Difference Electron Density Map Research Articles

Atomic resolution crystal structure of hydroxynitrile lyase from Hevea brasiliensis.

The X-ray crystal structure of native hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) has been determined at 1.1 A resolution. It refined to a final R of 11.5% for all data and an Rfree of 14.4%. The favorable data-to-parameter ratio at atomic resolution made the refinement of individual anisotropic displacement parameters possible. The data also allowed a clear distinction of the alternate orientations of all histidine and the majority of asparagine and glutamine side chains. A number of hydrogen atoms, including one on the imidazole of the mechanistically important His-235, became visible as peaks in a difference electron density map. The structure revealed a discretely disordered sidechain of Ser-80, which is part of the putative catalytic triad. Analysis of the anisotropy indicated an increased mobility of residues near the entrance to the active site and within the active site.

Difference density quality (DDQ): a method to assess the global and local correctness of macromolecular crystal structures.

Methods for the evaluation of the accuracy of crystal structures of proteins and nucleic acids are of general importance for structure-function studies as well as for biotechnological and biomedical research based upon three-dimensional structures of biomacromolecules. The structure-validation program DDQ (difference-density quality) has been developed to complement existing validation procedures. The DDQ method is based on the information present in a difference electron-density map calculated with the water molecules deliberately omitted from the structure-factor calculation. The quality of a crystal structure is reflected in this difference map by (i) the height of solvent peaks occurring at physical chemically reasonable positions with respect to protein and ligand atoms and (ii) the number and height of positive and negative 'shift' peaks next to protein atoms. The higher the solvent peaks and the lower the shift peaks, the better the structure is likely to be. Moreover, extraneous positive density due to an incomplete molecular model is also monitored, since this is another indicator of imperfections in the structure. Automated analysis of these types of features in difference electron densities is used to quantify the local as well as global accuracy of a structure. In the case of proteins, the DDQ structure-validation method is found to be very sensitive to small local errors, to omitted atoms and also to global errors in crystal structure determinations.

Imaging RNA and dynamic protein segments with low-resolution virus crystallography: experimental design, data processing and implications of electron density maps

Single crystal diffraction data were collected from virus crystals in the resolution range of 270 to 14 Å using a synchrotron X-ray source and a small-angle scattering instrument adapted for single crystal measurements. Reflections were measured from single crystals of the capsid of the double-stranded DNA bacteriophage HK97 and synthetic Flock House virus-like particles (sFHV). The quality of the low-resolution measurements was confirmed by excellent scaling statistics for both data sets. The sFHV amplitudes between 270 and 90 Å resolution were closely similar to independently measured solution scattering data, and to data calculated from the Fourier transform of a uniform density sphere of 315 Å diameter. A rotation function computed with the sFHV data between 70 and 20 Å resolution was readily interpretable. A uniform density sphere model was used to compute phases for measured amplitudes between 270 and 68 Å resolution. The calculated phases were refined and extended to 14 Å resolution with real space averaging employing an external mask shape defined by the high-resolution structure. The resulting electron density map displayed regions interpretable as loosely ordered RNA that connected ordered RNA segments seen in a published 3.0 Å resolution map. The published high-resolution electron density map lacked data inside 15 Å resolution and the interior of the particle in that map appeared hollow. Difference electron density maps corresponding to bulk RNA were computed by subtracting the contribution of the protein shell, based on the available high-resolution atomic model, from either the cryo-electron microscopy density or the low-resolution X-ray density. Features of the RNA were closely similar in the cryo-electron microscopy and X-ray maps, demonstrating the consistency of the two imaging methods. Electron density maps computed at 14 and 6 Å resolution with the X-ray amplitudes showed that RNA contributed little to the scattering beyond 14 Å resolution.

The 0.78 A structure of a serine protease: Bacillus lentus subtilisin.

Ultrahigh-resolution X-ray diffraction data from cryo-cooled, B. lentus subtilisin crystals has been collected to a resolution of 0.78 A. The refined model coordinates have a rms deviation of 0.22 A relative to the same structure determined at room temperature and 2.0 A resolution. Several regions of main-chain and side-chain disorder have been identified for 21 out of 269 residues in one polypeptide chain. Hydrogen atoms appear as significant peaks in the Fo - Fc difference electron density map, and carbon, nitrogen, and oxygen atoms can be differentiated. The estimated standard deviation (ESD) for all main-chain non-hydrogen bond lengths is 0.009 A and 0.5 degrees for bond angles based on an unrestrained full-matrix least-squares refinement. Hydrogen bonds are resolved in the serine protease catalytic triad (Ser-His-Asp). Electron density is observed for an unusual, short hydrogen bond between aspartic acid and histidine in the catalytic triad. The hydrogen atom, identified by NMR in numerous serine proteases, appears to be shared by the heteroatoms in the bond. This represents the first reported correlation between detailed chemical features identified by NMR and those in a cryo-cooled crystallographic structure determination at ultrahigh resolution. The short hydrogen bond, designated "catalytic hydrogen bond", occurs as part of an elaborate hydrogen bond network, involving Asp of the catalytic triad. While unusual, these features appear to have conserved analogues in other serine protease families although specific details differ from family to family.

