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Chemistry	100%

Selected Abstracts

On the use of logarithmic scales for analysis of diffraction data

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009
Alexandre Urzhumtsev
Predictions of the possible model parameterization and of the values of model characteristics such as R factors are important for macromolecular refinement and validation protocols. One of the key parameters defining these and other values is the resolution of the experimentally measured diffraction data. The higher the resolution, the larger the number of diffraction data Nref, the larger its ratio to the number Nat of non-H atoms, the more parameters per atom can be used for modelling and the more precise and detailed a model can be obtained. The ratio Nref/Nat was calculated for models deposited in the Protein Data Bank as a function of the resolution at which the structures were reported. The most frequent values for this distribution depend essentially linearly on resolution when the latter is expressed on a uniform logarithmic scale. This defines simple analytic formulae for the typical Matthews coefficient and for the typically allowed number of parameters per atom for crystals diffracting to a given resolution. This simple dependence makes it possible in many cases to estimate the expected resolution of the experimental data for a crystal with a given Matthews coefficient. When expressed using the same logarithmic scale, the most frequent values for R and Rfree factors and for their difference are also essentially linear across a large resolution range. The minimal R -factor values are practically constant at resolutions better than 3,Å, below which they begin to grow sharply. This simple dependence on the resolution allows the prediction of expected R -factor values for unknown structures and may be used to guide model refinement and validation. [source]

Application of normal-mode refinement to X-ray crystal structures at the lower resolution limit

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2009
Fengyun Ni
The structural refinement of large complexes at the lower resolution limit is often difficult and inefficient owing to the limited number of reflections and the frequently high-level structural flexibility. A new normal-mode-based X-ray crystallographic refinement method has recently been developed that enables anisotropic B -factor refinement using a drastically smaller number of thermal parameters than even isotropic refinement. Here, the method has been systematically tested on a total of eight systems in the resolution range 3.0,3.9,Å. This series of tests established the most applicable scenarios for the method, the detailed procedures for its application and the degree of structural improvement. The results demonstrated substantial model improvement at the lower resolution limit, especially in cases in which other methods such as the translation,libration,screw (TLS) model were not applicable owing to the poorly converged isotropic B -factor distribution. It is expected that this normal-mode-based method will be a useful tool for structural refinement, in particular at the lower resolution limit, in the field of X-ray crystallography. [source]

X-ray structure determination at low resolution

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2009
Axel T. Brunger
As an example of structure determination in the 3.5,4.5,Å resolution range, crystal structures of the ATPase p97/VCP, consisting of an N-terminal domain followed by a tandem pair of ATPase domains (D1 and D2), are discussed. The structures were originally solved by molecular replacement with the high-resolution structure of the N-D1 fragment of p97/VCP, whereas the D2 domain was manually built using its homology to the D1 domain as a guide. The structure of the D2 domain alone was subsequently solved at 3,Å resolution. The refined model of D2 and the high-resolution structure of the N-D1 fragment were then used as starting models for re-refinement against the low-resolution diffraction data for full-length p97. The re-refined full-length models showed significant improvement in both secondary structure and R values. The free R values dropped by as much as 5% compared with the original structure refinements, indicating that refinement is meaningful at low resolution and that there is information in the diffraction data even at ,4,Å resolution that objectively assesses the quality of the model. It is concluded that de novo model building is problematic at low resolution and refinement should start from high-resolution crystal structures whenever possible. [source]

Structure of acostatin, a dimeric disintegrin from Southern copperhead (Agkistrodon contortrix contortrix), at 1.7,Å resolution

