Biomedical subjects

R H Blessing

Publications and source records attributed to R H Blessing.

At least 19 recordsLinked to original sources

PubMed2001

Improvement of SAS triple invariant estimates for macromolecular direct-methods phasing.

Single-wavelength anomalous dispersion (SAS) data can in principle be phased by direct methods since a priori estimates of the three-phase structure invariants can be computed from these data. The mean phase error of the most reliable triple estimates for a small protein, however, is typically no better than 60 degrees, and does not bode well for applications to larger structures. A procedure is described that can substantially lower the error in these estimates and introduce a larger number of useful triple invariants into the phasing process. The mean phase error of the most reliable triples for a 2.5 A resolution data set from a Pt derivative of a 115-residue protein was reduced from 55 to 25 degrees by this method. It was also possible to identify a significant number of the poorest triple estimates, those with mean phase errors approaching 90 degrees, such that they could be reliably down-weighted or excluded from the phasing process.

Crystallography, X-Ray↗

PubMed2001

Map self-validation: a useful discriminator of phase correctness at low resolution.

A new map-validation procedure is based on the correlation-coefficient agreement between the observed structure-factor magnitudes and their extrapolated values from suitably modified electron-density maps from which they have been each in turn systematically excluded. The correlation coefficient tends to a maximum as the phase errors in a map are reduced. This principle was used to resolve the single-wavelength anomalous scattering (SAS) and single-derivative isomorphous replacement (SIR) phase ambiguity for a number of error-free trial structures. Applications employing real data sets tend to be more difficult owing to data incompleteness and errors affecting the construction of the Argand diagram.

Crystallography↗

PubMed2000

Accurate protein crystallography at ultra-high resolution: valence electron distribution in crambin.

The charge density distribution of a protein has been refined experimentally. Diffraction data for a crambin crystal were measured to ultra-high resolution (0.54 A) at low temperature by using short-wavelength synchrotron radiation. The crystal structure was refined with a model for charged, nonspherical, multipolar atoms to accurately describe the molecular electron density distribution. The refined parameters agree within 25% with our transferable electron density library derived from accurate single crystal diffraction analyses of several amino acids and small peptides. The resulting electron density maps of redistributed valence electrons (deformation maps) compare quantitatively well with a high-level quantum mechanical calculation performed on a monopeptide. This study provides validation for experimentally derived parameters and a window into charge density analysis of biological macromolecules.

Crystallography, X-Ray↗

PubMed2000

Bulk-solvent correction in direct-methods phasing.

It is shown that for crystals of large proteins at low diffraction resolution, with N approximately 10 000 independent non-H protein atoms and d(min) approximately 8 A, a simple bulk-solvent correction yields the Sayre equation in its classical form, F(h) = q summation operator(k)F(k)F(h - k). In the low-resolution protein case, the proportionality factor becomes q = 1/[( - rho(S))V], where V is the unit-cell volume, rho(S) is the assumed constant electron density in the solvent regions of the crystal and is the average electron density in the protein regions. The classical form of the tangent formula follows from the bulk-solvent-corrected Sayre equation and its validity at low resolution is verified in empirical calculations.

Adenosylhomocysteinase↗

PubMed2000

Optimizing DREAR and SnB parameters for determining Se-atom substructures.

The determination of the anomalous scattering substructure is the first essential step in any successful macromolecular structure determination using the multiwavelength anomalous diffraction (MAD) technique. The diffE method of calculating difference Es in conjunction with SnB has had considerable success in determining large Se-atom substructures. An investigation of the parameters used in both the data-reduction and error-analysis routines (DREAR) as well as the SnB phasing process itself was undertaken to optimize these parameters for more efficient use of the procedure. Two sets of selenomethionyl S-adenosylhomocysteine hydrolase MAD data were used as test data. The elimination of all erroneously large differences prior to phasing was found to be critical and the best results were obtained from accurate highly redundant intensity measurements. The high-resolution data collected in the typical MAD experiment are sufficient, but the inclusion of low-resolution data below 20 A improved the success rate considerably. Although the best results have been obtained from single-wavelength peak anomalous diffraction data alone, independent SnB analysis of data measured at other wavelengths can provide confirmation for questionable sites.

Computer Simulation↗

PubMed2000

Globbic approximation in low-resolution direct-methods phasing.

