摘要:

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 sequenti

ISSN:1399-0047    

DOI:10.1107/S0907444994000727

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

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 funct

ISSN:1399-0047    

DOI:10.1107/S0907444994002295

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

Ferricytochromescwere 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 ferricytochromesc: 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 groupP212121. 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

ISSN:1399-0047    

DOI:10.1107/S0907444994002568

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

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 a

ISSN:1399-0047    

DOI:10.1107/S0907444994002398

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

The structure of the copper protein plastocyanin from poplar leaves (Populus nigravar.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 programEREFresulted in lower root‐mean‐square deviations from ideal geometry (e.g.0.011 Å for bond lengths) but a higher residualR(0.153) than restrained least‐squares refinement using the programPROLSQ(0.014 Å, 0.132). Electron‐density difference maps in both refinements provided evidence for disorder at some side chains and solvent atoms, and thePROLSQrefinement 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 thePROLSQrefinement; 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 Cαatoms, 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

ISSN:1399-0047    

DOI:10.1107/S0907444994003021

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

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. TheRvalues 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 CO2molecule in the active s

ISSN:1399-0047    

DOI:10.1107/S0907444994001873

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

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 groupF432 with cell parametera= 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 CuKα 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 ferr

ISSN:1399-0047    

DOI:10.1107/S0907444994003227

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

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 groupP212121with cell dimensionsa= 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 theQmolecular axes of both tetramers are approximately coincident with the crystallographicaaxis, and the non‐crystallographic symmetry relating the two tetramers is approximately

ISSN:1399-0047    

DOI:10.1107/S0907444994001915

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

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 groupI222 with approximate cell dimensionsa= 89,b= 87 andc= 63 Å and one monomer in the asymmetric unit. The finalRfactor for Co‐substituted concanavalin A is 17.8% for 29 211 reflections withF>1.0σ(F) between 8.0 and 1.6 Å. For Ni‐substituted concanavalin A the finalRfactor is 15.9% for 16 128 reflections withF>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 whic

ISSN:1399-0047    

DOI:10.1107/S0907444994002143

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

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 clamLucina 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 invar

ISSN:1399-0047    

DOI:10.1107/S0907444994002556

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY