Crystallographic Data Sets Research Articles

Simultaneous Refinement of the Structure of BPTI Against NMR Data Measured in Solution and X-ray Diffraction Data Measured in Single Crystals

The structure of the bovine pancreatic trypsin inhibitor (BPTI) has been determined to high resolution by both NMR spectroscopy in solution and X-ray diffraction in crystals. The root-mean-square difference calculated between the two structures for the polypeptide backbone is 0·9 Å. Several amino acid side-chains, of which all but one are charged or polar, have different conformations. We find that by refining one structure simultaneously against both the NMR and crystallographic data sets, it can accommodate both. Different starting configurations were used, including the X-ray structure 5pti, an NMR conformer, and the X-ray structure in the full unit cell with extra solvent placed in the bulk solvent region. The X-ray structures quickly converged to accommodate the NMR data in addition to the crystallographic data. Starting from an NMR conformer, however, the convergence to accommodate the more abundant X-ray data in addition to the NMR data is much slower.

Refinement of the three-dimensional structure of PhoE porin at 3.2Å resolution as determined by electron crystallography

Density modification methods were used to improve the three-dimensional map of PhoE porin which has been obtained by electron crystallographic techniques. The electron crystallographic data set consists of diffraction amplitudes that extend to a resolution of∽ 2.8 Å and phase information derived from 22 tilted and 8 untilted images at 3.5 Å resolution. However the data set was restricted to tilt angles less than ∽ 60 degrees, and therefore there is a missing cone regionin our 3-D data set. Non-crystallographic symmetry averaging and solvent flattening techniques were used to improve and extend the phase information, allowing for the calculation of high resolution Coulomb potential maps of membrane-embedded PhoE. These maps were used as a basis for building a partial molecular model, which was subsequently used to supply phase information to be combined with the experimentally determined phases. The combined phases were then used to calculate improved maps. As the maps continue to improve, more of the backbone and some of the side chains were built into the maps.

Cloning heterologous genes into E. Coli for enzyme production and crystal growth: Problems of expression and microheterogeneity

Protein crystal growth is heavily dependant on provision of large amounts of very pure protein. For this reason, molecular cloning will be used increasingly to permit the study of proteins which cannot otherwise be prepared in sufficient amounts, or purity, or both. We have obtained a stable clone of the tryptophanyl-tRNA synthetase from Bacillus stearothermophilus that is active in enzyme production. This result entailed two unusual aspects of interest to those using molecular cloning for enzyme production and crystal growth: (1) The cloning steps required stringent selection procedures that may have selected an unspecified mutational event 5′ to the structural gene, because an as yet unknown flanking element of the B. stearothermophilus DNA produces a marked instability in plasmids containing the native DNA. (2) The homologous Escherichia coli trpS enzyme apparently interferes with crystallization of B. stearothermophilus tryptophanyl-tRNA synthetase purified from an E. coli strain. We have therefore deleted the E. coli chromosomal trpS gene by site-specific recombination of a recombinant lambda phage containing a marked deletion of the E. coli trpS gene. Enzyme prepared from this deletion strain crystallizes in a normal fashion, suitable for high-resolution X-ray crystallography studies. Crystallographic data sets from isomorphous crystals grown with native and cloned protein are identical to 3Åresolution to within normal scaling statistics.

Normal probability plot analysis of small samples

In using normal probability plots for comparing two sets of crystallographic data [Abrahams, S. C. & Keve, E. T. (1971), Acta Cryst. A27, 157] note should be taken of the fact that the expected values of normal order statistics are not given exactly by the percentage points of the normal distribution. This becomes an important consideration only for small samples. Tables of expected ranked exact moduli of normal observations, for sample sizes to 41, are presented: these are useful for half-normal probability plots.