Probabilistic direct-methods phasing theory, originally based on a uniform atomic distribution hypothesis, is shown to be adaptable to a non-uniform bulk-solvent-compensated globbic approximation for protein crystals at low resolution. The effective number n(g) of non-H protein atoms per polyatomic glob increases with decreasing resolution; low-resolution phases depend on the positions of only N(g) = N(a)/n(g) globs rather than N(a) atoms. Test calculations were performed with measured structure-factor data and the refined structural parameters from a protein crystal with approximately 10 000 non-H protein atoms per molecule and approximately 60% solvent volume. Low-resolution data sets with d(min) ranging from 15 to 5 A gave n(g) = ad(min) + b, with a = 1.0 A(-1) and b = -1.9 for the test case. Results of tangent-formula phase-estimation trials emphasize that completeness of the low-resolution data is critically important for probabilistic phasing.

Crystallization↗

PubMed2000

R6 hexameric insulin complexed with m-cresol or resorcinol.

The structures of three R(6) human insulin hexamers have been determined. Crystals of monoclinic m-cresol-insulin, monoclinic resorcinol-insulin and rhombohedral m-cresol-insulin diffracted to 1. 9, 1.9 and 1.78 A, respectively, and have been refined to residuals of 0.195, 0.179 and 0.200, respectively. In all three structures, a phenolic derivative is found to occupy the phenolic binding site, where it forms hydrogen bonds to the carbonyl O atom of CysA6 and the N atom of CysA11. Two additional phenolic derivative binding sites were identified within or between hexamers. The structures of all three hexamers are nearly identical, although a large displacement of the N-terminus of one B chain in both monoclinic structures results from coordination to a sodium ion which is located between symmetry-related hexamers. Other minor differences in structure arise from differences in packing in the monoclinic cell compared with the rhombohedral cell. Based upon the differences in conformation of the GluB13 side chains in T(6), T(3)R(f)(3) and R(6) hexamers, the deprotonation of these side chains appears to be associated with the T-->R conformational transition.

Cresols↗

PubMed2000

The first protein crystal structure determined from high-resolution X-ray powder diffraction data: a variant of T3R3 human insulin-zinc complex produced by grinding.

X-ray diffraction analysis of protein structure is often limited by the availability of suitable crystals. However, the absence of single crystals need not present an insurmountable obstacle in protein crystallography any more than it does in materials science, where powder diffraction techniques have developed to the point where complex oxide, zeolite and small organic molecular structures can often be solved from powder data alone. Here, that fact is demonstrated with the structure solution and refinement of a new variant of the T(3)R(3) Zn-human insulin complex produced by mechanical grinding of a polycrystalline sample. High-resolution synchrotron X-ray powder diffraction data were used to solve this crystal structure by molecular replacement adapted for Rietveld refinement. A complete Rietveld refinement of the 1630-atom protein was achieved by combining 7981 stereochemical restraints with a 4800-step (d(min) = 3.24 A) powder diffraction pattern and yielded the residuals R(wp) = 3.73%, R(p) = 2.84%, R(F)(2) = 8.25%. It was determined that the grinding-induced phase change is accompanied by 9.5 and 17.2 degrees rotations of the two T(3)R(3) complexes that comprise the crystal structure. The material reverts over 2-3 d to recover the original T(3)R(3) crystal structure. A Rietveld refinement of this 815-atom protein by combining 3886 stereochemical restraints with a 6000-step (d(min) = 3.06 A) powder diffraction pattern yielded the residuals R(wp) = 3.46%, R(p) = 2.64%, R(F)(2) = 7.10%. The demonstrated ability to solve and refine a protein crystal structure from powder diffraction data suggests that this approach can be employed, for example, to examine structural changes in a series of protein derivatives in which the structure of one member is known from a single-crystal study.

Humans↗

PubMed1999

Progress on the direct-methods solution of macromolecular structures using single-wavelength anomalous-dispersion (SAS) data.

In the past few years, a number of strategies have been outlined to resolve the SAS phase ambiguity given that unique estimates omega (h, k) of the triple invariants are available. A new least-squares method is described that can in principle resolve the phase ambiguity to determine macromolecular phases provided that omega (h, k) estimates are unbiased. Limitations of the method in practical applications are discussed. An example is given where the correct solution can be identified by use of the SAS tangent formula in the instance that traditional SAS phasing methods have lead to an incorrect heavy-atom substructure.

Anti-Bacterial Agents↗

PubMed1999

On 'globbicity' of low-resolution protein structures.

Using Harker's [Harker (1953). Acta Cryst. 6, 731-736] idea of spherically averaged polyatomic groups or 'globs' as the units of structure suitable for analyzing low-resolution diffraction data from protein crystals, 'globbic' scattering factors have been calculated for main-chain peptide units and amino-acid side-chain groups to 3 A resolution via Debye's [Debye (1915). Ann. Phys. (Leipzig), 46, 809-823] scattering formula. It is shown that the scattering factors are insensitive to intra-globbic conformational variation and can be approximated fairly well by a single-Gaussian formula, i.e. fg(s) = Zg exp(-1.7Zgs2), where s = (sin theta)/lambda and Zg is the total electron count for the atoms of the glob. Phase errors due to the globbic approximation and their effect on electron-density maps at 3.5 A resolution have been assessed via calculations for the crambin structure; this analysis indicates that the globbic scattering factors will be useful in efforts to develop procedures for direct-methods phasing of diffraction data to approximately 3.5 A resolution from protein crystals.

