摘要:

The molecular structure of calf γB‐crystallin (previously called γII), a lens‐specific protein, has been refined to a crystallographicRfactor of 18.1% for all reflection data, between 8.0 and 1.47 Å, 25 959hklmeasured at 293 (1) K. 230 water molecules have been defined by difference Fourier techniques and included in a restrained least‐squares refinement. Difference Fourier maps clearly indicated the presence of multiple sites for the sulfur atoms of Cys 18 and Cys 22 which were therefore given coupled second‐site occupancies during the refinement. The sulfur atom in the major position of Cys 22 is in the reduced state. Either of the Cys 18 sites can form a high‐energy disulfide bridge with the minor position of Cys 22. The position of the carboxy terminus and many other surface side chains have been further defined including the RGD signal peptide. The hydration of the backbone and the interdomain region has been analysed. 27 water molecules make extensive contacts to a single protein molecule and th

ISSN:1399-0047    

DOI:10.1107/S0907444992007601

出版商:International Union of Crystallography    

年代:1993

数据来源: WILEY

摘要:

Low‐humidity monoclinic lysozyme, resulting from a water‐mediated transformation, has one of the lowest solvent contents (22% by volume) observed in a protein crystal. Its structure has been solved by the molecular replacement method and refined to anRvalue of 0.175 for 7684 observed reflections in the 10–1.75 Å resolution shell. 90% of the solvent in the well ordered crystals could be located. Favourable sites of hydration on the protein surface include side chains with multiple hydrogen‐bonding centres, and regions between short hydrophilic side chains and the main‐chain CO or NH groups of the same or nearby residues. Major secondary structural features are not disrupted by hydration. However, the free CO groups at the C terminii and, to a lesser extent, the NH groups at the N terminii of helices provide favourable sites for water interactions, as do reverse turns and regions which connect β‐structure and helices. The hydration shell consists of discontinuous networks of water molecules, the maximum number of molecules in a network being ten. The substrate‐binding cleft is heavily hydrated, as is the main loop region which is stabilized by water interactions. The protein molecules are close packed in the crystals with a molecular coordination number of 14. Arginyl residues are extensively involved in intermolecular hydrogen bonds and water bridges. The water molecules in the crystal are organized into discrete clusters. A distinctive feature of the clusters is the frequent occurrence of three‐membered rings. The protein molecules undergo substantial rearrangement during the transformation from the native to the low‐humidity form. The main‐chain conformations in the two forms are nearly the same, but differences exist in the side‐chain conformation. The differences are particularly pronounced in relation to Trp 62 and Trp 63. The shift in Trp 62 is especially interesting as it is also known t

ISSN:1399-0047    

DOI:10.1107/S090744499200653X

出版商:International Union of Crystallography    

年代:1993

数据来源: WILEY

摘要:

The structure of aldose reductase, a monomeric enzyme of 314 amino acids which crystallizes in space groupP1 with four monomers per asymmetric unit, has been solved using a combination of single isomorphous replacement (SIR), solvent flattening and local symmetry averaging. The self rotation showed evidence of 222 local symmetry. The map calculated from the original single isomorphous replacement phases showed a clear solvent envelope but was uninterpretable. A first averaging attempt failed because the molecular envelope obtained from theSIRmap weighted with monomer correlation was too small and the averaging was biased by low‐resolution truncation. A second attempt with an enlarged envelope and including low‐resolution reflections succeeded in refining phases at 3.5 Å resolution but failed to extend them correctly. Rigid‐body refinement of a partial model based on the 3.5 Å map calculated from refined phases showed significant departures from the 222 symmetry. A third averaging attempt using the improved symmetry succeeded in producing a clear map with phases extended to 3.07 Å resolution. This map revealed a (β/α)8fold, not previously found in NADPH‐dependent enzymes. This work shows the importance of mask definition and local symmetry elements accuracy for averaging, and describes a method for improvin

ISSN:1399-0047    

DOI:10.1107/S090744499200773X

出版商:International Union of Crystallography    

年代:1993

数据来源: WILEY

摘要:

The crystal structure of a complex of ribonuclease fromStreptomyces aureofaciens(RNase Sa) with guanosine‐2′‐monophosphate (2′‐GMP) has been refined against synchrotron data recorded from a single crystal using radiation from beamline X31 at EMBL, Hamburg, and an imaging plate scanner. The crystals are in space groupP212121with cell dimensionsa= 64.7,b= 78.8 andc= 39.1 Å. The structure has two enzyme molecules in the asymmetric unit, complexed with 2′‐GMP inhibitor with occupancies of 1 and (different to the 3′‐GMP complex crystal structure where only one of the two independent RNase Sa molecules binds nucleotide), 492 associated water molecules and one sulfate ion, and was refined using all data between 10.0 and 1.7 Å to a final crystallographicRfactor of 13.25%. Binding of the base to the enzyme confirms the basis for the guanine specificity but the structural results still do not provide direct evidence of the identity and role of the particular residues involved in the catalytic process. New native RNase Sa data to 1.8 Å were recorded to provide a reference set measured under comparable experimental conditions. The crystals are in the same space group and have the same lattice as those of the 2′‐GMP complex. The native structure with 423 water molecules was refined in a similar manner to the complex to a finalRfactor of 13.87%. 1.77 Å resolution data were independently measured on a 2′‐GMP complex crystal at UCLA using an R‐AXIS II image plate scanner mounted on a conventional source. The cell dimensions were essentially the same as above. 2′‐GMP was bound more fully to moleculeAthan to moleculeBof the RNase Sa. The structure was refined to anRfactor of 14.64% with 388 water molecules. This work follows on from the structure determination of native RNase Sa and its complex with 3′‐GMP [Sevcik

ISSN:1399-0047    

DOI:10.1107/S0907444992007261

出版商:International Union of Crystallography    

年代:1993

数据来源: WILEY

摘要:

Ferredoxin I fromAzotobacter vinelandii(AvFdI) is an iron–sulfur protein composed of 106 amino acids, seven Fe atoms and eight inorganic S* atoms. A crystallographic redetermination of its structure showed the originally reported structure to be incorrect. We report here the crystal structure of AvFdI at pH 6.5. Extensive refinement has led to a finalRvalue of 0.170 for all 6986 non‐extinct reflections in the range 10–2.3 Å using a solvent model which includes 98 discrete solvent atoms with occupancies between 0.3 and 1.0 and an averageBvalue of 22.5 Å2. The first half of the peptide chain closely resembles that of the 55‐residue ferredoxin fromPeptococcus aerogenes(PaFd), while the remainder consists of three turns of helix and a series of loops which form a cap over part of the molecular core. Despite the similarities in structure and surroundings, the corresponding 4Fe4S* clusters in PaFd and AvFdI have strikingly different redox potentials; a possible explanation has been sought in the differing hydration models for th

ISSN:1399-0047    

DOI:10.1107/S0907444992007248

出版商:International Union of Crystallography    

年代:1993

数据来源: WILEY

摘要:

The hexagonal crystal form of the octamer d(GTGTACAC), grown in the presence of spermine, has unit‐cell dimensionsa=b= 32.18 andc= 78.51 Å, space groupP6122, with one DNA strand in the asymmetric unit. The structure has been refined starting with the earlier lower resolution model and using high‐resolution 1.4 Å data collected on a Siemens‐Xentronics area detector at 258 K. There were 4365 unique reflections greater than 2σ(F) in the resolution range 5–1.4 Å. The model was refitted into 3Fo− 2Fc. Sim‐weighted omit maps and difference maps were used to locate water molecules. The final model with 161 DNA atoms and 37 water molecules gave anRfactor of 19.8%. Crystals of the same octamer were also grown in the presence of spermidine instead of spermine, and refinement using nominal 1.45 Å resolution data, 3292 unique reflections, finalR= 19.1%, gave virtually identical DNA parameters. No bound spermine or spermidine was detected in either of these structure analyses. The electron density was clear for the DNA and showed holes in the center of the six‐membered rings of bases, and also in the center of some of the sugar rings. The high‐resolution structure has provided more precise DNA parameters and confirmed the features observed in the earlier 2 Å study including the packing‐induced distortion in the A7 (A15) sugar pucker from C(3′)‐endoand C(2′)‐endo. This change causes the end base pairs to bend away from the helix axis while the rest of the duplex is nearly linear. The hydration patterns in the deep and shallow grooves have been characterized. Chains of water molecules were found, but no rings. The familiar intermolecular contact region between the end base pair and the minor groove of a symmetry‐related duplex, involving four residues on one strand and two on the other, has been analyzed.

ISSN:1399-0047    

DOI:10.1107/S0907444992007522

出版商:International Union of Crystallography    

年代:1993

数据来源: WILEY

摘要:

The molecular structure of an iron‐containing 18 kDa fragment of duck ovotransferrin, obtained by proteolysis of the intact protein, has been elucidated by protein crystallographic techniques at 2.3 Å resolution. This structure supports a mechanism of iron uptake in the intact protein whereby the binding of the synergistic (bi)carbonate anion is followed by binding of the metal with the lobe in the open configuration. These stages are then followed by domain closure in which the aspartic acid residue plays a further key role, by forming an interdomain hydrogen‐bond interaction in addition to serving as a ligand to the iron. This essential dual role is highlighted by model building studies on the C‐terminal lobe of a known human variant. In this variant a mutation of a glycine by an arginine residue enables the aspartic acid to form an ion pair and reduce its effectiveness for both metal binding and domain closure. The X‐ray structure of the 18 kDa fragment strongly suggests that the histidine residue present at the iron binding site of the intact protein and arising from the second interdomain connecting strand has been removed during the preparati

ISSN:1399-0047    

DOI:10.1107/S0907444992012101

出版商:International Union of Crystallography    

年代:1993

数据来源: WILEY

摘要:

The white‐beam Laue‐diffraction method is a useful tool for rapid measurement of crystallographic intensities with synchrotron radiation. Considerations of the signal‐to‐noise ratio to be expected from scattering of X‐rays within a limited wavelength range suggest that it will pay to limit that range to something like an octave. This rule‐of‐thumb has the added advantage that there will be significantly fewer diffraction spots that are overlapping harmonics of one another. To maximize the number of reflections recorded in a single stationary‐crystal exposure, one should choose this octave of wavelengths in a region where the curvature of the Ewald sphere is greatest, that is at the longest wavelength allowable after other considerations are tak

ISSN:1399-0047    

DOI:10.1107/S0907444992012095

出版商:International Union of Crystallography    

年代:1993

数据来源: WILEY

摘要:

. Sulfur atoms, an integral part of many proteins, are possible candidates for anomalous scattering in phase determination by multiple‐wavelength methods. The main difficulty encountered is that a wavelength of about 5 Å is required to obtain a large anomalous signal from these atoms, leading to very large absorption effects. Initial experiments have been carried out using a synchrotron X‐ray source, evacuated beam tubes, a diffractometer inside a vacuum chamber, a special sample holder and a suitable scattering geometry. The results are encouraging, showing that Bragg reflections can be measured, and that changes in their intensities around the absorption edge are obse

ISSN:1399-0047    

DOI:10.1107/S0907444992011910

出版商:International Union of Crystallography    

年代:1993

数据来源: WILEY