned are reviewed here. In addition, an extensive compendium of quality criteria and quality-control methods that are or have been used to validate models of biomacromolecules has been compiled. The emphasis in this compendium is on the validation of protein crystal structures.

Single-wavelength anomalous diffraction phasing revisited

Abstract: Multiwavelength anomalous diffraction (MAD) phasing has become a routinely used tool for determining new macromolecular structures. The MAD method has stringent data-collection requirements, typically necessitating radiation-resistant crystals and access to a tunable synchrotron beamline. In cases where synchrotron time, monochromator tunability or radiation damage is a concern or where high-throughput structure determination is desired, phasing methods capable of producing interpretable electron-density maps from less data become attractive alternatives to MAD. The increasing availability of tunable synchrotron data-collection facilities prompted the authors to revisit single-wavelength anomalous diffraction (SAD) phasing used in conjunction with a phase-ambiguity resolving method such as solvent flattening. The anomalous diffraction from seven different selenomethionine-labelled protein crystals has been analysed and it is shown that in conjunction with solvent flattening, diffraction data from the peak anomalous wavelength alone can produce interpretable electron-density maps of comparable quality to those resulting from full MAD phasing. Single-wavelength anomalous diffraction (SAD) phasing can therefore be a time-efficient alternative to MAD. The data also show that radiation damage can have a significant effect on the quality of SAD/MAD diffraction data. These results may be useful in the design of optimal strategies for collection of the diffraction data.

Resolution measurement in structures derived from single particles

Abstract: Analytical expressions are derived and computer simulations are presented to assess the accuracy of procedures commonly used to estimate the resolution of three-dimensional (3D) structures derived from images of single protein molecules or complexes. It is shown that in the case of a low signal-to-noise ratio in the images, the Fourier ring correlation between two structures, each calculated using one half of the data, significantly overestimates the resolution when the two half data sets were aligned against the same reference structure. The overestimate arises because of a correlation between the noise components present in the images. The correlation is introduced by the alignment and becomes more serious as the signal-to-noise ratio is reduced. A reliable resolution measure is only obtained when the two half data sets are aligned against two independent reference structures. It is further shown that the noise correlation also significantly affects the spectral signal-to-noise ratio and the Q factor, making them unreliable measures of signal present in a 3D structure and in the original images, respectively. It is concluded that the alignment of images is always accompanied by a correlation of the noise and that this correlation is indistinguishable from a correlation arising from a signal.

The geometry of metal–ligand interactions relevant to proteins. II. Angles at the metal atom, additional weak metal–donor interactions

Abstract: Geometrical data which could be of relevance in the structure determination, structure refinement, assessment or understanding of metalloproteins have been extracted from the Cambridge Structural Database (CSD). The CSD contains crystallographic data from `small-molecule' structures determined by X-ray or neutron diffraction to an accuracy and precision much better than that of most current protein structure determinations. Structures of Mg, Mn, Fe, Cu and Zn complexes with ligands whose donor atoms may be only N, O, S or Cl have been selected and analysed in terms of the geometry of the metal coordination group – octahedral, tetrahedral, tetragonal pyramidal etc. The r.m.s. deviation of all the interbond angles around the metal atom provides a measure, δ, of the deviation from ideal geometry. Average values of δ are tabulated for the different metals in each type of complex. For simple non-chelated complexes of Mn, Fe and Zn, distortions of up to 5° in octahedral complexes and 10° in tetrahedral complexes are found to be normal and seem likely to be a consequence of packing effects, ligand bulk or intramolecular effects. Substantially larger distortions are found for some other metals and geometries and are common for chelated complexes. Brief comments on six-, seven- and eight-coordinate Ca complexes are included. Tables are also presented showing that for four- and five-coordinate complexes of Zn and Cu it is quite common to find additional weakly coordinated ligands, usually with N or O donor atoms and with M⋯N,O distances longer than a normal bond length but shorter than a van der Waals contact, e.g. in the range 2.4–3.0 A for Zn and 2.6–3.0 A for Cu. Although the contributions to bond valency or bonding energy of such interactions may not be great, their effect on geometry can be considerable; they can, for example, cause much larger distortions of tetrahedral Zn complexes than indicated above.

The porphobilinogen synthase family of ­metalloenzymes

Abstract: The porphobilinogen synthase (PBGS) family of enzymes catalyzes the first common step in the biosynthesis of the essential tetrapyrroles such as chlorophyll and porphyrin. Although PBGSs are highly conserved at all four levels of protein structure, there is considerable diversity in the use of divalent cations for the catalytically essential and allosteric roles. Assumptions regarding commonalities among the PBGS proteins coupled with the diversity of usage of metal ions has led to a confused literature. The recent publication of crystal structures for three PBGS proteins coupled with more than 50 individual PBGS sequences allows an evaluation of these assumptions. This topical review focuses on the usage of metals by the PBGS family of proteins. It raises doubt concerning a dogma that there has been an evolutionary shift between ZnII and MgII at one or more of the divalent metal-binding sites. It also raises the possibility that there may be up to four specific divalent metal ion-binding sites, each serving a unique function that can be alternatively filled by amino acids in some of the PBGSs.

Crystallization in cubo: general applicability to membrane proteins

Abstract: Obtaining well ordered crystals of membrane proteins is the single most serious stumbling block in the pursuit of their high-resolution structures. The applicability of lipidic cubic phase-mediated crystallization is demonstrated on a diverse set of bacterial membrane proteins: two photosynthetic reaction centres, a light-harvesting complex and two retinal proteins, halorhodopsin and bacteriorhodopsin. Despite marked differences in molecular dimensions, subunit composition and membrane origin, one single lipid, monoolein, is sufficient to form a crystallization matrix for all the aforementioned systems. Therefore, the lipidic cubic phase approach is proposed as a general method for crystallizing membrane proteins.

Objective comparison of protein structures: error-scaled difference distance matrices

Abstract: Understanding of macromolecular function in many cases relies on the comparison of related structural models. Commonly used least-squares superposition methods suffer from bias introduced into the comparison process by the subjective choice of atoms employed for the superposition. Difference distance matrices are a more objective means of comparing structures as they do not depend on a particular superposition scheme. However, they suffer from very high noise originating from coordinate errors. Modern refinement programs allow the rigorous estimation of standard uncertainties for individual atomic positions. These errors can be propagated through the calculation of a difference distance matrix allowing one to assess the significance level of structural differences. An algorithm is presented which produces an intuitive graphical representation of difference distance matrices after normalization to their error levels. Two examples where its application was revealing are described. Alternatives are suggested for cases where rigorous estimation of individual errors by the inversion of the full least-squares matrix is not feasible. The method offers an unbiased way to detect significant similarities and differences between related structures, as encountered in studies of complexes and mutants or when multiple models are obtained from experiments such as crystal structures involving non-crystallographic symmetry or different crystal modifications, or ensembles derived from NMR spectroscopy.

The Three-Dimensional Structure of a Trichoderma Reesei Beta-Mannanase from Glycoside Hydrolase Family 5

Abstract: The crystal structure of the catalytic core domain of β-­mannanase from the fungus Trichoderma reesei has been determined at a resolution of 1.5 A. The structure was solved using the anomalous scattering from a single non-isomorphous platinum complex with two heavy-metal sites in space group P21. The map computed with the experimental phases was enhanced by the application of an automated model building and refinement procedure using the amplitudes and experimental phases as observations. This approach is expected to be of more general application. The structure of the native enzyme and complexes with Tris–HCl and mannobiose are also reported: the mannobiose binds in subsites +1 and +2. The structure is briefly compared with that of the homologous β-­mannanase from the bacterium Thermomonospora fusca.

The oversampling phasing method.

Abstract: Sampling the diffraction pattern of a finite specimen more finely than the Nyquist frequency (the inverse of the size of the diffracting specimen) corresponds to surrounding the electron density of the specimen with a no-density region. When the no-density region is bigger than the electron-density region, sufficient information is recorded so that the phase information can be retrieved from the oversampled diffraction pattern, at least in principle. By employing an iterative algorithm, the phase information from the oversampled diffraction pattern of a micrometre-sized test specimen has been successfully retrieved. This method is believed to be able to open a door for high-resolution three-dimensional structure determination of complex and non-crystalline biological specimens, i.e. whole cells and sub-micrometre molecular clusters and micrometre-sized protein crystals. With the possible appearance in the future of X-ray free-electron lasers, it may become possible to image single molecules by recording diffraction patterns before radiation damage manifests itself.

Structure of Human Transthyretin Complexed with Bromophenols : A New Mode of Binding

Abstract: The binding of two organohalogen substances, pentabromophenol (PBP) and 2,4,6-tribromophenol (TBP), to human transthyretin (TTR), a thyroid hormone transport protein, has been studied by in vitro competitive binding assays and by X-­ray crystallography. Both compounds bind to TTR with high affinity, in competition with the natural ligand thyroxine (T4). The crystal structures of the TTR–PBP and TTR–TBP complexes show some unusual binding patterns for the ligands. They bind exclusively in the `reversed' mode, with their hydroxyl group pointing towards the mouth of the binding channel and in planes approximately perpendicular to that adopted by the T4 phenolic ring in a TTR–T4 complex, a feature not observed before. The hydroxyl group in the ligands, which was previously thought to be a key ingredient for a strong binding to TTR, does not seem to play an important role in the binding of these compounds to TTR. In the TTR–PBP complex, it is primarily the halogens which interact with the TTR molecule and therefore must account for the strong affinity of binding. The interactions with the halogens are smaller in number in TTR–TBP and there is a decrease in affinity, even though the interaction with the hydroxyl group is stronger than that in the TTR–PBP complex.

The structures of deoxy human haemoglobin and the mutant Hb Tyrα42His at 120 K

Abstract: The structures of deoxy human haemoglobin and an artificial mutant (Tyrα42→His) have been solved at 120 K. While overall agreement between these structures and others in the PDB is very good, certain side chains are found to be shifted, absent from the electron-density map or in different rotamers. Non-crystallographic symmetry (NCS) is very well obeyed in the native protein, but not around the site of the changed residue in the mutant. NCS is also not obeyed by the water molecule invariably found in the α-chain haem pocket in room-temperature crystal structures of haemoglobin. At 120 K, this water molecule disappears from one α chain in the asymmetric unit but not the other.

Crystallization, structure solution and refinement of hen egg-white lysozyme at pH 8.0 in the presence of MPD

Abstract: Hen egg-white lysozyme has been crystallized at slightly alkaline pH using 2-methyl-2,4-pentanediol (MPD) as the precipitant. The crystals are nearly isomorphous to crystals grown at acidic pH using sodium chloride as the precipitant. However, the growth kinetics differ markedly between the two conditions. The major reason for this is a molecule of MPD that binds tightly in between two lysozyme molecules and favors the growth of the crystals along the crystallographic c direction over growth perpendicular to it.

Modelling prior distributions of atoms for macromolecular refinement and completion.

Abstract: Until modelling is complete, macromolecular structures are refined in the absence of a model for some of the atoms in the crystal. Techniques for defining positional probability distributions of atoms, and using them to model the missing part of a macromolecular crystal structure and the bulk solvent, are described. The starting information may consist of either a tentative structural model for the missing atoms or an electron-density map. During structure completion and refinement, the use of probability distributions enables the retention of low-resolution phase information while avoiding premature commitment to uncertain higher resolution features. Homographic exponential modelling is proposed as a flexible, compact and robust parametrization that proves to be superior to a traditional Fourier expansion in approximating a model protein envelope. The homographic ­exponential model also has potential applications to ab initio phasing of Fourier amplitudes associated with macro­molecular envelopes.

General quadratic functions in real and reciprocal space and their application to likelihood phasing

Abstract: A general multivariate quadratic function of the structure factors is constructed and transformed to obtain a quadratic function of the continuous electron density. Two special cases, where structure factors are independent and where electron-density values are independent, are examined. These results are related to the new likelihood-based framework of Terwilliger [Terwilliger (1999), Acta Cryst. D55, pp. 1863–1871] for employing structural information which was previously exploited by means of conventional density-modification calculations. The treatment here involves different assumptions and highlights new features of Terwilliger's calculation. The generalization quadratic construction allows the generation of cross terms relating all reflections and electron densities. Other applications of this approach are considered.

The structure of human MRP8, a member of the S100 calcium-binding protein family, by MAD phasing at 1.9 Å resolution

Abstract: The structure of human MRP8 in the calcium-bound form was determined at 1.9 A resolution by X-ray crystallography. The structure was initially solved by MAD phasing of an ytterbium-substituted crystal and was refined against data obtained from a Ca2+-bound crystal. The dimeric form of MRP8 was stabilized by hydrophobic interactions between mutually wrapped helices. There were two EF-hand motifs per monomer and each EF-hand bound one Ca2+ with a different affinity [the affinity of the C-terminal EF-hand (EF-2) for Ca2+ was stronger than that of the N-terminal EF-hand (EF-1)]. Furthermore, replacement with Yb3+ occurred in the C-terminal EF-hand only, suggesting a more flexible nature forEF-2 than for EF-1. This, combined with previous observations that the helix in EF-2 (helix III) undergoes a large conformational change upon calcium binding, suggests that the C-terminal EF-hand (EF-2) plays a role as a trigger for Ca2+-induced conformational change.

Macromolecular structure determination by cryo-electron microscopy

Abstract: Recent advances in transmission electron microscopy (EM) hardware, low-temperature methods and image-processing software have made cryo-EM an important complement to X-­ray crystallography and NMR for macromolecular structure determination, particularly of large assemblies. This review provides a summary of the main advances and a survey of the capabilities of this approach.

Structure of human erythrocyte catalase.

Abstract: Catalase (E.C. 1.11.1.6) was purified from human erythrocytes and crystallized in three different forms: orthorhombic, hexagonal and tetragonal. The structure of the orthorhombic crystal form of human erythrocyte catalase (HEC), with space group P212121 and unit-cell parameters a = 84.9, b = 141.7, c = 232.5 A, was determined and refined with 2.75 A resolution data. Non-crystallographic symmetry restraints were employed and the resulting R value and Rfree were 0.206 and 0.272, respectively. The overall structure and arrangement of HEC molecules in the orthorhombic unit cell were very similar to those of bovine liver catalase (BLC). However, no NADPH was observed in the HEC crystal and a water was bound to the active-site residue His75. Conserved lattice interactions suggested a common growth mechanism for the orthorhombic crystals of HEC and BLC.

The first protein crystal structure determined from high-resolution X-ray powder diffraction data: a variant of T3R3 human insulin–zinc complex produced by grinding

Abstract: X-ray diffraction analysis of protein structure is often limited by the availability of suitable crystals. However, the absence of single crystals need not present an insurmountable obstacle in protein crystallography any more than it does in materials science, where powder diffraction techniques have developed to the point where complex oxide, zeolite and small organic molecular structures can often be solved from powder data alone. Here, that fact is demonstrated with the structure solution and refinement of a new variant of the T3R3 Zn–human insulin complex produced by mechanical grinding of a polycrystalline sample. High-resolution synchrotron X-ray powder diffraction data were used to solve this crystal structure by molecular replacement adapted for Rietveld refinement. A complete Rietveld refinement of the 1630-atom protein was achieved by combining 7981 stereochemical restraints with a 4800-step (dmin = 3.24 A) powder diffraction pattern and yielded the residuals Rwp = 3.73%, Rp = 2.84%, R_{F}^{2} = 8.25%. It was determined that the grinding-induced phase change is accompanied by 9.5 and 17.2° rotations of the two T3R3 complexes that comprise the crystal structure. The material reverts over 2–3 d to recover the original T3R3 crystal structure. A Rietveld refinement of this 815-atom protein by combining 3886 stereochemical restraints with a 6000-step (dmin = 3.06 A) powder diffraction pattern yielded the residuals Rwp = 3.46%, Rp = 2.64%, R_{F}^{2} = 7.10%. The demonstrated ability to solve and refine a protein crystal structure from powder diffraction data suggests that this approach can be employed, for example, to examine structural changes in a series of protein derivatives in which the structure of one member is known from a single-crystal study.

Structure of jack bean chitinase.

Abstract: The structure of jack bean chitinase was solved at 1.8 A resolution by molecular replacement. It is an α-helical protein with three disulfide bridges. The active site is related in structure to animal and viral lysozymes. However, unlike in lysozyme, the architecture of the active site suggests a single-step cleavage. According to this mechanism, Glu68 is the proton donor and Glu90 assists in the reaction by moving towards the substrate and recruiting a water molecule that acts as the nucleophile. In this model, a water molecule was found in contact with Glu90 O∊1 and Thr119 Oγ at a distance of 3.0 and 2.8 A, respectively. The model is in accordance with the observed inversion mechanism.

The refined structure of canavalin from jack bean in two crystal forms at 2.1 and 2.0 A resolution.

Abstract: The structure of canavalin was refined to 2.1 and 2.0 A resolution in cubic and hexagonal crystals of space group P213 and P63, respectively. The threefold molecular symmetry is expressed in the symmetry of both crystals, where each identical subunit is an asymmetric unit. The canavalin subunit consists of two very similar domains, each comprised of a core subdomain having Swiss-roll topology with a loop subdomain that contains helices. The refined canavalin models resolved the discrepancy in amino-acid registers of the secondary-structural elements compared with phaseolin. The presence of strand Z in both domains of canavalin was confirmed and a new helix in the loop between strands A and B of each domain was observed. The models were analyzed in terms of the duplicated vicilin domains. Three strictly conserved residues, two glycines and a proline, were identified. The similarity between entire vicilin molecules is greater than that between separate domains of canavalin and phaseolin. Homology modeling of the sucrose-binding protein (SBP) from soybean showed a plausible trimeric assembly of subunits similar to that of vicilins.

R6 hexameric insulin complexed with m-cresol or resorcinol.

Abstract: The structures of three R6 human insulin hexamers have been determined. Crystals of monoclinic m-cresol–insulin, monoclinic resorcinol–insulin and rhombohedral m-cresol–insulin diffracted to 1.9, 1.9 and 1.78 A, respectively, and have been refined to residuals of 0.195, 0.179 and 0.200, respectively. In all three structures, a phenolic derivative is found to occupy the phenolic binding site, where it forms hydrogen bonds to the carbonyl O atom of CysA6 and the N atom of CysA11. Two additional phenolic derivative binding sites were identified within or between hexamers. The structures of all three hexamers are nearly identical, although a large displacement of the N-terminus of one B chain in both monoclinic structures results from coordination to a sodium ion which is located between symmetry-related hexamers. Other minor differences in structure arise from differences in packing in the monoclinic cell compared with the rhombohedral cell. Based upon the differences in conformation of the GluB13 side chains in T6, T3R^{\rm f}_{3} and R6 hexamers, the deprotonation of these side chains appears to be associated with the T→R conformational transition.