摘要:

There are many examples in macromolecular crystallography where interest focuses on the differences between a previously determined `native' structure and a nearly isomorphous `variant'. In such cases, a useful approach to atomic refinement of the variant structure is through weighted least‐squares minimization of the residual between the observed and calculateddifferencesin amplitudes of structure factors, a strategy first used in the refinement of deoxycobalt hemoglobin [Fermi, Perutz, Dickinson&Chien (1982).J. Mol. Biol.155, 495–505] and termed `difference refinement'. For cases in which the modeling errors for the native and variant structures are correlated, theoretical arguments indicate that difference refinement should lead to improved estimates of structural differences when compared with conventional independent refinement. Tests employing simulated peptide data sets and real data from a wild‐type protein and a mutant show that difference refinement can substantially reduce errors in the differences between structures when compared with independent refinement. The algorithm is very easy to implement and does not increase the computational demands of refin

ISSN:1399-0047    

DOI:10.1107/S0907444994013247

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The three‐dimensional structure of a mutant human lysozyme, C77A‐a, in which the residue Cys77 is replaced by alanine, has been refined to anRvalue of 0.125 using 8230 reflections in the resolution range 10.0–1.8 Å. It has been shown that C77A‐a, in which the counterpart of Cys77 (Cys95) is modified with glutathione, has been shown to mimic an intermediate in the formation of the disulfide bond Cys77–Cys95 during the folding of human lysozyme [Hayano, Inaka, Otsu, Taniyama, Miki, Matsushima&Kikuchi (1993).FEBS Lett.328, 203–208]. An earlier structure demonstrates that its overall structure is essentially identical to that of the wild‐type protein and served as the starting model. The refined model includes atoms for all protein residues (1–130), 20 glutathione atoms and 113 water atoms. Further refinement shows more clearly the details of the protein, the bound glutathione molecule and solvent structure. However, the main‐chain folding and the atomic thermal factors of the loop region from Thr70 to Leu79 were highly affected by the binding of the glutathione molecule, as compared with those of the wild‐type protein. The bound glutathione shifted the main‐chain atoms from Va174 to Ala77 by more than 6.0 Å, and the temperature factors of the atoms in the loop region were quite high (more than 40 Å2), indicating that the backbone conformation of this region is highly flexible and that the loop region is not folded in the specific conformation observed in the wild‐type protein. These results strongly suggest that the loop structure in human lysozyme is folded later than the other regions of the proteinin vivo, as observed inin vitrofolding. Since the bound glutathione is efficiently and irreversibly dissociated by protein disulfide isomerase, the glutathione molecule may act as a protecting group to prevent the formation of an incorrect disulfide bond in th

ISSN:1399-0047    

DOI:10.1107/S0907444994013478

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The direct‐methods programSAYTANhas been applied to the known structure of 2‐Zn insulin with 806 atoms, excluding solvent, in the asymmetric unit. Useful sets of phases can be obtained and selected by figures of merit for data resolutions between 1.5 and 2.25 Å and these can be extended bySAYTANto give mean phase errors of 68° for more than 2000 reflections. A feature of the phases so found is that the phase errors decrease with increasing resolution – which is the opposite of the situation when phases are found by isomorphous‐replacement

ISSN:1399-0047    

DOI:10.1107/S090744499401351X

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The three‐dimensional structure of the diferric form of human lactoferrin has been refined at 2.2 Å resolution, using synchrotron data combined with a lower resolution (3.2 Å) diffractometer data set. Following restrained least‐squares refinement and model rebuilding the final model comprises 5330 protein atoms (691 residues), 2Fe3+and 2CO32−ions, 469 solvent molecules and 98 carbohydrate atoms (eight sugar residues). Root‐mean‐square deviations from standard geometry are 0.015 Å for bond lengths and 0.038  Å for angle (1–3) distances, and the final crystallographicR‐factor is 0.179 for all 39 113 reflections in the resolution range 8.0–2.2 Å. A close structural similarity is seen between the two lobes of the molecule, with differences mainly in loops and turns. The two binding sites are extremely similar, the only apparent differences being a slightly more asymmetric bidentate binding of the carbonate ion to the metal, and a slightly longer Fe—O bond to one of the Tyr ligands, in the N‐lobe site relative to the C‐lobe site. Distinct differences are seen in the interactions made by two cationic groups, Arg210 and Lys546, behind the iron site, and these may influence the stability of the two metal sites. Analysis of interdomain and interlobe interactions shows that these are few in number which is consistent with the known flexibility of the molecule with respect to domain and lobe movements. Internal water molecules are found in discrete sites and in two large clusters (in the two interdomain clefts) and one tightly bound water molecule is present 3.8 Å from the Fe atom in each lobe. The carbohydrate is weakly defined and has been modelled to a limited extent; two sugar residues of the N‐lobe glycan and six of the C‐lobe glycan. Only one direc

