Notes: Crystal structure solution by anomalous dispersion methods has been greatly facilitated using the rapidly tunable station 9.5 at the Daresbury SRS. Both SIROAS and MAD techniques, with IP data, have been used in the phasing of a brominated nucleotide and a seleno deaminase, respectively. The electron density maps in each case are interpretable. Throughput of projects could be improved upon with a better duty cycle detector. Another category of data collection is that at very high resolution. Detailed structure refinement pushes the limits of resolution and data quality. Station 9.5 has been used to collect high resolution (1.4 A(ring)) native data for the protein concanavalin A. This utilized very short wavelengths (0.7 A(ring)), the image plate, and crystal freezing. A total of 155 407 measurements from two crystals benefited from the on-line nature of the IP detector device, but a slow and quick pass are required to capture the full dynamic range of the data. There are data seen to 1.2 A(ring) and beyond for a pure Mn substituted form of the protein, but a higher intensity still is required to actually record these data. By comparison, trials at CHESS, on a multipole wiggler (station A1) with a CCD (without image intensifier) system, yield native concanavalin A data to 0.98 A(ring) and beyond. This demonstrates that the combination of yet higher intensity and the ease of use of a CCD offers worthwhile improvements; in this case an increase in the data by a factor of (1.4/0.98)3, thus at least doubling the data to parameter ratio for protein structure model refinement and potentially opening up direct structure determination of proteins of the size of concanavalin A (25 kDa).Finally, possibilities at ESRF and further detector developments, such as mosaic CCDs and scintillator coatings, offer further impetus for the field. These include more intense rapidly tunable beams for anomalous dispersion-based structure solution and "ideal'' higher resolution data collection and reactivity studies. ESRF BL19 is described; facilities on BL19 will include a system for freezing and storing crystals at cryogenic temperatures, so that data can be recorded from the same crystal on different runs. Overall, there have been tremendous strides made in this field in the last 15 years, and yet further improvements are to come. © 1995 American Institute of Physics.

Notes: Many years ago the idea of collecting voluminous quantities of weak reflection intensities from a protein crystal, at high resolution, was a particular challenge [J.R. Helliwell (1979) Daresbury Study Weekend DL/SCI R13, pp. 1–6]. The combination of insertion devices with very high x-ray fluxes at short x-ray wavelengths, sensitive CCD detectors, and freezing of crystals have provided the means to certainly match those best hopes. So much so that the data can best be described as ultrahigh resolution, at least as evidenced in our studies of the 25000 molecular weight plant protein concanavalin A. (The intrinsic property of this protein is to bind sugar molecules; it is implicated in cell-to-cell recognition processes and is widely used as a laboratory diagnostic tool.) At CHESS we have used a 0.9 A(ring) wavelength beam on station A1, fed by a 24 pole multipole wiggler. Both an imaging plate system and the Princeton 1k CCD detector [M. Tate et al., J. Appl. Cryst. 28, 196 (1995)] have been used on this experimental setup to collect diffraction data sets from frozen concanavalin A crystals (saccharide-free crystal form). The rapid readout of the CCD was most convenient compared with the image plate and its associated scanning and erasing. Moreover the data processing results towards the edges of the detectors, 0.98 A(ring), show that the CCD is much better than the image plate at recording these weaker data (Rmerge(I) 13% versus 44%, respectively). The poor performance of the image plate with weak signals has of course been documented by the Daresbury detector group [R. Lewis, J. Synchrotron Radiation 1, 43 (1994)]. However, the aperture of the CCD used was limiting here. Very recently, in another run at CHESS with the CCD on A1, we have been able to record diffraction data to 0.94 A(ring) by further offsetting the detector. We again found that the reflections are still strong at the edge. Clearly the use of even shorter wavelengths than 0.9 A(ring) would be very useful in matching the solid angle of the diffraction pattern to the available detector aperture, for a reasonable crystal-to-detector distance. In addition, absorption errors in the data can be simultaneously removed by such a strategy. Indeed, finely focused x-ray beams of, say 0.5 A(ring) wavelength, are especially well suited to high energy, low emittance synchrotron radition (SR) machines. Some initial tests carried out on CHESS station F2 with a 0.5 A(ring) wavelength beam and the CCD detector show an improvement in the R-merge(I) to 2 A(ring) resolution, in comparison to the data collected at 0.9 A(ring) wavelength (i.e., 2.3% versus 3.0%). In conclusion, the diffraction resolution limit (0.94 A(ring)) seen already in our concanavalin A studies can be further enhanced and is important for the most detailed molecular model refinement (and the testing of structure solving strategies), in conjunction with novel spectroscopic and theoretical studies. This paper builds upon the work of Deacon et al. [Rev. Sci. Instrum. 66, 1287 (1995)]. © 1996 American Institute of Physics.

Notes: The molecular structures of cobalt- and nickel-substituted concanavalin A have been refined at 1.6 and 2.0 Å resolution, respectively. Both metal derivatives crystallize in space group I222 with approximate cell dimensions a = 89, b = 87 and c = 63 Å and one monomer in the asymmetric unit. The final R factor for Co-substituted concanavalin A is 17.8% for 29 211 reflections with F 〉 1.0σ(F) between 8.0 and 1.6 Å. For Ni-substituted concanavalin A the final R factor is 15.9% for 16 128 reflections with F 〉 1.0σ(F) between 8.0 and 2.0 Å resolution. Both structures contain a transition-metal binding site and a calcium-binding site but, unlike Cd-substituted concanavalin A, do not have a third metal-binding site. The Co-substituted concanavalin A structure diffracts to the highest resolution of any concanavalin A structure reported to date. A comparison of the structures of Ni-, Co-, Cd-substituted and native concanavalin A gives an indication of coordinate errors, which is a useful baseline for comparisons with saccharide complexes of concanavalin A described in other work. We also give a detailed account of multiple conformations which were found for five side-chain residues.

Notes: The complex of methyl α-D-arabinofuranoside with concanavalin A crystallizes in the orthorhombic space group P21221 with cell dimensions a = 97.5, b = 87.0 and c = 61.5 Å. The asymmetric unit contains one dimer and the unit cell consists of two tetrahedral clusters of point-group symmetry 222. The crystals diffract to 2.0 Å resolution.

Notes: The solution of the cubic crystal form (a = 167.8 Å) of concanavalin A complexed with the monosaccharide methyl α-D-glucopyranoside is described. The space group has been determined as I213 rather than I23. The use of cadmium to replace cobalt at the transition metal-ion binding site and to replace calcium at its binding site proved to be crucial to the successful solution of the crystal structure. The relatively small isomorphous signals of 21 e− for the replacement of cobalt and 28 e− for the replacement of calcium, yielded interpretable difference Patterson maps. The electron-density map calculated in space group I213 at 5.4 Å resolution, based on phases derived from single- and double-substituted cadmium differences, revealed a classical concanavalin A tetramer of 222 point symmetry, as seen in all the known crystal structures of concanavalin A. Rigid-body refinement at 3.6 Å using the refined coordinates of saccharide-free concanavalin A converged to an R factor of 27.4%. A molecular-replacement analysis, consistent with this crystal structure, and initial experiences in the incorrect space group I23 are described as these also prove to be instructive.

Notes: Two tetrameric NADP+-dependent bacterial secondary alcohol dehydrogenases have been crystallized in the apo- and the holo-enzyme forms. Crystals of the holo-enzyme from the mesophilic Clostridium beijerinckii (NCBAD) belong to space group P212121 with unit-cell dimensions a = 90.5, b = 127.9, c = 151.4 Å. Crystals of the apo-enzyme (CBAD) belong to the same space group with unit-cell dimensions a = 80.4, b = 102.3, c = 193.5 Å. Crystals of the holo-enzyme from the thermophilic Thermoanaerobium brockii (NTBAD) belong to space group P61(5) (a = b = 80.6, c = 400.7 Å). Crystals of the apo-form of TBAD (point mutant GI98D) belong to space group P21 with cell dimensions a = 123.0, b = 84.8, c = 160.4 Å β = 99.5°. Crystals of CBAD, NCBAD and NTBAD contain one tetramer per asymmetric unit. They diffract to 2.0 Å resolution at liquid nitrogen temperature. Crystals of TBAD(GI98D) have two tetramers per asymmetric unit and diffract to 2.7 Å at 276 K. Self-rotation analysis shows that both enzymes are tetramers of 222 symmetry.

Notes: The three-dimensional structure of the complex between methyl α-D-mannopyranoside and concanavalin A has been refined at 2.0 Å resolution. Diffraction data were recorded from a single crystal (space group P212121, a = 123.7, b = 128.6, c = 67.2 Å) using synchrotron radiation at a wavelength of 1.488 Å. The final model has good geometry and an R factor of 19.9% for 58 871 reflections (82% complete), within the resolution limits of 8 to 2 Å, with F 〉 1.0σ(F). The asymmetric unit contains four protein subunits arranged as a dimer of dimers with approximate 222 point symmetry. Each monomer binds one saccharide molecule. Each sugar is bound to the protein by hydrogen bonds and van der Waals contacts. Although the four subunits are not crystallographically equivalent, the protein–saccharide interactions are nearly identical in each of the four binding sites. The differences that do occur between the four sites are in the structure of the water network which surrounds each saccharide; these networks are involved in crystal packing. The structure of the complex is compared with a refined saccharide-free concanavalin A structure. The saccharide-free structure is composed of crystallographically identical subunits, again assembled as a dimer of dimers, but with exact 222 symmetry. In the saccharide complex the tetramer association is different in that the monomers tend to separate resulting in fewer intersubunit interactions. The average temperature factor of the mannoside complex is considerably higher than that of the saccharide-free protein. The binding site in the saccharide-free structure is occupied by three ordered water molecules and the side chain of Asp71 from a neighbouring molecule in the crystal. These occupy positions similar to those of the four saccharide hydroxyls which are hydrogen bonded to the site. Superposition of the saccharide-binding site from each structure shows that the major changes on binding involve expulsion of these ordered solvents and the reorientation of the side chain of Tyrl00. Overall the surface accessibility of the saccharide decreases from 370 to 100 Å2 when it binds to the protein. This work builds upon the earlier studies of Derewenda et al. [Derewenda, Yariv, Helliwell, Kalb (Gilboa), Dodson, Papiz, Wan & Campbell (1989). EMBO J. 8, 2198–2193] at 2.9 Å resolution, which was the first detailed study of lectin–saccharide interactions.

Notes: The three-dimensional structure of cadmium-substituted concanavalin A has been refined using X-PLOR. The R factor on all data between 8 and 2 Å is 17.1%. The protein crystallizes in space group I222 with cell dimensions a = 88.7, b = 86.5 and c = 62.5 Å and has one protein subunit per asymmetric unit. The final structure contains 237 amino acids, two Cd ions, one Ca ion and 144 water molecules. One Cd ion occupies the transition-metal binding site and the second occupies an additional site, the coordinates of which were first reported by Weinzierl & Kalb [FEBS Lett. (1971), 18, 268–270]. The additional Cd ion is bound with distorted octahedral symmetry and bridges the cleft between the two monomers which form the conventional dimer of concanavalin A. This study provides a detailed analysis of the refined structure of saccharide-free concanavalin A and is the basis for comparison with saccharide complexes reported elsewhere.

Notes: . A low-resolution partial structure of bacterioferritin was solved using a combination of molecular replacement and rigid-body refinement methods. Modification of bacterioferritin crystals by soaking in tetrachloroplatinate results in a phase transition from tetragonal symmetry (space group P42212) to a pseudo-cubic one (approximate space group I432). Helical parts of human H ferritin structure stripped of side chains beyond the Cβ atoms were used as the model. An electron-density map of the refined model revealed a region of extended density which by its shape and position in a pocket between helices was identified as haem. Inclusion of haem in the refinement showed that it can occupy only one of two symmetry-related sites near a twofold axis of the molecule.

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.