Ab Initio Phasing Research Articles

Cloning, expression, purification, crystallization and preliminary crystallographic analysis of MsDpo4: a Y-family DNA polymerase from Mycobacterium smegmatis.

The expression of error-prone DNA polymerases belonging to the Y-family is upregulated in prokaryotes under adverse conditions to facilitate adaptive mutagenesis. However, it has been suggested that representatives of this family in mycobacteria do not participate in adaptive mutagenesis. These studies raise the possibility that mycobacterial representatives might be devoid of biochemical activity. In order to determine whether this possible loss of activity is a consequence of significant changes in the structure of these enzymes, structural studies on a representative enzyme from Mycobacterium smegmatis, MsDpo4, were initiated. The protein crystallized in space group P6(1)22 or P6(5)22. The Matthews coefficient was 4.0 Å3 Da(-1) assuming the presence of one molecule in the asymmetric unit; the corresponding solvent content was 69%. X-ray diffraction data were collected to a minimum Bragg spacing of 2.6 Å and crystals of selenomethionine-labelled MsDpo4 have been prepared for ab initio phasing.

Analysis of phases in the structure determination of an icosahedral virus.

The constraints imposed on structure-factor phases by noncrystallographic symmetry (NCS) allow phase improvement, phase extension to higher resolution and hence ab initio phase determination. The more numerous the NCS redundancy and the greater the volume used for solvent flattening, the greater the power for phase determination. In a case analyzed here the icosahedral NCS phasing appeared to have broken down, although later successful phase extension was possible when the envelope around the NCS region was tightened. The phases from the failed phase-determination attempt fell into four classes, all of which satisfied the NCS constraints. These four classes corresponded to the correct solution, opposite enantiomorph, Babinet inversion and opposite enantiomorph with Babinet inversion. These incorrect solutions can be seeded from structure factors belonging to reciprocal-space volumes that lie close to icosahedral NCS axes where the structure amplitudes tend to be large and the phases tend to be 0 or π. Furthermore, the false solutions can spread more easily if there are large errors in defining the envelope designating the region in which NCS averaging is performed.

Solubility Products for Precipitate Phases in Steels from First-principles Calculations

ABSTRACTThe work presented gives an insight into using formation enthalpies determined from ab initio calculations for computing solubility products in steels. The role of enthalpy and entropy contributions to the solubility product is discussed. As an illustration of the method, we present solubility products for observed stoichiometric precipitate phases in ferrite from first-principles calculations and in austenite as obtained from the combined approach based on ab initio and experimental phase diagram analysis. The results are compared with experimental data where available.

PVMR: assembling small helix fragments as structural solutions for molecular replacement

A new real-space implementation of the molecular-replacement method is described. The method locates the search model in the target crystal by maximizing the matching between the search-model vectors and the Patterson self and cross vectors. In previous work, a new rotation function was introduced for the molecular-replacement method [Jiang (2008). Acta Cryst. D64, 561-566]. This rotation function is calculated by matching the search model directly with both the Patterson self and cross vectors in real space. All the matches are summed and averaged to enhance the overall signal-to-noise ratio for a given orientation of the search model. Recently, to avoid the dependence of the weights derived from the linear regression on the properties of the search model and the target crystal structure, such as secondary structures, space groups and cell parameters, a dynamic correlation coefficient has been designed and used as the total rotation function score [Jiang & Ding (2010). Chin. Phys. B, 19, 106101]. This work further extends this idea to the implementation of translation search. A new real- or direct-space translation function has been implemented by matching the cross vectors between the symmetry mates of the search model to the Patterson cross vectors. This method enables effective searching for small helix fragments in the target crystal. Although the solution model assembled by using multiple fragments of helix is insufficient to start ab initio phasing of the target crystal, it can be used to identify the known protein folds in the Protein Data Bank that are homologous to the target structure. It can also be combined with other experimental and theoretical models to screen and select for better search models for molecular replacement.

Ab initio phase diagram of ultracold 87Rb in a one-dimensional two-colour superlattice

We investigate the ab initio phase diagram of ultracold 87Rb atoms in a one-dimensional two-colour superlattice. Using single-particle band structure calculations we map the experimental setup onto the parameters of the Bose–Hubbard model. This ab initio ansatz allows us to express the phase diagrams in terms of the experimental control parameters, i.e. the intensities of the lasers that form the optical superlattice. In order to solve the many-body problem for experimental system sizes we adopt the density-matrix renormalization-group algorithm. A detailed study of convergence and finite-size effects for all observables is presented. Our results show that all relevant quantum phases, i.e. superfluid, Mott insulator and quasi Bose glass, can be accessed through intensity variation of the lasers alone. However, it turns out that the phase diagram is strongly affected by the longitudinal trapping potential.

ATAT@WIEN2k: An interface for cluster expansion based on the linearized augmented planewave method

We have developed an interface between the all-electron density functional theory code WIEN2k, and the MIT Ab-initio Phase Stability (MAPS) code of the Alloy-Theoretic Automated Toolkit (ATAT). WIEN2k is an implementation of the full-potential linearized augmented planewave method which yields highly accurate total energies and optimized geometries for any given structure. The ATAT package consists of two parts. The first one is the MAPS code, which constructs a cluster expansion (CE) in conjunction with a first-principles code. These results form the basis for the second part, which computes the thermodynamic properties of the alloy. The main task of the CE is to calculate the many-body potentials or effective cluster interactions (ECIs) from the first-principles total energies of different structures or supercells using the structure-inversion technique. By linking MAPS seamlessly with WIEN2k we have created a tool to obtain the ECIs for any lattice type of an alloy. We have chosen fcc Al–Ti and bcc W–Re to evaluate our implementation. Our calculated ECIs exhibit all features of a converged CE and compare well with literature results.

First principles calculations of theσ andχ phases in the Mo–Re and W–Re systems

The total energies of all the ordered configurations of theσ andχ phases have been calculated by using first principles methodology in both Mo–Re andW–Re systems. These two complex structures possess 5 and 4 inequivalent sitesgenerating 32 and 16 different ordered configurations, respectively, for a binaryA–B system. The converged total energies of all the fully relaxed structures have been used tocompute the occupancy of the inequivalent sites as a function of compositionand temperature by using the Bragg–Williams approximation in the completecomposition range. It is shown that the configurational entropy stabilizes theσ andχ phases in the Mo–Re and W–Re systems. The results evidence the preference of Re forlower coordination number site occupancy and are in very good agreement with theexperimental measurements. Tentative ab initio phase diagrams have also been drawn.

The kinematic convergent-beam electron diffraction method for nanocrystal structure determination

Kinematic convergent-beam electron diffraction (KCBED) is a technique capable of probing and solving the atomic structure of isolated nanocrystals as small as a few nanometers in diameter. The method was evaluated by solving the structure of a spinel crystal from three-dimensional diffraction data. The charge-flipping algorithm was used for ab initio phasing and SHELX was used for structure refinement. We discuss the relative merits of the KCBED and precession methods for nanocrystallography.

Crystallographic ab initio protein structure solution below atomic resolution

Ab initio macromolecular phasing has been so far limited to small proteins diffracting at atomic resolution (beyond 1.2 A) unless heavy atoms are present. We describe a general ab initio phasing method for 2 A data, based on combination of localizing model fragments such as small á-helices with Phaser and density modification with SHELXE. We implemented this approach in the program Arcimboldo to solve a 222-amino-acid structure at 1.95 A.

EDM–DEDM and protein crystal structure solution

Electron-density modification (EDM) procedures are the classical tool for driving model phases closer to those of the target structure. They are often combined with automated model-building programs to provide a correct protein model. The task is not always performed, mostly because of the large initial phase error. A recently proposed procedure combined EDM with DEDM (difference electron-density modification); the method was applied to the refinement of phases obtained by molecular replacement, ab initio or SAD phasing [Caliandro, Carrozzini, Cascarano, Giacovazzo, Mazzone & Siliqi (2009), Acta Cryst. D65, 249-256] and was more effective in improving phases than EDM alone. In this paper, a novel fully automated protocol for protein structure refinement based on the iterative application of automated model-building programs combined with the additional power derived from the EDM-DEDM algorithm is presented. The cyclic procedure was successfully tested on challenging cases for which all other approaches had failed.

Structure of a fatty acid-binding protein fromBacillus subtilisdetermined by sulfur-SAD phasing using in-house chromium radiation

Sulfur single-wavelength anomalous dispersion (S-SAD) and halide-soaking methods are increasingly being used for ab initio phasing. With the introduction of in-house Cr X-ray sources, these methods benefit from the enhanced anomalous scattering of S and halide atoms, respectively. Here, these methods were combined to determine the crystal structure of BsDegV, a DegV protein-family member from Bacillus subtilis. The protein was cocrystallized with bromide and low-redundancy data were collected to 2.5 A resolution using Cr Kalpha radiation. 17 heavy-atom sites (ten sulfurs and seven bromides) were located using standard methods. The anomalous scattering of some of the BsDegV S atoms and Br atoms was weak, thus neither sulfurs nor bromides could be used alone for structure determination using the collected data. When all 17 heavy-atom sites were used for SAD phasing, an easily interpretable electron-density map was obtained after density modification. The model of BsDegV was built automatically and a palmitate was found tightly bound in the active site. Sequence alignment and comparisons with other known DegV structures provided further insight into the specificity of fatty-acid selection and recognition within this protein family.