Crystallography, X-Ray↗

PubMed1998

The use of SnB to determine an anomalous scattering substructure.

The positions of eight Se atoms in a selenomethionyl 35 kDa protein were determined at 2.0 and 2.5 A resolution using the direct-methods program SnB. Data at the selenium peak, edge and remote wavelengths were measured and processed independently. Anomalous difference E magnitudes at each wavelength were derived by two different procedures: renormalized diffE values were calculated according to the equation diffE = ¿sigma[(f + f')2 + f"2]||E+|-|E-||¿1/2/2q(sigma f"2)1/2, where q is a least-squares fitted renormalization function of sinstraight theta/lambda such that = 1.0; and difference E magnitudes were calculated from DeltaF2. Locally normalized E magnitudes corresponding to |FA| were also derived from the combination of the data at all three wavelengths through the use of the MADSYS program suite. Each of the independent sets of anomalous difference E magnitudes was capable of producing the correct solution, as did the E data obtained from the FA data. Higher success rates with SnB were observed for the 2.0 A peak and edge diffE data.

Complement C3d↗

PubMed1998

The 1.1 A resolution crystal structure of [Tyr15]EpI, a novel alpha-conotoxin from Conus episcopatus, solved by direct methods.

Conotoxins are valuable probes of receptors and ion channels because of their small size and highly selective activity. alpha-Conotoxin EpI, a 16-residue peptide from the mollusk-hunting Conus episcopatus, has the amino acid sequence GCCSDPRCNMNNPDY(SO3H)C-NH2 and appears to be an extremely potent and selective inhibitor of the alpha3beta2 and alpha3beta4 neuronal subtypes of the nicotinic acetylcholine receptor (nAChR). The desulfated form of EpI ([Tyr15]EpI) has a potency and selectivity for the nAChR receptor similar to those of EpI. Here we describe the crystal structure of [Tyr15]EpI solved at a resolution of 1.1 A using SnB. The asymmetric unit has a total of 284 non-hydrogen atoms, making this one of the largest structures solved de novo by direct methods. The [Tyr15]EpI structure brings to six the number of alpha-conotoxin structures that have been determined to date. Four of these, [Tyr15]EpI, PnIA, PnIB, and MII, have an alpha4/7 cysteine framework and are selective for the neuronal subtype of the nAChR. The structure of [Tyr15]EpI has the same backbone fold as the other alpha4/7-conotoxin structures, supporting the notion that this conotoxin cysteine framework and spacing give rise to a conserved fold. The surface charge distribution of [Tyr15]EpI is similar to that of PnIA and PnIB but is likely to be different from that of MII, suggesting that [Tyr15]EpI and MII may have different binding modes for the same receptor subtype.

Amino Acid Sequence↗

PubMed1997

On integrating the techniques of direct methods with anomalous dispersion. IV. A simplified perturbation treatment for SAS phasing.

Results from probabilistic theory for the single-wavelength anomalous-scattering (SAS) Friedel pair, two-phase structure invariants, psi H = phi H + phi-H, are used to show that the SAS three-phase structure invariants, psi HK = phi H + phi K + phi-H-K, tend to positive values that are easily estimated. Appropriate averages of the estimates provide SAS perturbation corrections in the form of positive origin shifts for the probability distribution of psi HK values and for the tangent formula. The theoretical probabilistic results are verified by empirical statistical analyses of model-calculated phases and experimentally measured structure-factor magnitudes for a small-molecule and a protein crystal structure.

Chemistry, Physical↗

PubMed1996

Structure of tris(cyclohexylammonium) phosphoenolpyruvate monohydrate and crystal chemistry of phosphoenolpyruvates.

The crystal structure of (C6H11NH3+)3. Pep3-.H2O, where Pep3- = (O-)2P(O)-O-C(CH2)-CO2-, is reported and the systematic structural variations among 19 crystallographic occurrences of H3Pep, H2Pep-, HPep2- and Pep3- species, which are important phosphate donors in the ATP cycle of bioenergetics, are reviewed. Tris(cyclohexylammonium) phosphoenolpyruvate monohydrate, (C6H11NH3+)3.-[O3POC(CH2)CO2]3-.H2O, M(r) = 483.6, m.p. 418-420K; T = 296(1)K; orthorhombic, P2(1)2(1)2(1); a = 16.7042(5), b = 24.4881 (6), c = 6.38910 (10) A; V = 2613.49(11) A3, Z = 4, Dx = 1.23, Dm = 1.22 mg mm-3, mu = 0.14 mm-1 for lambda(MoK alpha) = 0.7107 A; F(000) = 1056 e; R(magnitude of F) = 0.0608 for 6056 hkl and hkl data with (sin theta)/lambda < or = 0.65 A-1.