ISSN:1399-0047    

DOI:10.1107/S0907444994013521

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The structure of a monomeric hemichrome form of an invertebrate hemoglobin, Hb‐C chain, fromCaudina arenicolahas been refined to anRvalue of 0.16 using the data from 5.0 to 2.5 Å resolution (R= 0.21 from 10.0 to 2.5 Å resolution). Hb‐C crystallizes in space groupP2lwith cell constantsa= 45.74,b= 45.23 andc= 40.92 Å and β = 104.4° with two monomers packed in the unit cell (Vm= 2.34 Å3 Da−1). The phases were determined by the multiple isomorphous replacement method with Hg2+the major derivative. The structure consists of 157 amino acids with N‐ and C‐terminal regions and eight α‐helices forming a heme pocket. The unique feature of this structure is the hemichrome form with the proximal and distal histidines coordinated to the heme Fe atom, which is nearly in the plane of the porphyrin ring. A total of 111 solvent molecules were added to the structure using difference density peaks of at least 3σ over background. Interestingly, all the heme groups present in th

ISSN:1399-0047    

DOI:10.1107/S0907444994013958

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The structure of hexagonal turkey egg‐white lysozyme (TEWL) has been determined and refined at 1.65 Å resolution. The crystals were grown from a 150 mMpotassium thiocyanate solution at pH 4.5 and belong to space groupP6122 with unit‐cell dimensionsa=b= 70.96,c= 83.01 Åα = β = 90, γ = 120°. The crystals were isomorphous with those of hexagonal pH 8.0 TEWL. The coordinates of PDB entry code 3LZ2 were therefore used as the initial model and subjected to rigid‐body refinement, simulated annealing and least‐squares refinement to a final residual of 0.20. The root‐mean‐square deviations from the ideal bond distances and angles were 0.016 Å and 2.2°, respectively. During the refinement, 86 water molecules and one thiocyanate ion were located in the structure. The thiocyanate ion lies close to the interface between two symmetry‐related molecules. The S atom of the ion forms two direct intermolecular contacts with Argl4 and interacts indirectlyviaa network of water molecules to Arg5 of a symmetry‐related molecule. The structure provides direct evidence for the mode of thiocyanate binding to arginine residues and suggests a possible mechanism for the efficiency of thiocyanate

ISSN:1399-0047    

DOI:10.1107/S0907444994013612

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

This paper describes the first successful full‐matrix least‐squares (FMLS) refinement of a protein structure. The example used is crambin which is a small hydrophobic protein (4.7 kDa, 46 residues). It proves the feasibility of refining such large molecules by this classic method, routinely applied to small molecules. The final structure with 381 non‐H protein atoms (54 protein atoms in alternative positions), 367 H atoms, 162 water molecules (combined occupancy 93) and one disordered ethanol molecule converged to a standard unweighted crystallographicR‐factor ofR= 9.0% when refined againstFwith reflections stronger thanF>2σ(F) andR= 9.5% when refined againstF2. The programsRFINE[Finger&Prince (1975).Natl Bur. Stand.(US)Tech. Note854.A System of FortranIVComputer Programs for Crystal Structure Computations] andSHELXL93 [Sheldrick (1993).SHELXL93.Program for Crystal Structure Refinement, Univ. of Göttingen, Germany] were used for FMLS refinement with the high‐resolution low‐temperature (0.83 Å, 130 K) data set of a mixed‐sequence form of crambin. A detailed analysis of the models obtained in FMLS andPROLSQ[restrained least squares or RLS; Teeter, Roe&Heo (1993).J. Mol. Biol.230, 292–311] refinements with the same data set is presented. The differences between the models obtained by both FMLS and RLS refinements are systematic but negligible and advantages and shortcomings of both methods are discussed. The final structure has very good geometry, fully comparable to the geometry of other structures in this resolution range. Ideal values used inPROLSQand those by Engh&Huber [Engh&Huber (1991).Acta Cryst.A47, 392–400] differ significantly from this refinement and we recommend a new standard. FMLS refinement constitutes a sensitive tool to detect and model disorder in highly refined protein structures. We describe the modeling of temperature factors by the TLS method [Schomaker&Trueblood (1968).Acta Cryst.B24, 63–76]. Rigid body–TLS refinements led to a better understanding of different modes of vibrations of the molecule. Refinements usingF2orFprotocols converged and reached slightly different minima. Despite theoretical support forF2‐based refinement, we recommend

