Ab Initio Direct Methods Research Articles

Ab initio structure solution of a proteolytic fragment using ARCIMBOLDO.

Crystal structure determination requires solving the phase problem. This can be accomplished using ab initio direct methods for small molecules and macromolecules at resolutions higher than 1.2 Å, whereas macromolecular structure determination at lower resolution requires either molecular replacement using a homologous structure or experimental phases using a derivative such as covalent labeling (for example selenomethionine or mercury derivatization) or heavy-atom soaking (for example iodide ions). Here, a case is presented in which crystals were obtained from a 30.8 kDa protein sample and yielded a 1.6 Å resolution data set with a unit cell that could accommodate approximately 8 kDa of protein. Thus, it was unclear what had been crystallized. Molecular replacement with pieces of homologous proteins and attempts at iodide ion soaking failed to yield a solution. The crystals could not be reproduced. Sequence-independent molecular replacement using the structures available in the Protein Data Bank also failed to yield a solution. Ultimately, ab initio structure solution proved successful using the program ARCIMBOLDO, which identified two α-helical elements and yielded interpretable maps. The structure was the C-terminal dimerization domain of the intended target from Mycobacterium smegmatis. This structure is presented as a user-friendly test case in which an unknown protein fragment could be determined using ARCIMBOLDO.

066 Crystal structure of the Rhagium Inquisitor antifreeze protein

Some insects use antifreeze proteins to overwinter. The structure-function relationships and mechanism of action of these proteins are not well understood. We report the crystal structure of an antifreeze protein from the longhorn beetle Rhagium inquisitor , determined at 1.15-A resolution using ab initio direct methods. The structure is the largest determined ab initio without heavy atoms, and reveals a compressed β -solenoid fold in which the top and bottom sheets are held together by a silk-like interdigitation of short side chains. The crystal structure shows the presence of ice-like waters that match several planes of hexagonal ice. Source of funding: NIH grant P01 GM022778. Conflict of interest: None declared. wuyi.meng@yale.edu

Continuous β-turn fold of an alternating alanyl/homoalanyl peptide nucleic acid

The crystal structure of the PNA (peptide nucleic acid) oligomer H-Lys-HalG-AlaG-HalC-AlaG-HalC-AlaC-Lys-NH(2) (PNA1, amino acids with D-configuration are underlined, Ala = alanyl, Hal = homoalanyl) has been determined by ab initio direct methods and refined against 1.0 Å data. The asymmetric unit consists of a tetrameric cage with almost ideal Watson-Crick C-G base pairing of all the guanine and cytosine side-chain substituents. Each PNA strand has a 90° β-turn every second residue, stabilized by three hydrogen bonds between the backbone amides. The first, second, fifth and sixth bases stack on one side of the monomer and pair with the corresponding complementary bases of a second monomer to form a dimer. The two remaining bases on each side of the resulting dimer form Watson-Crick pairs with the complementary bases of a second dimer, leading to a unique cage structure. The extra methylene groups in the homoalanyl residues enable stacking of the bases with an optimal distance between base-planes but also with an appreciable lateral displacement (slide).

Structures of viscotoxins A1 and B2 from European mistletoe solved using native data alone

Crystals of the cytotoxic thionin proteins viscotoxins A1 and B2 extracted from mistletoe diffracted to high resolution (1.25 and 1.05 A, respectively) and are excellent candidates for testing crystallographic methods. Ab initio direct methods were only successful in solving the viscotoxin B2 structure, which with 861 unique non-H atoms is one of the largest unknown structures without an atom heavier than sulfur to be solved in this way, but sulfur-SAD phasing provided a convincing solution for viscotoxin A1. Both proteins form dimers in the crystal and viscotoxin B2 (net charge +4 per monomer), but not viscotoxin A1 (net charge +6), is coordinated by sulfate or phosphate anions. The viscotoxin A1 crystal has a higher solvent content than the viscotoxin B2 crystal (49% as opposed to 28%) with solvent channels along the crystallographic 4(3) axes.

Structure of the lipopeptide antibiotic tsushimycin

The amphomycin derivative tsushimycin has been crystallized and its structure determined at 1.0 A resolution. The asymmetric unit contains 12 molecules and with 1300 independent atoms this structure is one of the largest solved using ab initio direct methods. The antibiotic is comprised of a cyclodecapeptide core, an exocyclic amino acid and a fatty-acid residue. Its backbone adopts a saddle-like conformation that is stabilized by a Ca2+ ion bound within the peptide ring and accounts for the Ca2+-dependence of this antibiotic class. Additional Ca2+ ions link the antibiotic molecules to dimers that enclose an empty space resembling a binding cleft. The dimers possess a large hydrophobic surface capable of interacting with the bacterial cell membrane. The antibiotic daptomycin may exhibit a similar conformation, as the amino-acid sequence is conserved at positions involved in Ca2+ binding.

Use of an ion-binding site to bypass the 1000-atom limit to structure determination by direct methods.

Proteins with more than 1000 non-H atoms and without heavy-atom prosthetic groups are very difficult to solve by ab initio direct methods. T4 lysozyme is being used to explore these limits. The protein has 1309 non-H atoms, seven S atoms, no disulfide bonds and no heavy-atom prosthetic group. It is recalcitrant to structure determination by direct methods using X-ray diffraction data to 0.97 A. It is shown here that it is possible to obtain a truly ab initio structure determination of a variant of the protein that has an Rb+ (Z = 37) binding site. Using diffraction data to 1.06 A resolution, the direct-methods programs SIR2002 and ACORN independently solved the structure in about 20 h. The bound Rb+, which contributes about 1.7% of the total scattering, does not appear to distort the structure or to inhibit refinement (R factor 12.1%). The phases obtained via SIR2002 or ACORN are in good agreement with those from a reference structure obtained from conventional molecular-substitution and refinement procedures (average error in the figure-of-merit-weighted phases of less than 25 degrees). Thus, proteins with more than 1000 atoms that include halide-binding or other such sites may be amenable to structure determination by ab initio direct methods. The direct-methods approaches are also compared with structure determination via use of the anomalous scattering of the Rb+ ion. As shown by examples, high-resolution structures determined by direct methods can be useful in highlighting regions of strain in the protein, including short hydrogen bonds and non-planar peptide groups.

The Application of Ab Initio Direct Methods with Electron Diffraction for Solving Zeolite Structures

Abstract Recently a number of reports have been made in which direct methods (DM) were used to solve bulk inorganic crystal structures with electron diffraction (ED) data, either ab initio or in a combined approach using phase information from images. This paper reports ab initio application of DM to experimental ED data from the zeolite mordenite. Data from mordenite [001] were taken using a JEOL 3010 TEM at 300 kV. For collecting data, a small condenser and spot size were used to obtain a defocused probe 50 nm in size positioned over the edge of a thin area. Patterns were recorded with a CCD camera. Fig. 1 compares experiment with modeled kinematical data based on a published structure. Phase determination by DM was performed with the minimum relative entropy approach. As a test, ab initio DM was performed on modeled kinematical data.The result, shown in Fig. 2, is not distinguishable from a picture of the projected potential.

Direct-methods determination of an RNA/DNA hybrid decamer at 1.15 A resolution.

For the first time, ab initio direct methods have been used to solve the crystal structure of an RNA/DNA hybrid decamer. The RNA/DNA sequence corresponds to the leftmost two-thirds of the polypurine tract (PPT), the primer for second-strand DNA synthesis by HIV-1 reverse transcriptase (RT). Direct methods using Shake-and-Bake (SnB) yielded solutions for the RNA/DNA decamer molecule using 1.15 A data, which is just on the resolution edge of what might work with direct methods. Atomic positions for 96% of the entire molecule, containing 514 non-H atoms including three Ca(2+) ions, were easily interpreted from a Fourier map based on the 'Shake-and-Bake' minimal function and CROQUE phase-refinement program. Only six atoms, primarily in the sugar linkage, were missing in this Fourier map. At present, the R factor of the model is 0.143 (R(free) = 0.186) for the 562 non-H atom sites located. The conformation of the RNA/DNA helix is A-form, with a typical A-helix minor-groove width. This paper presents the methodology used in solving this structure.

Ab initio solution and refinement of two high-potential iron protein structures at atomic resolution.

The crystal structure of the reduced high-potential iron protein (HiPIP) from Chromatium vinosum has been redetermined in a new orthorhombic crystal modification, and the structure of its H42Q mutant has been determined in orthorhombic (H42Q-1) and cubic (H42Q-2) modifications. The first two were solved by ab initio direct methods using data collected to atomic resolution (1.20 and 0. 93 A, respectively). The recombinant wild type (rc-WT) with two HiPIP molecules in the asymmetric unit has 1264 protein atoms and 335 solvent sites, and is the second largest structure reported so far that has been solved by pure direct methods. The solutions were obtained in a fully automated way and included more than 80% of the protein atoms. Restrained anisotropic refinement for rc-WT and H42Q-1 converged to R(1) = summation operator||F(o)| - |F(c)|| / summation operator|F(o)| of 12.0 and 13.6%, respectively [data with I > 2sigma(I)], and 12.8 and 15.5% (all data). H42Q-2 contains two molecules in the asymmetric unit and diffracted only to 2.6 A. In both molecules of rc-WT and in the single unique molecule of H42Q-1 the [Fe(4)S(4)](2+) cluster dimensions are very similar and show a characteristic tetragonal distortion with four short Fe-S bonds along four approximately parallel cube edges, and eight long Fe-S bonds. The unique protein molecules in H42Q-2 and rc-WT are also very similar in other respects, except for the hydrogen bonding around the mutated residue that is at the surface of the protein, supporting the hypothesis that the difference in redox potentials at lower pH values is caused primarily by differences in the charge distribution near the surface of the protein rather than by structural differences in the cluster region.

Direct methods structure determination from synchrotron powder diffraction data of a new clathrasil, TMA Silicate

The structure of as-synthesized TMA Silicate, an aluminosilicate synthesized with the tetramethyl ammonium (TMA) structure directing agent, was solved using intensities extracted from high-resolution synchrotron powder diffraction data and ab initio direct methods. The trial topology was improved by DLS refinement, and the structure was confirmed by successful Rietveld refinement. The space group is C2/m, and lattice parameters are a = 13.35211(14) A ̊ ; b = 13.05531(12) A ̊ ; c = 12.53013(13) A ̊ ; β = 113.285(1) °. The framework topology of as-synthesized TMA Silicate consists of 4-connected T-atoms in 4-, 5-, 6- and 8-rings. Two types of cages are interconnected by pores no larger than 6-rings. The larger cage is a peanut-shaped 30-hedron [4 85 126 108] that is the fundamental polyhedral building unit. The three-dimensional structure results from sharing the 4-, 5- and 6-ring faces between adjoining large cages. The three-dimensional structure also contains a smaller 10-hedron cage [4 45 46 2]. Both cages have crystallographic 2/m site symmetry. Tetramethyl ammonium cations are located in each lobe of the peanut-shaped cages. Because the largest opening to any cage is through 6-ring pores, which are too small to let the TMA pass, as-synthesized TMA Silicate can be classified as a clathrasil.

95/05526 Fuels' social coast: Evidence from all electric-generating firms

The structure of as-synthesized TMA Silicate, an aluminosilicate synthesized with the tetramethyl ammonium (TMA) structure directing agent, was solved using intensities extracted from high-resolution synchrotron powder diffraction data and ab initio direct methods. The trial topology was improved by DLS refinement, and the structure was confirmed by successful Rietveld refinement. The space group is C2/m, and lattice parameters are a = 13.35211(14) Å; b = 13.05531(12) Å; c = 12.53013(13) Å; β = 113.285(1) °. The framework topology of as-synthesized TMA Silicate consists of 4-connected T-atoms in 4-, 5-, 6- and 8-rings. Two types of cages are interconnected by pores no larger than 6-rings. The larger cage is a peanut-shaped 30-hedron [485126108] that is the fundamental polyhedral building unit. The three-dimensional structure results from sharing the 4-, 5- and 6-ring faces between adjoining large cages. The three-dimensional structure also contains a smaller 10-hedron cage [445462]. Both cages have crystallographic 2/m site symmetry. Tetramethyl ammonium cations are located in each lobe of the peanut-shaped cages. Because the largest opening to any cage is through 6-ring pores, which are too small to let the TMA pass, as-synthesized TMA Silicate can be classified as a clathrasil.

95/05525 Fresh links in the fabrication chain

The structure of as-synthesized TMA Silicate, an aluminosilicate synthesized with the tetramethyl ammonium (TMA) structure directing agent, was solved using intensities extracted from high-resolution synchrotron powder diffraction data and ab initio direct methods. The trial topology was improved by DLS refinement, and the structure was confirmed by successful Rietveld refinement. The space group is C2/m, and lattice parameters are a = 13.35211(14) Å; b = 13.05531(12) Å; c = 12.53013(13) Å; β = 113.285(1) °. The framework topology of as-synthesized TMA Silicate consists of 4-connected T-atoms in 4-, 5-, 6- and 8-rings. Two types of cages are interconnected by pores no larger than 6-rings. The larger cage is a peanut-shaped 30-hedron [485126108] that is the fundamental polyhedral building unit. The three-dimensional structure results from sharing the 4-, 5- and 6-ring faces between adjoining large cages. The three-dimensional structure also contains a smaller 10-hedron cage [445462]. Both cages have crystallographic 2/m site symmetry. Tetramethyl ammonium cations are located in each lobe of the peanut-shaped cages. Because the largest opening to any cage is through 6-ring pores, which are too small to let the TMA pass, as-synthesized TMA Silicate can be classified as a clathrasil.

Ab initio direct methods: practical advice for getting beyond the first 300 atoms.

Not all crystallographic structural investigations are amenable to a phasing solution by direct methods alone. Guideline procedures are outlined which are intended to help the evaluation of whether direct-methods procedures may be expected to phase diffraction data for large molecular structures. This analysis is directed at three separate levels of inquiry: (1) How good are the primary data and can E values be derived to represent a point-atom structure. (2) How well do the data interact through phase relationships and may they be expected to produce a stable phasing solution. (3) What is the prognosis for finding recognizable solutions. Data are presented from the post-mortem analyses of a number of large, difficult-to-solve, structures to illustrate each of these points. Direct-methods practitioners are to be encouraged that crystal structures having more than 300 atoms per asymmetric unit may occasionally be determined utilizing present methodologies provided that an a priori prognosis for obtaining a solution is favorably high, adequate computational resources are available, and sufficient persistent effort is applied.