High-pressure protein crystallography (HPPX): instrumentation, methodology and results on lysozyme crystals.

A new set-up and associated methodology for the collection of angle-dispersive diffraction data from protein crystals submitted to high hydrostastic pressure have been developed on beamline ID30 at the ESRF. The instrument makes use of intense X-rays of ultra-short wavelength emitted by two collinear undulators, and combines a membrane-driven diamond-anvil cell mounted on a two-axis goniometer and an imaging-plate scanner. Sharp and clean diffraction pictures from tetragonal crystals of hen egg-white lysozyme (tHEWL) and orthorhombic crystals of bovine erythrocyte Cu, Zn superoxide dismutase (SOD) were recorded at room temperature and pressures up to 0.915 and 1.00 GPa, respectively. The compressibility of tHEWL was determined from unit-cell parameters determined at 24 different pressures up to 0.915 GPa. High-pressure diffraction data sets from several crystals of tHEWL were collected and analyzed. Merging of data recorded on different crystals at 0.30 and 0.58 GPa produced two sets of structure amplitudes with good resolution, completeness, redundancy and R(sym) values. A third set at 0.69 GPa was of a similar quality except a lower completeness. The three structures have been refined. The pressure-induced loss of crystalline order in a tHEWL crystal beyond 0.82 GPa was captured through a series of diffraction pictures.

Low-resolution phase information in multiple-wavelength anomalous solvent contrast variation experiments.

The basic theory and principles of the multiple-wavelength anomalous solvent-contrast (MASC) method are introduced as a contrast-variation technique for generating low-resolution crystallographic phase information on the envelope of a macromolecule. Experimental techniques and practical considerations concerning the choice of anomalous scatterer, sample preparation and data acquisition are discussed. Test cases of crystals of three proteins of differing molecular weights from 14 kDa through to 173 kDa are illustrated. Methods for extracting the moduli of the anomalous structure factors from the MASC data are briefly discussed and the experimental results are compared with the known macromolecular envelopes. In all cases, the lowest resolution shells exhibit very large anomalous signals which diminish at higher resolution, as expected by theory. However, in each case the anomalous signal persists at high resolution, which is strong evidence for ordered sites of the anomalous scatterers. For the smaller two of these proteins the heavy-atom parameters could be refined for some of these sites. Finally, a novel method for phasing the envelope structure-factor moduli is presented. This method takes into account the relatively low number of observations at low resolution and describes the macromolecular envelope with a small number of parameters by presuming that the envelope is a compact domain of known volume. The parameterized envelope is expressed as a linear combination of independent functions such as spherical harmonics. Phasing starts from solutions of a sphere in the unit cell after positional refinement from random trials and the parameters describing the envelope are then refined against the data of structure-factor moduli. The preliminary results using simulated data show that the method can be used to reconstruct the correct macromolecular envelope and is able to discriminate against some false solutions.

Multiwavelength anomalous solvent contrast (MASC): derivation of envelope structure-factor amplitudes and comparison with model values.

A previous article [Fourme et al. (1995). J. Synchrotron Rad. 2, 36-48] presented the theoretical foundations of MASC, a new contrast-variation method using multiwavelength anomalous scattering, and reported the first experimental results. New experiments have been conducted both at the ESRF (Grenoble, France) and at LURE-DCI (Orsay, France), using cryocooled crystals of three proteins of known structures and very different molecular weights. Amplitudes of {GammaT(h)}, the 'normal' structure factors of the anomalously scattering part of the crystal including the solvent zone and the ordered anomalous scattering sites (if any), have been extracted from multiwavelength data. In the very low resolution range (d >/= 20 A), the agreement between experimental {GammaT(h)} and model values calculated from the bulk solvent is all the more satisfactory since the molecular weight of the protein is high. For spacings between 10 and 20 A, the agreement between experimental {GammaT(h)} and model values is also satisfactory if one takes into account ordered anomalous scatterer sites. Such sites have been found in the three cases.

A zipper-like duplex in DNA: the crystal structure of d(GCGAAAGCT) at 2.1 A resolution.

BACKGROUND: The replication origin of the single-stranded (ss)DNA bacteriophage G4 has been proposed to fold into a hairpin loop containing the sequence GCGAAAGC. This sequence comprises a purine-rich motif (GAAA), which also occurs in conserved repetitive sequences of centromeric DNA. ssDNA analogues of these sequences often show exceptional stability which is associated with hairpin loops or unusual duplexes, and may be important in DNA replication and centromere function. Nuclear magnetic resonance (NMR) studies indicate that the GCGAAAGC sequence forms a hairpin loop in solution, while centromere-like repeats dimerise into unusual duplexes. The factors stabilising these unusual secondary structure elements in ssDNA, however, are poorly understood. RESULTS: The nonamer d(GCGAAAGCT) was crystallised as a bromocytosine derivative in the presence of cobalt hexammine. The crystal structure, solved by the multiple wavelength anomalous dispersion (MAD) method at the bromine K-edge, reveals an unexpected zipper-like motif in the middle of a standard B-DNA duplex. Four central adenines, flanked by two sheared G.A mismatches, are intercalated and stacked on top of each other without any interstrand Watson-Crick base pairing. The cobalt hexammine cation appears to participate only in crystal cohesion. CONCLUSIONS: The GAAA consensus sequence can dimerise into a stable zipper-like duplex as well as forming a hairpin loop. The arrangement closes the minor groove and exposes the intercalated, unpaired, adenines to the solvent and DNA-binding proteins. Such a motif, which can transform into a hairpin, should be considered as a structural option in modelling DNA and as a potential binding site, where it could have a role in DNA replication, nuclease resistance, ssDNA genome packaging and centromere function.

Exploring hydrophobic sites in proteins with xenon or krypton.

X-ray diffraction is used to study the binding of xenon and krypton to a variety of crystallised proteins: porcine pancreatic elastase; subtilisin Carlsberg from Bacillus licheniformis; cutinase from Fusarium solani; collagenase from Hypoderma lineatum; hen egg lysozyme, the lipoamide dehydrogenase domain from the outer membrane protein P64k from Neisseria meningitidis; urate-oxidase from Aspergillus flavus, mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis and the ligand-binding domain of the human nuclear retinoid-X receptor RXR-alpha. Under gas pressures ranging from 8 to 20 bar, xenon is able to bind to discrete sites in hydrophobic cavities, ligand and substrate binding pockets, and into the pore of channel-like structures. These xenon complexes can be used to map hydrophobic sites in proteins, or as heavy-atom derivatives in the isomorphous replacement method of structure determination.

Molecular structure of the lipoamide dehydrogenase domain of a surface antigen from Neisseria meningitidis.

The protein p64k from the surface of the Neisseria meningitidis bacteria has been characterized as a two-domain protein. It contains a dihydrolipoamide dehydrogenase domain of 482 residues, involving a FAD prosthetic group as a cofactor, and a smaller lipoic acid binding domain of 86 residues. The two domains are joined by a flexible segment rich in alanine and proline residues. The structure of the dihydrolipoamide dehydrogenase domain was determined by X-ray diffraction. It was solved by a combination of molecular replacement and multiple isomorphous replacement techniques and refined to 2.7 A resolution. In the crystal, the recombinant p64k mimics the functional homo-dimer by using one of the crystallographic 2-fold axes. The reactive disulphide bridge Cys161-Cys166 is in the oxidised state and the FAD is bound in an extended conformation. This main domain contains the major antigenic determinant of the protein, an extended loop of 32 residues at the surface of the protein. A mis-attribution at residue 553 in the sequence has been detected by inspection of electron density maps and the geometry. However, when compared to the other dihydrolipoamide dehydrogenases, there are some significant differences: (1) an unusual number of cis-proline residues and (2) a new motif built around a 2-fold axis by the sulphur atoms of residues Met558, Cys560 and their symmetry related equivalents.

High-pressure krypton gas and statistical heavy-atom refinement: a successful combination of tools for macromolecular structure determination.

The noble gas krypton is shown to bind to crystallized proteins in a similar way to xenon [Schiltz, Prangé & Fourme (1994). J. Appl. Cryst. 27, 950-960]. Preliminary tests show that the major krypton binding sites are essentially identical to those of xenon. Noticeable substitution is achieved only at substantially higher pressures (above 50 x 10(5) Pa). As is the case for xenon, the protein complexes with krypton are highly isomorphous with the native structure so that these complexes can be used for phase determination in protein crystallography. Krypton is not as heavy as xenon, but its K-absorption edge is situated at a wavelength (0.86 A) that is readily accessible on synchrotron radiation sources. As a test case, X-ray diffraction data at the high-energy side of the K edge were collected on a crystal of porcine pancreatic elastase (molecular weight of 25.9 kDa) put under a krypton gas pressure of 56 x 10(5) Pa. The occupancy of the single Kr atom is approximately 0.5, giving isomorphous and anomalous scattering strengths of 15.2 and 1.9 e, respectively. This derivative could be used successfully for phase determination with the SIRAS method (single isomorphous replacement with anomalous scattering). After phase improvement by solvent flattening, the resulting electron-density map is of exceptionally high quality, and has a correlation coefficient of 0.85 with a map calculated from the refined native structure. Careful data collection and processing, as well as the correct statistical treatment of isomorphous and anomalous signals have proven to be crucial in the determination of this electron-density map. Heavy-atom refinement and phasing were carried out with the program SHARP, which is a fully fledged implementation of the maximum-likelihood theory for heavy-atom refinement [Bricogne (1991). Crystallographic Computing 5, edited by D. Moras, A. D. Podjarny & J. C. Thierry, pp. 257-297. Oxford: Clarendon Press]. It is concluded that the use of xenon and krypton derivatives, when they can be obtained, associated with statistical heavy-atom refinement will allow one to overcome the two major limitations of the isomorphous replacement method i.e. non-isomorphism and the problem of optimal estimation of heavy-atom parameters.

Protein Crystallography at Ultra-Short Wavelengths: Feasibility Study of Anomalous-Dispersion Experiments at the Xenon K-edge.

A protein crystallography experiment at the xenon K-edge (lambda = 0.358 A) has been successfully carried out at the materials science beamline (BL2/ID11) of the ESRF. The samples used in this methodological study were crystals of porcine pancreatic elastase, a 26 kDa protein of known structure. The diffraction data are of excellent quality. The combination of isomorphous replacement and anomalous dispersion of a single xenon heavy-atom derivative allowed accurate phase determination and the computation of a high-quality electron density map of the protein molecule. This is the first fully documented report on a complete protein crystallography experiment, from data collection up to phase determination and calculation of an electron density map, carried out with data obtained at ultra-short wavelengths. Experimental considerations as well as possible advantages and drawbacks of protein crystallography at very short and ultra-short wavelengths are discussed.

Crystallization of the MS2 translational repressor alone and complexed to bromouridine.

The coat protein from the MS2 bacteriophage plays a dual role by encapsidating viral RNA and also by binding RNA as a translational repressor. In order to study the isolated dimer in a conformation not influenced by capsid interactions, a mutant molecule was crystallized that is defective in capsid assembly but is an active repressor. The unassembled dimer crystallized in the space group P21212 with a = 76.2, b = 55.7, and c = 28.4 A. In these crystals, monomers were related by twofold symmetry. When this dimer was co-crystallized with 5-bromouridine, crystals formed in space group R3 with a = b = 155.9 A, c = 29.9 A, gamma = 120 degrees; the dimer was the asymmetric unit.

The Perfection of Protein Crystals Probed by Direct Recording of Bragg Reflection Profiles with a Quasi-Planar X-ray Wave.

Profiles of Bragg reflections from earth-grown crystals of lysozyme from hen egg-white and collagenase from Hypoderma lineatum were directly recorded with a quasi-planar X-ray wave. One crystal of each protein was chosen for a detailed investigation. Each sample is shown to consist of only a few (three and two, respectively) highly ordered domains, misoriented with respect to each other by a few arc s. The smallest rocking widths were observed for the large domain of the collagenase sample (FWHM corrected for instrumental broadening: 0.0016 degrees for a strong reflection at 3 A resolution). With appropriate improvements, this method might become a quantitative tool for characterizing the perfection of crystals from biological macromolecules.

The catalytic site of serine proteinases as a specific binding cavity for xenon.

BACKGROUND: Under moderate pressure, xenon can bind to proteins and form weak but specific interactions. Such protein-xenon complexes can be used as isomorphous derivatives for phase determination in X-ray crystallography. RESULTS: Investigation of the serine proteinase class of enzymes shows that the catalytic triad, the common hydrolytic motif of these enzymes, is a specific binding site for one xenon atom and shows high occupancy at pressures below 12 bar. Complexes of xenon with two different serine proteinases, elastase and collagenase, were analyzed and refined to 2.2 A and 2.5 A resolution, respectively. In both cases, a single xenon atom with a low temperature factor is located in the active site at identical positions. Weak interactions exist with several side chains of conserved amino acids at the active site. Xenon binding does not induce any major changes in the protein structure and, as a consequence, crystals of the xenon complexes are highly isomorphous with the native protein structures. Xenon is also found to bind to the active site of subtilisin Carlsberg, a bacterial serine proteinase, that also has a catalytic triad motif. CONCLUSIONS: As the region around the active site shows conserved structural homology in all serine proteinases, it is anticipated that xenon binding will prove to be a general feature of this class of proteins.

The Multiwavelength Anomalous Solvent Contrast (MASC) Method in Macromolecular Crystallography.

The wavelength dependence of anomalous scattering of X-rays, due to atoms randomly dispersed in the solvent phase of a macromolecular crystal, is a way of producing solvent-density contrast variation with perfect isomorphism. The largest contrast variations are obtained by tuning the X-ray wavelength near an absorption edge of the anomalous-scattering species. In this method, which we call MASC, the anomalous partial structure is an extended uniform electron density, in contrast to the few punctual ordered scatterers in the multiwavelength anomalous-dispersion (MAD) method. MASC is, in principle, applicable to the determination of the molecular envelope and of low-resolution structure-factor phases. Structure factors (lambda)F(+/-h) leads to a set of equations which can be solved to give |G(h)| and |(0)F(h)|, the modulus of the envelope and of the total ;normal' structure factors, respectively, and Deltavarphi = (varphi(0)(F)-varphi(G)). The moduli {|G|} behave like structure-factor amplitudes from small-molecule crystals, and the estimation of their phases can be carried out by statistical direct methods. Then, the phase of (0)F(h) and finally the conventional (e.g. in vacuum) protein structure factor F(p)(h) can be determined. As in the MAD method, the strength of MASC signals can be quantified by Bijvoet and dispensive ratios, for which practical expressions are derived in the case of zero contrast. The behaviour of these ratios at increasing resolution is discussed, using approximations for |G(h)| and |Delta(h)| , respectively, derived from Porod's law and assuming a random distribution of atoms in the solvent excluding volume. Expected values of anomalous ratios are calculated for a hypothetical MASC experiment based on the known three-dimensional structure of kallikrein A, using a solvent with 3.5 M ammonium selenate to ensure zero contrast, and wavelength tuning near the Se K-absorption edge. The main steps of a MASC experiment are discussed in the context of a MAD-like data collection optimized for accurate measurements of intensities of anomalous pairs at low resolution. Finally, the results of preliminary experiments on two protein crystals are reported. The first, a partial single-wavelength data collection, used anomalous scattering of selenium at the K edge and gave anomalous ratios with the expected behaviour. The second one, at three wavelengths, used anomalous scattering of ytterbium at the L(III) edge. In this case, effects from solvent as well as from ordered lanthanide ions were demonstrated.

Crystallization and preliminary X-ray investigation of a recombinant outer membrane protein from Neisseria meningitidis.

A protein constituent of the outer membrane from Neisseria meningitidis (hereafter called P64K) has been crystallized using the hanging drop technique. Crystals are tetragonal with unit cell dimensions a = b = 136.84 A and c = 78.44 A, compatible with a single monomer of 64 kDa in the asymmetric unit. When exposed to high intensity synchrotron radiation, these crystals diffract X-rays to at least 2.9 A resolution, indicating that a high resolution structure analysis is feasible.

Crystallization and preliminary X-ray analysis of leukemia inhibitory factor.

Leukemia inhibitory factor (LIF) is a polyfunctional molecule with significant and diverse biological activities. LIF is a glycoprotein secreted by a number of different cell types in vitro. It is induced in fibroblasts, lymphocytes, monocytes and astrocytes by various inducers such as serum, TNF, interleukin-IP and EGF. Due to extensive and variable glycosylation the molecular weight can range from 38 to 67 kDA. The biological functions of LIF are mediated through a receptor and a signal transducer, gp130, which is also used by factors like interleukin-6 (IL-6), cilliary neurotropic factor (CNTF), and oncostatin M (OSM). Here, we report the crystallization of the non-glycosylated human-like LIF expressed in E. coli. The present crystals diffract to 2.0 A using synchrotron radiation. They belong to the monoclinic space group C2, and the cell dimensions are a = 61.5 A, b = 45.3 A, c = 77.7 A and beta = 112.3 degrees.

Derivation by statistical methods of phase information from multiple-wavelength anomalous diffraction data. Basic questions, 'best' electron-density map, implementation and tests.

A probability distribution of the structure factor is established from the analysis of the effects of errors involved in the multiple-wavelength anomalous diffraction (MAD) method. This probability distribution, derived from those of the intensities, is two-dimensional for acentric reflections and uni-dimensional for centric reflections. It permits, using the centroid of the distribution, the calculation of the modulus and the phase of the 'best' structure factor. The procedure for extracting the phase and its figure of merit is presented. Tests performed on simulated data show the contribution of this method with respect to other methods which use a distribution of only the phase as a function of the error of closure.

X-ray structure of nucleoside diphosphate kinase.

The X-ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta-sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro----Ser substitution in Killer of prune (K-pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three-dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.

Structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by MAD phasing.

Calcium-dependent (C-type) animal lectins participate in many cell surface recognition events mediated by protein-carbohydrate interactions. The C-type lectin family includes cell adhesion molecules, endocytic receptors, and extracellular matrix proteins. Mammalian mannose-binding proteins are C-type lectins that function in antibody-independent host defense against pathogens. The crystal structure of the carbohydrate-recognition domain of a rat mannose-binding protein, determined as the holmium-substituted complex by multiwavelength anomalous dispersion (MAD) phasing, reveals an unusual fold consisting of two distinct regions, one of which contains extensive nonregular secondary structure stabilized by two holmium ions. The structure explains the conservation of 32 residues in all C-type carbohydrate-recognition domains, suggesting that the fold seen here is common to these domains. The strong anomalous scattering observed at the Ho LIII edge demonstrates that traditional heavy atom complexes will be generally amenable to the MAD phasing method.

Crystallization and preliminary X-ray studies of human vascular anticoagulant protein.

The human vascular anticoagulant protein, a 36 kDa member of the annexin/lipocortin family, has been crystallized using polyethylene glycol 20,000, by the vapour diffusion method. The crystals are monoclinic, space group P2(1), cell dimensions a = 83.9 A, b = 80.9 A, c = 71.4 A, beta = 108.7 degrees and diffract to at least 2.2 A resolution.

Conserved residues of tumour necrosis factor and lymphotoxin constitute the framework of the trimeric structure.

Four distinct areas of primary sequence conservation between known tumour necrosis factor and lymphotoxin polypeptides from various species can be recognized. When these amino acid sequences are highlighted in the three-dimensional structure, all are found in the same region, constituting the framework of the trimeric structure.