ISSN:1399-0047    

DOI:10.1107/S0907444994014484

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

β‐Lactamases are bacterial enzymes which catalyse the hydrolysis of the β‐lactam ring of penicillins, cephalosporins and related compounds, thus inactivating these antibiotics. The crystal structure of the TEM1 β‐lactamase has been determined at 1.9 Å resolution by the molecular‐replacement method, using the atomic coordinates of two homologous β‐lactamase refined structures which show about 36% strict identity in their amino‐acid sequences and 1.96 Å r.m.s. deviation between equivalent Cα atoms. The TEM1 enzyme crystallizes in space groupP212121and there is one molecule per asymmetric unit. The structure was refined by simulated annealing to anR‐factor of 15.6% for 15 086 reflections withI≥ 2σ(I) in the resolution range 5.0–1.9 Å. The final crystallographic structure contains 263 amino‐acid residues, one sulfate anion in the catalytic cleft and 135 water molecules per asymmetric unit. The folding is very similar to that of the other known class A β‐lactamases. It consists of two domains, the first is formed by a five‐stranded β‐sheet covered by three α‐helices on one face and one α‐helix on the other, the second domain contains mainly α‐helices. The catalytic cleft is located at the interface between the two domains. We also report the crystallographic study of the TEM S235A mutant. This mutation of an active‐site residue specifically decreases the acylation rate of cephalosporins. This TEM S235A mutant crystallizes under the same conditions as the wild‐type protein and its structure was refined at 2.0 Å resolution with anRvalue of 17.6%. The major modification is the appearance of a water molecule near the mutated residue, which is incompatible with the OG 235 present in the wild‐type enzyme, and causes very small

ISSN:1399-0047    

DOI:10.1107/S0907444994014496

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

A fast and simple method has been developed for theab initiocalculation of low‐resolution solvent envelopes for macromolecular structures. In essence, a sphere of point scatterers is moved through the asymmetric unit cell in a part random, part systematic search for the configuration which corresponds to the lowestRvalue. The spheres correspond to the solvent regions in the cell. The program has been shown to work successfully for a number of test structures in a variety of space groups. No prior knowledge of the structures is needed, and c.p.u. requirements are extremely modes

ISSN:1399-0047    

DOI:10.1107/S0907444994015040

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The crystal structures of 88 and 79% relative humidity forms of ribonuclease A, resulting from water‐mediated transformations, have been refined employing the restrained least‐squares method using X‐ray data collected on an area detector toR= 0.173 for 15 326 observed reflections in the 10–1.5 Å resolution shell andR= 0.176 for 8534 observed reflections in the 10–1.8 Å shell, respectively. The comparison of these structures with those of the native, the phosphate‐bound and the sulfate‐bound forms demonstrates that the mobility of the ribonuclease A molecule involves hinge‐bending movement of the two domains and local flexibility within them, particularly at the termini of regular secondary structures and in loops. The comparison also leads to the identification of 31 invariant water molecules in the hydration shell of the enzyme, many of which are involved in holding different parts of the molecule together and in stabilizing local structure. The conformational changes that accompany the partial removal of the surrounding water, particularly those observed in the 79% form, could be similar to those that occur

ISSN:1399-0047    

DOI:10.1107/S0907444994014794

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY