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PubMed2006

Considerations for the refinement of low-resolution crystal structures.

It is often assumed that crystal structures have to be obtained at sufficiently high resolution in order to perform macromolecular refinement. In several recent structures, the threshold of what is considered ;acceptable' has been pushed to lower diffraction resolutions. Here, considerations and modifications to standard refinement protocols are described that were used to solve and refine a particular set of low-resolution structures for the ATPase p97/VCP. It was found that reasonable R(free) values and good geometry can be achieved upon refinement that includes experimental phase information along with judicious use of restraints at diffraction limits as low as 4.7 A. At this resolution, the topology and the backbone-chain trace are mostly defined, some side-chain positions can be unambiguously assigned and ligands within known binding sites can be identified. Furthermore, large conformational changes can be discerned when structures in different states are available, information that is not easily obtainable by other means.

Adenosine Triphosphatases↗

PubMed1994

Structure of copper- and oxalate-substituted human lactoferrin at 2.0 A resolution.

The three-dimensional structure of human dicupric monooxalate lactoferrin, Cu(2)oxLf, has been determined to 2.0 A resolution, using X-ray diffraction data collected by diffractometry to 2.5 A resolution, and oscillation photography on a synchrotron source to 2.0 A resolution. Difference electron-density maps calculated between Cu(2)oxLf and both dicupric lactoferrin, Cu(2)Lf, and diferric lactoferrin, Fe(2)Lf, showed that the oxalate had replaced a carbonate in the C-terminal binding site, and that, relative to Cu(2)Lf, there were no significant differences in the N-terminal site. The structure was then refined crystallographically by restrained least-squares methods. The final model, in which the r.m.s. deviation in bond distances is 0.017 A, contains 5314 protein atoms (691 residues), two Cu(2+) ions, one bicarbonate ion, one oxalate ion, 325 solvent molecules and one sugar residue. The crystallographic R factor of 0.193 is for 46 134 reflections in the range 8.0 to 2.0 A resolution. The oxalate ion is coordinated to copper in a 1,2-bidentate fashion, and the added bulk of the anion results in the rearrangement of the side chains of nearby arginine and tyrosine residues. No other major alterations in the molecule can be observed, the overall protein structure being the same as that for Cu(2)Lf and Fe(2)Lf.

Journal Article↗

PubMed1994

Extension of the diffraction resolution of crystals.

The diffraction resolution of crystals of the guanine nucleotide-exchange factor complex, EF-Tu-Ts, has been extended from 5.0 to 2.5 A by lowering the solvent content in the crystals as well as the temperature of data collection. The common form of EF-Tu-Ts crystal belongs to space group P2(1)2(1)2(1) with a = 81.1, b = 109.9, c = 207.5 A and has a solvent content of 61%. The crystals diffract to a resolution of 5.0 A at 293 K and 4.0 A at 273 K. When cryoprotective agents are slowly diffused into the crystals, the cell constants shrink to a = 74.4, b = 109.9, c = 198.7 A and the solvent content falls to 55%. After the cryoprotective agent has been added, the crystals diffract to 2.7 A resolution at 293 or 273 K and 2.5 A at 250 K. X-ray diffraction data, collected before and after the transformation of individual EF-Tu-Ts crystals, demonstrate that a large percentage of the improvement in diffraction resolution is due solely to the addition of cryoprotective agents. The transfer procedures for the successful introduction of cryoprotective agents into EF-Tu-Ts crystals as well as the general applicability to other crystal systems will be discussed.

Journal Article↗

PubMed1995

Refinement of purothionins reveals solute particles important for lattice formation and toxicity. Part 2: structure of beta-purothionin at 1.7 A resolution.