Effect of Fluorine Substitution on the Energies of Small Ring Compounds

The effect of fluorine substitution on the energies of small ring compounds has been examined via ab initio calculations at the MP2/6-31G*, MP2/6-311+G*, and B3LYP/6-311+G* theoretical levels, along with correction for differences in zero-point energies. The introduction of a fluorine into a cyclopropane ring leads to destabilization, which results from the higher s character in the orbitals forming the bonds to a substituent. On the other hand, this is not seen with cyclopropene. The effect of 3-substituents on cyclopropene was examined by studying cyclopropenone, cyclopropenethione, methylenecyclopropene, and 3,3-difluorocyclopropene. The stabilization was largest and about equal with the O and S substituents. Smaller effects were observed with CH2 and F2 substituents. The nature of the effects was studied making use of electron density difference maps. The structures and energies of the series of C4H4 and C4F4 derivatives were studied at the above theoretical levels. In most cases, fluorine substitution led to stabilization with respect to but-3-en-1-yne, but with tetrahedrane there was considerable destabilization. Fluorine substitution leads to destabilization of cyclobutyne with respect to cyclobutene.

The GTP effector site of ornithine decarboxylase from Lactobacillus 30a: kinetic and structural characterization.

A nucleotide effector site of the biodegradative form of ornithine decarboxylase from Lactobacillus 30a (OrnDC L30a) has been identified. OrnDC L30a activity at pH 8.0, where the enzyme is normally inactive, is stimulated by GTP and dGTP and to a lesser extent by GDP but not by ATP, CTP, or UTP. The pH profile indicates that activation by GTP is reflected by an increase in kcat/KM,orn (above pH 6.8), while Vmax remains constant over the pH range 4.0-9. 0. Scatchard plot analysis shows that GTP binds to OrnDC L30a at both pH 5.8 (KD = 0.11 microM) and pH 8.0 (KD = 1.6 microM), but unexpectedly, half-site binding is observed at the higher pH. The OrnDC L30a dodecamer dissociates into dimers at high pH in the presence or absence of GTP. The GTP binding site was located in difference electron density maps using low-resolution X-ray data. This represents a new type of GTP binding site. A model explaining the activation of OrnDC L30a by GTP is presented.

Structural Aspects of Cyclopropyl Conjugation: Experimental Studies and Ab Initio Calculations

AbstractLow temperature crystal structural studies of small cyclopropyl‐conjugated unsaturated hydrocarbons reveal significant differences in bond lengths between carbon atoms mainly within the three‐membered rings but also for adjacent CC single bonds. The strongest influence of conjugation was observed for spiro[2.4]hepta‐4,6‐diene (1), where the difference in the lengths of three‐membered ring bonds is 0.050 Å (X‐ray). The same effect is also obvious from ab initio calculations at HF/6‐31G(d) (to a minor extent) and MP2/6‐31G(d) levels, as shown by a difference analysis of bond lengths which was performed in order to cancel out systematic errors and differences for both methods. Static difference electron density maps were derived from the experimental data by multipole refinements. These maps showed shifts of electron density to exocyclic locations within the planes of the three‐membered rings and significant bond ellipticities for the adjacent single bonds. For 1,1‐diethenylcyclopropane (4) a nonsymmetric conformation was found in the crystal lattice, very close to the global minimum structure calculated by ab initio methods.

Structure of Poly d(AI)·Poly d(CT) in Two Different Packing Arrangements

X-ray diffraction analysis of poly d(AI)poly·d(CT) in oriented and polycrystalline fibers has revealed the DNA structure to be a 10-fold, right-handed, antiparallel, Watson-Crick base paired double helix in two distinct packing arrangements corresponding to one and two helices, respectively, in the unit cell. The helix pitch is 32.1 Å and 32.4 Å in the two cases, almost 1.5 Å shorter than in classical B-DNA. The resulting B-DNA geometry, described in terms of a tetranucleotide repeat which is conformationally similar to B-DNA, has its minor groove closely shut and major groove correspondingly widened, thus striking a sharp morphological contrast to B-DNA. According to difference electron density maps, a spine of hydration along the minor groove connects both strands and provides structural stability; ordered sodium ions and water molecules are actively involved in bridging the phosphate groups of neighboring helices. The crystallographic R-values for these two allomorphs are 0.26 and 0.20, respectively, for data up to 3. 0 Å resolution.