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2008
Natalia Moiseeva
Disintegrins are a family of small (4,14,kDa) proteins that bind to another class of proteins, integrins. Therefore, as integrin inhibitors, they can be exploited as anticancer and antiplatelet agents. Acostatin, an ,, heterodimeric disintegrin, has been isolated from the venom of Southern copperhead (Agkistrodon contortrix contortrix). The three-dimensional structure of acostatin has been determined by macromolecular crystallography using the molecular-replacement method. The asymmetric unit of the acostatin crystals consists of two heterodimers. The structure has been refined to an Rwork and Rfree of 18.6% and 21.5%, respectively, using all data in the 20,1.7,Å resolution range. The structure of all subunits is similar and is well ordered into N-terminal and C-terminal clusters with four intramolecular disulfide bonds. The overall fold consists of short ,-sheets, each of which is formed by a pair of antiparallel ,-strands connected by ,-turns and flexible loops of different lengths. Conformational flexibility is found in the RGD loops and in the C-terminal segment. The interaction of two N-terminal clusters via two intermolecular disulfide bridges anchors the ,, chains of the acostatin dimers. The C-terminal clusters of the heterodimer project in opposite directions and form a larger angle between them in comparison with other dimeric disintegrins. Extensive interactions are observed between two heterodimers, revealing an ,,,, acostatin tetramer. Further experiments are required to identify whether the ,,,, acostatin complex plays a functional role in vivo. [source]

Structure of ribosomal protein L1 from Methanococcus thermolithotrophicus.

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6-2 2002
Functionally important structural invariants on the L1 surface
The crystal structure of ribosomal protein L1 from the archaeon Methanococcus thermolithotrophicus has been determined at 2.7,Å resolution. The crystals belong to space group P212121, with unit-cell parameters a = 67.0, b = 70.1, c = 106.3,Å and two molecules per asymmetric unit. The structure was solved by the molecular-replacement method with AMoRe and refined with CNS to an R value of 18.9% and an Rfree of 25.4% in the resolution range 30,2.7,Å. Comparison of this structure with those obtained previously for two L1 proteins from other sources (the bacterium Thermus thermophilus and the archaeon M. jannaschii) as well as detailed analysis of intermolecular contacts in the corresponding L1 crystals reveal structural invariants on the molecular surface which are probably important for binding the 23S ribosomal RNA and protein function within the ribosome. [source]

Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5,Å resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sites

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2001
Andrzej Wojtczak
The first observation of the unique environment for thyroxine (T4) binding in tetrameric rat transthyretin (rTTR) is reported as determined by X-ray diffraction. These data revealed different modes of hormone binding in the two unique hormone-binding sites in the rat TTR tetramer channel. Differences in the orientation of thyroxine and the position of water molecules in the two binding sites further suggest a mechanism for the docking pathway of the hormone into the channel of TTR. Crystals of the rat transthyretin,thyroxine complex are isomorphous with those reported for apo rTTR and crystallized in the tetragonal space group P43212 with four independent TTR monomeric subunits in the asymmetric part of the crystal lattice. Data were collected to 2.5,Å resolution and the structure was refined to R = 20.9% for 15,384 data in the resolution range 12,2.5,Å. Similar to human TTR, the rat protein is also a 54,000,Da tetramer with four identical polypeptide chains of 127 amino-acid residues. Of the 22 amino-acid residues which differ between the human and rat sequences, none are in the thyroxine-binding domains. Analysis of these structural data reveals that the tertiary structure is similar to that of hTTR, with only small differences in the flexible loop regions on the surface of the structure. Conformational changes of the amino acids in the channel result in a hydrogen-bonded network that connects the two binding domains, in contrast to the hydrogen bonds formed along the tetramer interface in the apo transthyretin structure. These changes suggest a mechanism for the signal transmission between thyroxine-binding domains. [source]

Structure of buffalo lactoferrin at 3.3,Å resolution at 277,K

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2000
S. Karthikeyan
The three-dimensional structure of diferric buffalo lactoferrin has been determined at 3.3,Å resolution. The structure was solved by molecular replacement using the coordinates of diferric human lactoferrin as a search model and was refined by simulated annealing (X-PLOR). The final model comprises 5316 protein atoms for all 689 residues, two Fe3+ and two CO ions. The final R factor was 21.8% for 11,711 reflections in the resolution range 17.0,3.3,Å. The folding of buffalo lactoferrin is essentially similar to that of the other members of the transferrin family. The significant differences are found in the dimensions of the binding cleft and the interlobe orientation. The interlobe interactions are predominantly hydrophobic in nature, thus facilitating the sliding of two lobes owing to external forces. The interdomain interactions are comparable in the N and C lobes. [source]