The crystal structure of beta-purothionin (beta-PT) has been determined at 1.7 A resolution. beta-PT and previously solved alpha(l)-PT belong to a family of membrane-active plant toxins homologous to crambin. (beta-PT crystallizes in the same space group as alpha(l)-PT (1422) but with the c axis 3 A longer than (alpha(l)-PT. The unit-cell dimensions of beta-PT crystals are a = b = 53.94 and c = 72.75 A. Two data sets were collected on a multiwire area detector, each with R(sym) around 6.0%, and were merged to get a single data set at 1.7 A, (R(merge) = 9.6%). The X-ray structure of alpha(l)-PT was used to build a starting model for beta-PT. The beta-PT model was refined using the program PROLSQ from 10 to 1.7 A resolution to an R-factor of 19.8% with very good geometry. The final structure contains 439 atoms including 337 protein atoms, 77 waters, two acetates, two glycerols and one phosphate. The high-resolution structure of the beta-PT agreed well with that of the lower resolution alpha(l)-PT structure only after the latter was extensively rerefined. Both refinements revealed phosphate and glycerol molecules which are important in lattice formation. The binding of phosphate and glycerol molecules to purothionins (PT) was confirmed by NMR and was implicated in the biological activity of toxins. Modeling of phospholipid binding to PT based on glycerol and phosphate-binding site could shed light on the lytic toxicity of this protein-toxin family. Although the structures of (alpha(l)-PT and beta-PT preserve the overall fold of crambin, they exhibit key differences that are directly relevant to the toxicity of thionins.

Journal Article↗

PubMed1996

Ribonuclease from Streptomyces aureofaciens at atomic resolution.

Crystals of ribonuclease from Streptomyces aureofaciens diffract to atomic resolution at room temperature. Using synchrotron radiation and an imaging-plate scanner, X-ray data have been recorded to 1.20 A resolution from a crystal of native enzyme and to 1.15 A from a crystal of a complex with guanosine-2'-monophosphate. Refinement with anisotropic atomic temperature factors resulted in increased accuracy of the structure. The R factors for the two structures are 10.6 and 10.9%. The estimated r.m.s. error in the coordinates is 0.05 A, less than half that obtained in the previous analysis at 1.7 A resolution. For the well ordered part of the main chain the error falls to below 0.02 A as estimated from inversion of the least-squares matrix. The two independent molecules in the asymmetric unit allowed detailed analysis of peptide planarity and some torsion angles. The high accuracy of the analysis revealed density for a partially occupied anion in the nucleotide binding site of molecule A in the native structure which was not seen at lower resolution. The anisotropic model allowed correction of the identity of the residue at position 72 from cysteine to threonine. Cys72 SG had been modelled in previous analyses with two conformations. The solvent structure was modelled by means of an automated procedure employing a set of objective criteria. The solvent structure for models refined using different programs with isotropic and anisotropic description of thermal motion is compared.

Journal Article↗

PubMed1996

Refinement and structural analysis of bovine cytochrome b5 at 1.5 A resolution.

The structure of bovine liver cytochrome b(5), a soluble 93-residue proteolytic fragment of a 16 kDa membrane-bound hemoprotein, initially solved at 2.0 A resolution, has been refined at 1.5 A using data collected on a diffractometer. Refinement to 2.0 A resolution used the Hendrickson-Konnert procedure PROLSQ and was then extended to 1.5 A resolution using the program PROFFT. Only residues 3-87 could be identified in the model and these residues together with 93 water molecules gave an agreement factor of R = 0.161 for data in the resolution range 1.5-5 A. The structure was finally refined using the program X-PLOR, which enabled alternate conformers to be modelled for several surface side chains. Residues 1 and 2 at the amino terminus of the protein and residue 88 near the carboxyl terminus could be identified from these electron-density maps. However the remaining disordered carboxy-terminal residues could not successfully be included in the model. A total of 117 solvent molecules were included in the final refinement to give R = 0.164 for the data between 1.5 and 10 A.

Journal Article↗

PubMed1996

How to escape from model bias with a high-resolution native data set - structure determination of the PcpA-S6 subunit III.