Structural Aspects of Cyclopropyl Homoconjugation: Experimental Studies and Ab Initio Calculations

AbstractLow‐temperature crystal structural studies of a series of saturated and unsaturated bicyclo[2.2.1]heptadiene, heptene and heptane compounds with 7‐spirocyclopropyl substitution reveal significant differences in the bond lengths of the three‐membered rings and in the CC single bonds of the bicyclic fragment. A complex interplay of strain and different types of conjugation influence the molecular structure of the bicycloheptadiene derivative 1, where the difference in the length of the three‐membered ring bonds is 0.040 Å and all CC single bonds in the bicyclic fragment are lengthened significantly. Ab initio calculations at the HF/6‐31G(d) (to a minor extent) and MP2/6‐31G(d) levels are in good agreement with the experimental data. Calculated charge distributions and dipole moments further support the relevance of cyclopropyl homoconjugation in the investigated prototype of Walsh and through‐space π‐orbital interaction. Static difference electron density maps have been derived from the experimental data by multipole refinements which showed exocyclic shifts of electron density in the planes of the three‐membered rings and significant bond ellipticities at the CC single bonds in the unsaturated bicyclic units.

On the nature of the [formula omitted] and [formula omitted] surface bonding

The nature of sulfate-Ag(111) and sulfate-Au(111) surface bonding has been investigated at the SCF + MP2 level of theory. Convergence of binding energy with cluster size is investigated and, unlike neutral adsorbates, large clusters are required in order to obtain reliable binding energies. In the most stable adsorption mode, sulfate binds to the surface via three oxygen atoms (C 3v symmetry) with a binding energy of 159.3 kcal/mol on Ag(111) and 143.9 kcal/mol on Au(111). The geometry of adsorbed sulfate was optimized at the SCF level. While the bond length between sulfur and the oxygens coordinated to the surface increases, the sulfur-uncoordinated oxygen bond length decreases. This weakening and strengthening of the bonds, respectively, is consistent with bond order conservation in adsorbates on metal surfaces. Although a charge transfer of 0.4 electrons towards the metal is observed, the adsorbate remains very much sulfate-like. The molecular orbital analysis indicates that there is also some charge back-donation towards unoccupied orbitals of sulfate. This results in an increased electron density around sulfur as revealed in the electron density difference maps. Analysis of the Laplacian of the charge density of free sulfate provides a suitable framework to understand the nature of the different charge transfer processes and allows us to establish some similarities with the CO- and SO 2-metal bondings.

Analysis of the structure of chemically synthesized HIV-1 protease complexed with a hexapeptide inhibitor. Part I: Crystallographic refinement of 2 A data.

The structure of a complex between a hexapeptide-based inhibitor, MVT-101, and the chemically synthesized (Aba 67,95,167,195; Aba: L-alpha-amino-n-butyric acid) protease from the human immunodeficiency virus (HIV-1), reported previously at 2.3 A has now been refined to a crystallographic R factor of 15.4% at 2.0 A resolution. Root mean square deviations from ideality are 0.18 A for bond lengths and 2.4 degrees for the angles. The inhibitor can be fitted to the difference electron density map in two alternative orientations. Drastic differences are observed for positions and interactions at P3/S3 and P3'/S3' subsites of the two orientations due to different crystallographic environments.

The role of hydrogens in stabilizing organic ions

The changes in electron density that result from processes in which ions are formed were examined using both electron density difference maps and calculations of electron populations using Bader's theory of atoms in molecules. The processes include the loss of an electron from a neutral precursor forming a radical cation, the loss of an electron from a free radical forming a carbocation, the addition of a proton to ammonia, the addition of an electron to a free radical forming a carbanion, and the loss of a proton from a neutral precursor forming an anion. In the reactions forming cations, the new positive charge resides mainly at the hydrogens, and the heavy atoms generally gain electron density. The anion-forming reactions lead to negative charge being shared among atoms but, again, much of the charge appears at the hydrogens. σ–π polarization is an important feature of the charge distribution in all of the ions. There is an advantage to placing the charge at the periphery of an ion since this will minimize its electrostatic energy. Key words: electron density, anions, cations, radical cations, σ–π polarization.

