Luis A. González-Ramírez

Granada Crystallisation Box: a new device for protein crystallisation by counter-diffusion techniques.

Granada Crystallisation Box (GCB) is a new crystallisation device designed to perform counter-diffusion experiments. Here we describe the device and its use for protein crystallisation purposes. GCB allows one to explore and exploit the coupling between crystallisation and diffusion. The role of viscous fluids, gels and/or microgravity can be enhanced by using capillary volumes, creating a perfect diffusive mass transport scenario. The use of capillaries also reduces the consumption of macromolecules and avoids the handling of crystals for X-ray diffraction data collection.

Crystallization and preliminary crystallographic studies of the recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114.

Dihydropyrimidinases are involved in the reductive pathway of pyrimidine degradation, catalysing the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamoyl beta-amino acids. This enzyme has often been referred to as hydantoinase owing to its industrial application in the production of optically pure amino acids starting from racemic mixtures of 5-monosubstituted hydantoins. Recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114 (SmelDhp) has been expressed, purified and crystallized. Crystallization was performed using the counter-diffusion method with capillaries of 0.3 mm inner diameter. Crystals of SmelDhp suitable for data collection and structure determination were grown in the presence of agarose at 0.1%(w/v) in order to ensure mass transport controlled by diffusion. X-ray data were collected to a resolution of 1.85 A. The crystal belongs to the orthorhombic space group C222(1), with unit-cell parameters a = 124.89, b = 126.28, c = 196.10 A and two molecules in the asymmetric unit. A molecular-replacement solution has been determined and refinement is in progress.

Structural Study of the Type II 3-Dehydroquinate Dehydratase from Actinobacillus Pleuropneumoniae

The structure of the type II dehydroquinate dehydratase (DHQase) from Actinobacillus pleuropneumoniae, the third enzyme of the shikimate pathway, has been determined. Crystals diffracting to 1.7 A were obtained in space and on earth using the counter-diffusion technique. The structure was solved using molecular replacement and refined to high resolution. The overall structure of the dodecameric enzyme is described and compared with structures of DHQases from other bacteria. DHQases contain a flexible loop that presumably closes over the active site upon substrate binding. The enzyme can exist in an open or closed conformation. The present structure displays the open conformation, with a sulfate anion bound in the active site. The availability of this structure opens a route to structure-based antibiotics targetting this pathogenic bacterium.

Soaking: the effect of osmotic shock on tetragonal lysozyme crystals

Protein crystals crack when they are soaked in a solution with ionic strength sufficiently different from the environment in which they grew. It is demonstrated for the case of tetragonal lysozyme that the forces involved and the mechanisms that lead to the formation of cracks are different for hypertonic and hypotonic soaking. Tetragonal lysozyme crystals are very sensitive to hypotonic shocks and, after a certain waiting time, cracks always appear with a characteristic pattern perpendicular to the crystallographic c axis. Conversely, a hypertonic shock is better withstood: cracks do not display any deterministic pattern, are only visible at higher differences in ionic strength and after a certain time a phenomenon of crystal reconstruction occurs and the cracks vanish. At the lattice level, the unit-cell volume expands in hypotonic shock and shrinks under hypertonic conditions. However, the compression of the unit cell is anisotropic: the c axis is compressed to a minimum, beyond which it expands despite the unit-cell volume continuing to shrink. This behaviour is a direct consequence of the positive charge that the crystals bear and the existence of channels along the crystallographic c axis. Both features are responsible for the Gibbs-Donnan effect which limits the free exchange of ions and affects the movement of water inside the channels and bound to the protein.

Structure of concanavalin A at pH 8: bound solvent and crystal contacts

Concanavalin A has been crystallized in the presence of the ligand (6-S-beta-D-galactopyranosyl-6-thio)-cyclomaltoheptaose. The crystals are isomorphous to those reported for ConA complexed with peptides at low resolution (3.00-2.75 angstroms). The structure was solved at 1.9 angstroms, with free R and R values of 0.201 and 0.184, respectively. As expected, no molecules of the ligand were bound to the protein. Soaking in the cryobuffer left its fingerprint as 25 molecules of glycerol in the bound solvent, most of them at specific positions. The fact that a glycerol molecule is located in the sugar-binding pocket of each of the four subunits in the asymmetric unit and another is located in two of the peptide-binding sites suggests a recognition phenomenon rather than a displacement of water molecules by glycerol. Crystal contact analysis shows that a relation exists between the residues that form hydrogen bonds to other asymmetric units and the space group: contact Asp58-Ser62 is a universal feature of ConA crystals, while Ser66-His121, Asn69-Asn118 and Tyr100-His205 contacts are general features of the C222(1) crystal form.