The structure of procarboxypeptidase A-S6 subunit III, a truncated zymogen E, has been determined by molecular replacement using as search model porcine elastase 1 which, as revealed by crystallographic analysis, contained about 20% of the amino acids in a radically different orientation. Two monoclinic crystal forms were used: the first one diffracts to 2.3 A resolution and contains one molecule per asymmetric unit; the second diffracts to 1.7 A resolution and contains two molecules per asymmetric unit. Molecular replacement and conventional X-PLOR refinement led to a model for which 20% of the chain was ill defined in both crystal forms. To remove the bias introduced by the initial model, an automated refinement procedure [Lamzin & Wilson (1993). Acta Cryst. D49, 129-147] was applied successfully to the second crystal form, which diffracts to high resolution. The resulting dramatic improvement of the electron-density map led to extensive rebuilding of some surface loops. The reliability of the modified model was confirmed by refinement of the first crystal form. For the two forms, the final R factor is 18.8% for data between 8.0 and 2.0 A resolution, and 18.4% for data between 8.0 and 1.7 A, respectively.

Journal Article↗

PubMed1996

Phasing proteins at low resolution.

A method for obtaining phases of low-order reflections is presented. It is based on four observations: (1) the electron density inside proteins is smooth and uniform at low resolution. (2) Since all proteins have almost the same density, the total volume of the protein is known if the molecular weight is known. (3) The overall shape of many proteins is fairly spherical. (4) The total scattering from a sphere of uniform density is in phase with a point scatterer at its centre of gravity, up to a well defined cross-over. After the first cross-over the total protein molecule scatters out of phase with its centre. If the centre of the protein can be found, the phases of typically the ten lowest resolution reflections can be very accurately determined. The method works, provided low-order reflections can be measured accurately and the centre of gravity can be well positioned from these data. The correctly phased low-resolution reflections may be used as a starting set for phase extension. By combining the measured amplitudes with these phases we believe that the size and low-resolution shape of an unknown protein, i.e. the envelope of the molecule, can be obtained.

Journal Article↗

PubMed1998

Transferability of multipole charge-density parameters: application to very high resolution oligopeptide and protein structures.

Crystallography at sub-atomic resolution permits the observation and measurement of the non-spherical character of the electron density (parameterized as multipoles) and of the atomic charges. This fine description of the electron density can be extended to structures of lower resolution by applying the notion of transferability of the charge and multipole parameters. A database of such parameters has been built from charge-density analysis of several peptide crystals. The aim of this study is to assess for which X-ray structures the application of transferability is physically meaningful. The charge-density multipole parameters have been transferred and the X-ray structure of a 310 helix octapeptide Ac-Aib2-L-Lys(Bz)-Aib2-L-Lys(Bz)-Aib2-NHMe refined subsequently, for which diffraction data have been collected to a resolution of 0.82 A at a cryogenic temperature of 100 K. The multipoles transfer resulted in a significant improvement of the crystallographic residual factors wR and wR free. The accumulation of electrons in the covalent bonds and oxygen lone pairs is clearly visible in the deformation electron-density maps at its expected value. The refinement of the charges for nine different atom types led to an additional improvement of the R factor and the refined charges are in good agreement with those of the AMBER molecular modelling dictionary. The use of scattering factors calculated from average results of charge-density work gives a negligible shift of the atomic coordinates in the octapeptide but induces a significant change in the temperature factors (DeltaB approximately 0.4 A2). Under the spherical atom approximation, the temperature factors are biased as they partly model the deformation electron density. The transfer of the multipoles thus improves the physical meaning of the thermal-displacement parameters. The contribution to the diffraction of the different components of the electron density has also been analyzed. This analysis indicates that the electron-density peaks are well defined in the dynamic deformation maps when the thermal motion of the atoms is moderate (B typically lower than 4 A2). In this case, a non-truncated Fourier synthesis of the deformation density requires that the diffraction data are available to a resolution better than 0.9 A.

Crystallography, X-Ray↗

PubMed1998

Structure of dimeric and monomeric erabutoxin a refined at 1.5 A resolution.

Erabutoxin a has been crystallized in its monomeric and dimeric forms. The structures were refined at 1.50 and 1.49 A resolution, respectively, using synchrotron radiation data. The crystals belong to space group P212121, with cell dimensions a = 49.84, b = 46.62, c = 21.22 A for the monomer and a = 55.32, b = 53.54, c = 40.76 A for the dimer. Using starting models from earlier structure determinations, the monomeric structure refined to an R value of 16.7% (8004 unique reflections, 17.0-1.50 A resolution range), while the dimeric structure has been solved by the molecular-replacement method with a final R value of 16.9% (19 444 unique reflections, 17.4-1.49 A resolution range). The high-resolution electron-density maps clearly revealed significant discrete disorder in the proteins and allowed an accurate determination of the solvent structure. For the monomer, the side chains of six residues were modelled with alternate conformers and 106 sites for water molecules and one site for a sulfate ion were included in the final model, whereas for the dimer, 206 sites for water molecules were included and both C-terminal residues together with the side chains of 11 residues adopted alternative conformations. A comparison was made with earlier structure determinations. The features of the solvent structure of the erabutoxin molecules are discussed in detail.

Crystallization↗

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↗

PubMed1999

Protein side-chain conformation: a systematic variation of chi 1 mean values with resolution - a consequence of multiple rotameric states?

A systematic variation with resolution of the mean values of the gauche-, trans and gauche+ chi1 rotamers in protein structures determined by X-ray crystallography has been observed. Further analysis revealed that these correlations differ considerably between residue types, being highly significant for some residue types (e.g. Ser, Thr, Leu, Lys) and absent for others (e.g. aromatics). For the individual residue types which exhibited the trend most strongly, these changes were accompanied by corresponding systematic variations in the percentage relative populations in the three energy wells. Examination of a uniformly sized subset of monomers showed that this effect, while attenuated, was still present, and was thus not entirely a consequence of the change in size and surface area which also correlates with resolution. An analysis of B values in the disfavoured high-energy barrier region between the rotameric wells showed a pronounced tendency towards larger than average values. As a plausible hypothesis, it is suggested here that these observations can be accounted for by the presence of multiple rotameric states. The averaged electron density produced by dual occupancy at low resolution giving an averaged conformation is resolved at high resolution into its individual components.

Asparagine↗

PubMed1999

Atomic resolution structure of biotin-free Tyr43Phe streptavidin: what is in the binding site?

The streptavidin-biotin system is an example of a high-affinity protein-ligand pair (Ka approximately 10(13) mol-1). The thermodynamic and structural properties have been extensively studied as a model system for protein-ligand interactions. Here, the X-ray crystal structure of a streptavidin mutant of a residue hydrogen bonding to biotin [Tyr43Phe (Y43F)] is reported at atomic resolution (1.14 A). The biotin-free structure was refined with anisotropic displacement parameters (SHELXL97 program package). The high-resolution data also allowed interpretation of side-chain and residue disorder in 41 residues where alternate conformations were refined. The Y43F mutation is unambiguously observed in difference maps, although only a single O atom per monomer is altered. The atomic resolution enabled the identification of 2-methyl-2, 4-pentanediol (MPD) molecules in the biotin-binding pocket for the first time. Electron density for MPD was observed in all four subunit binding sites of the tetrameric protein. This was not possible with data at lower resolution (1.8-2.3 A) for wild-type streptavidin or mutants in the same crystal form using MPD in the crystallization. The impact of MPD binding on these studies is discussed.

Binding Sites↗

PubMed2000

Towards the charge-density study of proteins: a room-temperature scorpion-toxin structure at 0.96 A resolution as a first test case.

The number of protein structures refined at a resolution higher than 1.0 A is continuously increasing. Subatomic structures may deserve a more sophisticated model than the spherical atomic electron density. In very high resolution structural studies (d < 0.5 A) of small peptides, a multipolar atom model is used to describe the valence electron density. This allows a much more accurate determination of the anisotropic thermal displacement parameters and the estimate of atomic charges. This information is of paramount importance in the understanding of biological processes involving enzymes and metalloproteins. The structure of the scorpion Androctonus australis Hector toxin II has been refined at 0.96 A resolution using synchrotron diffraction data collected at room temperature. Refinement with a multipolar electron-density model in which the multipole populations are transferred from previous peptide studies led to the observation of valence electrons on covalent bonds of the most ordered residues. The refined net charges of the peptide-bond atoms were of the correct sign but were underestimated. Such protein-structure refinements against higher resolution data collected at cryogenic temperature will enable the calculation of experimental atomic charges and properties such as electrostatic potentials.

Crystallography, X-Ray↗

PubMed2006

On the use of CCD area detectors for high-resolution specular X-ray reflectivity.