Crystallography, X-Ray↗

PubMed1996

O-phospho-DL-threonine and O-phospho-L-threonine compared with their serine analogs.

In crystals of O-phospho-DL-threonine and O-phospho-L-threonine, the molecules are zwitterions HO3-POCH-(CH3)CH(NH3+)CO2H linked by three-dimensional networks of strong P-O-H...O = P, C-O-H...O = P, N-H...O = P and N-H...O = C hydrogen bonds with (O...O) = 2.55 (3) A and (N...O) = 2.84 (4) A. Both the molecular conformations and the nearest-neighbor hydrogen-bonded surroundings are very similar in the racemic and enantiomeric crystals of the threonine compounds, but earlier studies of crystals of the analogous serine compounds have shown that the serine zwitterions HO3-POCH2CH(NH3+)CO2H have different conformations about the C beta-O gamma-P phosphate ester bonds and different hydrogen-bonded surroundings.

Crystallization↗

PubMed1995

On the differences between X-ray and neutron thermal vibration parameters.

For crystal structures analyzed by both X-ray and neutron diffraction, the anisotropic mean-square displacement parameters of the non-H atoms are sometimes found to differ significantly. The differences can usually be adjusted by either: (1) an isotropic factor q, defined by UijX = qUijN, to correct for a temperature difference between the two experiments; (2) anisotropic factors qij, defined by UijX = qijUijN, to correct for a temperature difference and different anisotropic diffraction effects of absorption, extinction, thermal diffuse scattering, multiple reflection, or systematic measuring errors in the two experiments; (3) anisotropic diffraction correction terms delta Uij, defined by UijX = UijN + delta Uij; (4) the sum of an isotropic temperature correction and anisotropic diffraction corrections, defined by UijX = qUijN + delta Uij. Correction parameters q, qij and delta Uij are easily calculated by linear least-squares fit, and the corrections from (3) or (4) seem to be the most reliable. Corrections calculated from X-ray and neutron Uij's of the non-H atoms of a crystal can be useful for adjusting the neutron Uij's of the H atoms for adoption, along with the neutron coordinates of the H atoms, as fixed parameters in an X-ray analysis of the electron density distribution.

Crystallography, X-Ray↗

PubMed1995

Computational studies of crystalline H3PO4.

A polarized split-valence wavefunction was computed for the H3PO4 molecule at its neutron crystallographic valence geometry, and the wavefunction was used to map the molecular electron-density distribution and to simulate X-ray crystal structure factors for both static, at-rest and dynamic thermally averaged structures. The thermal vibrational averaging was approximated using anisotropic mean-square atomic displacements from approximately 300 K neutron diffraction data. The simulated X-ray data were used to test pseudoatom multipole modeling of the valence electron-density distribution, in particular, radial modeling of the M valence shell of the P atom, and deconvolution of the nonspherical density features from anisotropic vibrational smearing.

Crystallography, X-Ray↗

PubMed1995

Experimental electron density in crystalline H3PO4.

X-ray diffraction data for H3PO4 crystals have been measured to dmin = 0.46 A resolution, and used to model the electron-density distribution with the hydrogen structure of the crystals adopted from an earlier neutron diffraction analysis. The molecule is asymmetric in the crystal with site symmetry 1 (C1), but the local symmetries of the pseudoatomic densities are, within experimental error, equivalent as they would be under idealized 3m (C3v) molecular symmetry. Although the experimental analysis entailed substantial problems with absorption and extinction corrections, the static deformation density from the experiment agrees very well with that from a polarized split-valence molecular orbital wavefunction for an isolated molecule with the crystallographic molecular geometry. Hydrogen bonding in the crystal polarizes the molecule's P==O acceptor group towards P(+)--O-, and appears to relocalize the lone-pair density of the P--OH donor groups. Crystal data: anhydrous orthophosphoric acid, H3PO4, M(r) = 98.00, room temperature, P2(1)/c, a = 5.7572 (13), b = 4.8310 (17), c = 11.5743 (21) A, beta = 95.274 (12) degrees, V = 320.55 (25) A3, Z = 4, dx = 2.030 mg mm-3, mu = 0.660 mm-1 for lambda(Mo K alpha) = 0.7107 A, F(000) = 200 e-, R(parallel F) = 0.026 for 3512 unique reflections.

Crystallography, X-Ray↗