X-ray analysis of bilirubin oxidase from Myrothecium verrucaria at 2.3,Å resolution using a twinned crystal

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010
Kimihiko Mizutani
Bilirubin oxidase (BOD), a multicopper oxidase found in Myrothecium verrucaria, catalyzes the oxidation of bilirubin to biliverdin. Oxygen is the electron acceptor and is reduced to water. BOD is used for diagnostic analysis of bilirubin in serum and has attracted considerable attention as an enzymatic catalyst for the cathode of biofuel cells that work under neutral conditions. Here, the crystal structure of BOD is reported for the first time. Blue bipyramid-shaped crystals of BOD obtained in 2-methyl-2,4-pentanediol (MPD) and ammonium sulfate solution were merohedrally twinned in space group P63. Structure determination was achieved by the single anomalous diffraction (SAD) method using the anomalous diffraction of Cu atoms and synchrotron radiation and twin refinement was performed in the resolution range 33,2.3,Å. The overall organization of BOD is almost the same as that of other multicopper oxidases: the protein is folded into three domains and a total of four copper-binding sites are found in domains 1 and 3. Although the four copper-binding sites were almost identical to those of other multicopper oxidases, the hydrophilic Asn residue (at the same position as a hydrophobic residue such as Leu in other multicopper oxidases) very close to the type I copper might contribute to the characteristically high redox potential of BOD. [source]

Crystallization and preliminary X-ray analysis of ZHE1, a hatching enzyme from the zebrafish Danio rerio

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009
Akitoshi Okada
The hatching enzyme of the zebrafish, ZHE1 (29.3,kDa), is a zinc metalloprotease that catalyzes digestion of the egg envelope (chorion). ZHE1 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. Two diffraction data sets with resolution ranges 50.0,1.80 and 50.0,1.14,Å were independently collected from two crystals and were merged to give a highly complete data set over the full resolution range 50.0,1.14,Å. The space group was assigned as primitive orthorhombic P212121, with unit-cell parameters a = 32.9, b = 62.5, c = 87.4,Å. The crystal contained one ZHE1 molecule in the asymmetric unit. [source]

X-ray diffraction analysis of a human tRNAGly acceptor-stem microhelix isoacceptor at 1.18,Å resolution

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2009
André Eichert
Interest has been focused on comparative X-ray structure analyses of different tRNAGly acceptor-stem helices. tRNAGly/glycyl-tRNA synthetase belongs to the so-called class II system, in which the tRNA identity elements consist of simple and unique determinants that are located in the tRNA acceptor stem and the discriminator base. Comparative structure investigations of tRNAGly microhelices provide insight into the role of tRNA identity elements. Predominant differences in the structures of glycyl-tRNA synthetases and in the tRNA identity elements between prokaryotes and eukaryotes point to divergence during the evolutionary process. Here, the crystallization and high-resolution X-ray diffraction analysis of a human tRNAGly acceptor-stem microhelix with sequence 5,-G1C2A3U4U5G6G7 -3,, 5,-C66C67A68A69U70G71C72 -3, is reported. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 37.32, b = 37.61, c = 30.47,Å, , = 112.60° and one molecule per asymmetric unit. A data set was collected using synchrotron radiation and data were processed within the resolution range 50.0,1.18,Å. The structure was solved by molecular replacement. [source]

Overexpression, purification, crystallization and preliminary X-ray cystallographic studies of a proline-specific aminopeptidase from Aneurinibacillus sp. strain AM-1

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2006
Makoto Akioka
To elucidate the structure and molecular mechanism of a characteristic proline-specific aminopeptidase produced by the thermophile Aneurinibacillus sp. strain AM-1, its gene was cloned and the recombinant protein was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.8,Å resolution from the recombinant aminopeptidase crystal. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 93.62, b = 68.20, c = 76.84,Å. A complete data set was also obtained from crystals of SeMet-substituted aminopeptidase. Data in the resolution range 20,2.1,Å from the MAD data set from the SeMet-substituted crystal were used for phase determination. [source]