The use and application of charge coupled device (CCD) area detectors for high-resolution specular X-ray reflectivity is discussed. Direct comparison of high-resolution specular X-ray reflectivity data measured with CCD area detectors and traditional X-ray scintillator ('point') detectors demonstrates that the use of CCD detectors leads to a substantial (approximately 30-fold) reduction in data acquisition rates because of the elimination of the need to scan the sample to distinguish signal from background. The angular resolution with a CCD detector is also improved by a factor of approximately 3. The ability to probe the large dynamic range inherent to high-resolution X-ray reflectivity data in the specular reflection geometry was demonstrated with measurements of the orthoclase (001)- and alpha-Al2O3 (012)-water interfaces, with measured reflectivity signals varying by a factor of approximately 10(6) without the use of any beam attenuators. Statistical errors in the reflectivity signal are also derived and directly compared with the repeatability of the measurements.

Equipment Design↗

PubMed1998

On high-resolution reciprocal-space mapping with a triple-crystal diffractometer for high-energy X-rays.

High-energy X-rav diffraction by means of triple-crystal techniques is a powerful tool for investigating dislocations and strain in bulk materials. Radiation with an energy typically higher than 80 keV combines the advantage of low attenuation with high resolution at large momentum transfers. The triple-crystal diffractometer at the High Energy Beamline of the European Synchrotron Radiation Facility is described. It is shown how the transverse and longitudinal resolution depend on the choice of the crystal reflection, and how the orientation of a reciprocal-lattice distortion in an investigated sample towards the resolution element of the instrument can play an important role. This effect is demonstrated on a single crystal of silicon where a layer of macro pores reveals satellites around the Bragg reflection. The resulting longitudinal distortion can be investigated using the high transverse resolution of the instrument when choosing an appropriate reflection.

Journal Article↗

PubMed1989

High resolution NMR spectroscopy using a recursive algorithm.

When the DFT (discrete Fourier transform) is used as a method for obtaining NMR (nuclear magnetic resonance) spectrum, resolution of the obtained NMR spectrum depends clearly on spin-spin relaxation times of the nucleus and the sampled number of NMR signal. That is, the NMR spectrum is affected by decay of NMR signal due to the relaxation times and errors associated with discrete approximation of NMR spectrum. As a results, its resolution is degraded and the spectrum disagrees with the density distribution of the nucleus. In the present paper, we propose a new NMR spectrum estimation method for reducing their effects, demonstrating a high resolution NMR spectrum which is closer to the density distribution than that of the DFT. The proposed method is derived from applying the Kalman filter or the recursive least-square method to a NMR signal model based on Bloch equation. Under various conditions, the proposed method is compared with the DFT using a proton 1H NMR signal observed from mayonnaise in a 2 [T] static magnetic field. The results show that if spin-spin relaxation times are known previously, the proposed method can provide a higher resolution NMR spectrum than the DFT. In addition, it is shown that the method can reduce the discrete approximation errors of NMR spectrum. Finally, we demonstrate that if frequency components of sources of NMR signal are present in a limited range in frequency domain of interest, the method can magnify NMR spectrum with a sampling time and interval fixed.

Algorithms↗

PubMed2004

Effect of skull resistivity on the spatial resolutions of EEG and MEG.

The resistivity values of the different tissues of the head affect the lead fields of electroencephalography (EEG). When the head is modeled with a concentric spherical model, the different resistivity values have no effect on the lead fields of the magnetoencephalography (MEG). Recent publications indicate that the resistivity of the skull is much lower than what was estimated by Rush and Driscoll. At the moment, this information on skull resistivity is, however, slightly controversial. We have compared the spatial resolution of EEG and MEG for cortical sources by calculating the half-sensitivity volumes (HSVs) of EEG and MEG as a function of electrode and magnetometer distance, respectively, with the relative skull resistivity as a parameter. Because the spatial resolution is related to the HSV, these data give an overview of the effect of these parameters on the spatial resolution of both techniques. Our calculations show that, with the new information on the resistivity of the skull, in the spherical model for cortical sources the spatial resolution of the EEG is better than that of the MEG.

Brain↗