Structure of a bovine thrombin-hirudin51-65 complex determined by a combination of molecular replacement and graphics. Incorporation of known structural information in molecular replacement.

Crystals of the bovine thrombin-hirudins(51-65) complex have space group P6(1)22 with cell constants a = 116.4, and c = 200.6 A and two thrombin molecules in the asymmetric unit. Only one thrombin molecule could be located by generalized molecular replacement; the second was fit visually as a rigid body to an improved electron-density difference map. The structure was refined to R = 0.192 with two B values per residue (main chain and side chain) at 3.2 A. The polar interactions of the peptides with the exosite of thrombin show differences consistent with the known flexibility in the interactions of the C-terminal peptide of hirudin with thrombin. The hirudin peptide in complex 2 has a higher temperature factor as compared with peptide 1 which may be correlated partly with a larger number of short-range electrostatic interactions between peptide 1 and thrombin and partly with the fact that thrombin 2 is epsilon-thrombin which is cleaved at Thr149A near the peptide binding site. Later, using this structure as a test case, it was shown that the position for the second thrombin could also be determined by a novel modification of the molecular-replacement method in which the contribution of the known molecule is subtracted from the structure factors. This approach is facile and applicable to any crystal containing two or more macromolecules in the asymmetric unit in which some but not all of the molecules can be determined by molecular replacement.

Single-crystal structure analysis and energy minimizations of a MFI-type zeolite at low p-dichlorobenzene sorbate loading

The crystal structure of a low-loaded adsorption complex of H-ZSM-5 with p-dichlorobenzene has been studied by single-crystal X-ray diffraction. The controversy in the literature, concerning the preferred location of the p-dichlorobenzene molecule, is explained by different interpretations of the difference electron-density map representing the electron density of the absorbed molecule. The crystal studied contains 2.56 (2) p-dichlorobenzene molecules per unit cell. The absorbed molecules prefer the position at the intersection of channels. Energy calculations, using the Biosym Catalysis and Sorption Software, strongly support this interpretation. In nearly all calculations the orientation of the relaxed molecule is close to the orientation found from the X-ray analysis. A second rotational position at the intersection, suggested from calculations in a fully relaxed structure, resembles the location of the p-xylene molecule at the intersection of channels in the high-loaded H-ZSM-5/8p-xylene complex. The unit cell of Si11.96Al0.04O24.0.32C6H4Cl2(+ 0.04H+), Mr = 767.42, is orthorhombic, Pnma, with a = 20.009 (3), b = 19.909 (4), c = 13.366 (2) Å, V = 5324 (2) Å3, Z = 8, Dx = 1.922 Mg m−3 and μ(Mo Kα) = 0.734 mm−1. The final R(wR) is 0.044 (0.048), w = 1/σ 2(F), for 5306 observed reflections with I> 2.0σ(I) measured at 293 K.

Structure and kinetics of the beta-lactamase mutants S70A and K73H from Staphylococcus aureus PC1.

Two mutant beta-lactamases from Staphylococcus aureus PC1 which probe key catalytic residues have been produced by site-directed mutagenesis. In the S70A enzyme, the nucleophilic group that attacks the beta-lactam carbonyl carbon atom was eliminated. Consequently, the kcat values for hydrolysis of benzylpenicillin and nitrocefin have been reduced by 10(4)-10(5) compared with the wild-type enzyme. The crystal structure of S70A beta-lactamase has been determined at 2.1 A resolution. With the exception of the mutation site, the structure is identical to that of the native enzyme. The residual activity is attributed either to mistranslation that leads to production of wild-type enzyme and/or to remaining features of the active site that stabilize the tetrahedral transition state. Soaking of the crystals with ampicillin or clavulanate, followed by flash-freezing, has been carried out and the structures examined at 2.0 A resolution. For both experiments, the difference electron density maps revealed buildup of density in the active site that presumably corresponds to beta-lactam binding. However, neither electron density is sufficiently clear for defining the atomic details of the bound compounds. The K73H beta-lactamase has been prepared to test the possible role of Lys73 in proton transfer. It exhibits no detectable activity toward benzylpenicillin, and 10(5)-fold reduction of kcat for nitrocefin hydrolysis compared with the wild-type enzyme. No significant recovery of activity has been measured when the pH was varied between 5.0 and 8.0. The crystal structure of K73H beta-lactamase has been determined at 1.9 A resolution. While the overall structure is similar to that of the native enzyme, the electrostatic interactions between His73 and neighboring residues indicate that the imidazole ring is positively charged. In addition, the hydroxyl group of Ser70 adopts a position that is incompatible with nucleophilic attack on substrates. A crystal soaked with ampicillin was flash-frozen, and diffraction data were collected at 2.1 A resolution. The electron density map showed no indication of substrate binding.

Crystal Structure of S-Adenosylmethionine Synthetase

Crystal Structure of S-Adenosylmethionine Synthetase

Sodium ions and water molecules in the structure of poly(dA)·poly(dT)

Analysis of X-ray diffraction data from a polycrystalline and well oriented fiber of the sodium salt of poly(dA).poly(dT) shows that this B'-DNA corresponds to a right-handed antiparallel tenfold double-helix of pitch 32.4 A, with C2'-endo furanose rings in both strands. The helix contrasts itself from B-DNA in terms of a very narrow minor groove. Difference electron-density maps have revealed that a continuous spine of water molecules, two per base pair, propagates along this groove with the same symmetry as the DNA and establishes new links between the two strands. In addition to this hydrated DNA helix, the monoclinic unit cell (space group P2(1)) accommodates about 20 sodium ions and 12 water molecules in the vicinity of phosphate groups. These structured guest molecules provide an intricate network of bridges, ranging in size from a single sodium ion to a multiple sodium-water-water-sodium unit, connecting phosphate groups belonging to adjacent DNA helices. The crystallographic R value for this structure is 0.23 for a total of 102 reflections extending out to 3.2 A resolution.

An Aminobenzyldioxocyclam Nickel(II) Complex

The structure was solved by the Patterson method and suc- cessive Fourier electron density difference maps, and refined by full-matrix least-squares procedures with anisotropic tem- perature factors for all non-H atoms. The positions of all H atoms were generated geometrically in the final stages of re- finement. Anomalous-dispersion corrections were taken from Cromer & Liberman (1970) for non-H atoms and from Stew- art, Davidson & Simpson (1965) for H atoms. Programs used were SDP (Enraf-Nonius, 1985), SHELX76 (Sheldrick, 1976) and ORTEP (Johnson, 1965). This work has received partial support from CNPq, FAPESP and FINEE which are hereby gratefully ac- knowledged. Abstract The title compound, (6-(4-aminobenzyl)- 1,11-diaza-4,8- diazanidacyclotetradecane-5,7-dione(2- )-/~4N 1,4,8,11 ) _ nickel(II), (Ni(C17H25N502)), has mirror symmetry with Ni--N(amide) and Ni--N(amine) bond distances of 1.862(5) and 1.925 (5)/~, respectively. The aromatic ring of the side chain is tipped 21.3 (3) ° towards the plane containing the Ni and four N atoms. The distance from the Ni atom to the center of the aromatic ring is 3.551 (5) A.

The molecular and solid‐state structure of borazine has been determined at 115 and 160 K by single‐crystal X‐ray crystallography. The molecular structure has C2 symmetry with small deviations from the expected D3h symmetry. The mean value of the BN bond length is 1.429(1) A and the interbond angles of the six‐membered ring are 117.1(1)° at the boron atoms and 122.9(1)°

Borazin im festen Zustand: Verdient es, als “anorganisches Benzol” bezeichnet zu werden?

Crystal Structure of Unmodified tRNAGln Complexed with Glutaminyl-tRNA Synthetase and ATP Suggests a Possible Role for Pseudo-Uridines in Stabilization of RNA Structure

tRNA(2Gln) made in vitro by transcription with T7 RNA polymerase does not contain the pseudouridines at positions 38, 39, and 55, the 4-thiouridine at position 8, or any of the methylated bases found in the tRNA(2Gln) made in vivo. Cocrystals of unmodified tRNA(2Gln) complexed with glutaminyl-tRNA synthetase from Escherichia coli are isomorphous with those of the complex with modified tRNA(2Gln). A difference electron density map between the complexes with modified and unmodified tRNAs calculated at 2.5-A resolution shows no differences in the protein or tRNA structures, except for some very small shifts in atoms contacting the thiol at the 4 position of uridine 8 that are required to accommodate the smaller oxygen in the unmodified tRNA. Perhaps the most functionally significant change in the unmodified tRNA is the absence of the specifically bound water molecules that are observed to cross-link the N5 of the pseudo-uridines to their 5' phosphate. This suggests a possible role for pseudouridinylation in stabilization of the tRNA through water-mediated linking of these modified bases to the backbone, which is consistent with the lower thermal stability of the unmodified tRNA. An identical water-bridging structure is possible at four of the five other psuedo-uridines in known tRNA structures.