Crystallization and diffraction patterns of the oxy and cyano forms of the Lucina pectinata haemoglobins complex

The native oxygen-carrier haemoglobins complex (HbII-III) is composed of haemoglobin II (HbII) and haemoglobin III (HbIII), which are found in the ctenidia tissue of the bivalve mollusc Lucina pectinata. This protein complex was isolated and purified from its natural source and crystallized using the vapour-diffusion and capillary counter-diffusion methods. Oxy and cyano derivatives of the complex crystallized using several conditions, but the best crystals in terms of quality and size were obtained from sodium formate pH 5 using the counter-diffusion method in a single capillary. Crystals of the oxy and cyano complexes, which showed a ruby-red colour and nonsingular prismatic shapes, scattered X-rays to resolution limits of 2.15 and 2.20 A, respectively, using a 0.886 A synchrotron-radiation source. The crystals belonged to the tetragonal system, space group P4(2)2(1)2, with unit-cell parameters a = b = 74.07, c = 152.07 and a = b = 73.83, c = 152.49 A for the oxy and cyano complexes, respectively. The asymmetric unit of both crystals is composed of a single copy of the heterodimer, with Matthew coefficients (V(M)) of 3.08 and 3.06 A(3) Da(-1) for the oxy and cyano complexes, respectively, which correspond to a solvent content of approximately 60.0% by volume.

Crystallization and preliminary crystallographic studies of an active-site mutant hydantoin racemase from Sinorhizobium meliloti CECT4114.

A recombinant active-site mutant of hydantoin racemase (C76A) from Sinorhizobium meliloti CECT 4114 (SmeHyuA) has been crystallized in the presence and absence of the substrate D,L-5-isopropyl hydantoin. Crystals of the SmeHyuA mutant suitable for data collection and structure determination were grown using the counter-diffusion method. X-ray data were collected to resolutions of 2.17 and 1.85 A for the free and bound enzymes, respectively. Both crystals belong to space group R3 and contain two molecules of SmeHyuA per asymmetric unit. The crystals of the free and complexed SmeHyuA have unit-cell parameters a = b = 85.43, c = 152.37 A and a = b = 85.69, c = 154.38 A, crystal volumes per protein weight (V(M)) of 1.94 and 1.98 A3 Da(-1) and solvent contents of 36.7 and 37.9%, respectively.

Two‐step counterdiffusion protocol for the crystallization of haemoglobin II from Lucina pectinata in the pH range 4–9

Lucina pectinata haemoglobin II (HbII) transports oxygen in the presence of H(2)S to the symbiotic system in this bivalve mollusc. The composition of the haem pocket at the distal site includes TyrB10 and GlnE7, which are very common in other haem proteins. Obtaining crystals of oxyHbII at various pH values is required in order to elucidate the changes in the conformations of TyrB10 and GlnE7 and structural scenarios induced by changes in pH. Here, the growth of crystals of oxyHbII using the capillary counterdiffusion (CCD) technique at various pH values using a two-step protocol is reported. In the first step, a mini-screen was used to validate sodium formate as the best precipitating reagent for the growth of oxyHbII crystals. The second step, a pH screen typically used for optimization, was used to produce crystals in the pH range 4-9. Very well faceted prismatic ruby-red crystals were obtained at all pH values. X-ray data sets were acquired using synchrotron radiation of wavelength 0.886 A (for the crystals obtained at pH 5) and 0.908 A (for those obtained at pH 4, 8 and 9) to maximum resolutions of 3.30, 1.95, 1.85 and 2.00 A for the crystals obtained at pH 4, 5, 8 and 9, respectively. All of the crystals were isomorphous and belonged to space group P4(2)2(1)2.

CRISTALES: a world to discover. An exhibition for schools and universities

The exhibition CRISTALES: a world to discover is a teaching/outreach activity whose main goals are to increase awareness of the importance of crystallography and its role in everyday life in modern society, motivate young people, and promote education and research in crystallography. CRISTALES is designed to inspire the audience with a careful design and a view of crystallography that places the emphasis not only on the most important contributions of crystallography to societys welfare, including new materials and biomedical research, but also on those aspects of crystallography related to art and the mind. This article describes the simplest version of the exhibition, composed of 14 posters that have been created specifically for schools and universities. Each poster displays an image that is both aesthetically powerful and scientifically intriguing, so as to provoke the curiosity of the students. The posters also contain a brief text explaining the image and its relation to crystallography and a QR code that links the poster to a web page containing further information.

Vapour Diffusion Route to Mineralize Graphene and Polymer Surfaces with Calcium Phosphate Intended for Biomedical Applications

After successful application of the sitting drop vapour diffusion route to deposit calcium phosphate layers on mica muscovite sheets, in this work we have extended the method to mineralize polymer surfaces (OSTE+) and bidimensional materials (graphene), aimed to prepare implants or scaffolds for nonload bearing applications in the medical sector. Thin octacalcium phosphate/apatite layers have been deposited on mica muscovite sheets. Thin apatite/octacalcium phosphate layers deposited on OSTE+ polymer lames. When graphene nanoflakes where used as support, this technique yielded apatite/graphene nanocomposites.