Ultralow-resolutionab initiophasing of filamentous proteins: crystals from a six-Ig fragment of titin as a case study

Low-resolution diffraction data (resolution below 12 angstroms) from crystals of a filamentous six-Ig fragment of titin, I65-I70, were used in ab initio phasing with the aim of calculating its lattice packing and molecular envelope. Filamentous molecules, characterized by marked anisometry and idiosyncratic crystal lattices, have not been addressed before using this methodology. In this study, low-resolution phasing (19-122 angstroms) successfully identified the region of the unit cell occupied by the molecule. Phase extension to a higher resolution (12 angstroms) yielded regions of high density that corresponded either to the positions of individual Ig domains or to zones of dense intermolecular contacts, hindering the identification of individual domains and the interpretation of electron-density maps in terms of a molecular model. This problem resulted from the acutely uneven packing of the molecules in the crystal and it was further accentuated by the presence of partially disordered regions in the molecule. Addition of low-resolution reflections with phases computed ab initio to those obtained experimentally using MIRAS improved the initial electron-density maps of the atomic model, demonstrating the generic utility of low-resolution phases for the structure-elucidation process, even when individual molecules cannot be resolved in the lattice.

IL MILIONE: a suite of computer programs for crystal structure solution of proteins

IL MILIONEis a suite of computer programs devoted to protein crystal structure determination by X-ray crystallography. It may be used in the following key activities. (a)Ab initiophasing,viaPatterson or direct methods. The program may succeed even with structures with up to 6000 non-H atoms in the asymmetric unit, provided that atomic resolution is available, and with data at quasi-atomic resolution (1.4–1.5 Å). (b) Single or multiple isomorphous replacement, single- or multiple-wavelength anomalous diffraction, and single or multiple isomorphous replacement with anomalous scattering techniques. In the first step the program finds the heavy-atom/anomalous scatterer substructure, then automatically uses the above information to phase protein reflections. Phase extension and refinement are performed by electron density modification techniques. (c) Molecular replacement. The orientation and the location of the protein molecules are foundviareciprocal space methods. Phase extension and refinement are performed by electron density modification techniques. All the programs integrated intoIL MILIONEare controlled by means of a user-friendly graphical user interface, which is used to input data and to monitor intermediate and final results by means of real-time updated messages, diagrams and histograms.

Characterization of the Cytochrome c Oxidase Assembly Factor Cox19 of Saccharomyces cerevisiae

Cox19 is an important accessory protein in the assembly of cytochrome c oxidase in yeast. The protein is functional when tethered to the mitochondrial inner membrane, suggesting its functional role within the intermembrane space. Cox19 resembles Cox17 in having a twin CX(9)C sequence motif that adopts a helical hairpin in Cox17. The function of Cox17 appears to be a Cu(I) donor protein in the assembly of the copper centers in cytochrome c oxidase. Cox19 also resembles Cox17 in its ability to coordinate Cu(I). Recombinant Cox19 binds 1 mol eq of Cu(I) per monomer and exists as a dimeric protein. Cox19 isolated from the mitochondrial intermembrane space contains variable quantities of copper, suggesting that Cu(I) binding may be a transient property. Cysteinyl residues important for Cu(I) binding are also shown to be important for the in vivo function of Cox19. Thus, a correlation exists in the ability to bind Cu(I) and in vivo function.

Crystallographic study and comparisons of compoundsA2Sr2B2C2Ox

It is sometimes difficult and/or time-consuming to obtain crystals of suitable size for single crystal X-ray diffraction measurements. However if a microcrystalline material can be obtained, it is still possible to perform powder diffraction experiments. Although the collapse of the three-dimensional reciprocal space into a one-dimensional powder diffraction pattern gives rise to a severe loss of information, the availability of high-resolution powder diffractometers and synchrotron sources enables the recording of high-quality powder patterns from proteins. From these spectra, a set of valid intensities can be extracted and/or Rietveld refinement can be carried out [1-2]. It is thus possible to investigate the binding of ligands or heavy atoms to biological macromolecules [3]. We present here results obtained recently in this field. Emphasis will be put on low-resolution phase determination by the methods of isomorphous replacement and contrast variation. By using extracted intensities from a series of powder diffraction patterns, the location of heavy-atom is possible and a low resolution structure envelope could even been obtained for some small proteins. We present here data for PPE (porcine pancreatic elastase) in which the radiation-induced anisotropic lattice expansion was used as a way to uncorrelate the intensities of overlapping peaks. On the other hand, contrast variation by solvent exchange is commonly used in small-angle scattering studies, both with X-rays and neutrons, and allows ab initio phasing andmolecular envelope determination [2]. We present here our first results on using the solvent contrast variation method in powder diffraction studies.

Ab initio phasing of X-ray powder diffraction patterns by charge flipping

Determining crystal structures from powder X-ray diffraction data remains a challenging problem in materials science. By embedding a Le-Bail-like procedure within the recently discovered charge-flipping phasing algorithm, an extremely simple, fast and effective ab initio method has been developed to determine phases directly from indexed powder diffraction patterns. The algorithm solves the degeneracy problem by applying spherical averaging for overlapping Bragg reflections, while solving the phase problem by using the Oszlányi-Süto charge-flipping algorithm. The processes of peak decomposition and phasing are integrated within the same iteration, and a dynamic support is used. The Fienup hybrid input-output algorithm is also incorporated to minimize stagnation. The ability of the algorithm to find structure-factor phases rapidly is found to assist with the fundamental problem of degeneracy (overlapping reflections) which is intrinsic to powder diffraction data. Space-group and chemical-composition information are not needed as inputs, and can be determined from the result. The method is illustrated using several experimental powder patterns of indifferent quality.

Low-resolutionab initiophasing ofSarcocystis murislectin SML-2

Structural analysis of the lectin SML-2 faced difficulties when applying standard crystallographic phasing methods. The connectivity-based ab initio phasing method allowed the computation of a 16 A resolution Fourier synthesis and the derivation of primary structural information. It was found that SML-2 crystals have three dimers in the asymmetric part of the unit cell linked by a noncrystallographic symmetry close to translation by (0, 0, 1/3). A clear identification of the noncrystallographic twofold axis explains the space-group transformation from the primitive P2(1)2(1)2(1) to the C-centred C222(1) observed during annealing procedures within an N(2) cryostream for cocrystals of SML-2 and galactose. Related packing considerations predict a possible arrangement of SML-2 molecules in a tetragonal unit cell. Multiple noncrystallographic symmetries and crystal forms provide a basis for further image improvements.

Diffractive electron imaging of nanoparticles on a substrate

The observation of the detailed atomic arrangement within nanostructures has previously required the use of an electron microscope for imaging. The development of diffractive (lensless) imaging in X-ray science and electron microscopy using ab initio phase retrieval provides a promising tool for nanostructural characterization. We show that it is possible experimentally to reconstruct the atomic-resolution complex image (exit-face wavefunction) of a small particle lying on a thin carbon substrate from its electron microdiffraction pattern alone. We use a modified iterative charge-flipping algorithm and an estimate of the complex substrate image is subtracted at each iteration. The diffraction pattern is recorded using a parallel beam with a diameter of approximately 50 nm, illuminating a gold nanoparticle of approximately 13.6 nm diameter. Prior knowledge of the boundary of the object is not required. The method has the advantage that the reconstructed exit-face wavefunction is free of the aberrations of the objective lens normally used in the microscope, whereas resolution is limited only by thermal vibration and noise.

Preparation, crystallization and X-ray diffraction analysis to 1.5 Å resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation

Cysteine dioxygenase (CDO; EC 1.13.11.20) is an approximately 23 kDa non-heme iron metalloenzyme that is responsible for the oxidation of cysteine by O2, yielding cysteinesulfinate. CDO catalyzes the first step in the conversion of cysteine to taurine, as well as the first step in the catabolism of cysteine to pyruvate plus sulfate. Recombinant rat CDO was heterologously expressed, purified and crystallized. The protein was expressed as a fusion protein bearing a polyhistidine tag to facilitate purification, a thioredoxin tag to improve solubility and a factor Xa cleavage site to permit removal of the entire N-terminus, leaving only the 200 amino acids inherent to the native protein. A multi-step purification scheme was used to achieve >95% purity of CDO. The optimal CDO crystals diffracted to 1.5 A resolution and belonged to space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 57.55, c = 123.06 A, alpha = beta = gamma = 90 degrees. CDO shows little homology to any other proteins; therefore, the structure of the enzyme will be determined by ab initio phasing using a selenomethionyl derivative.

Overcoming the false-minima problem in direct methods: structure determination of the packaging enzyme P4 from bacteriophage phi13.

The problems encountered during the phasing and structure determination of the packaging enzyme P4 from bacteriophage phi13 using the anomalous signal from selenium in a single-wavelength anomalous dispersion experiment (SAD) are described. The oligomeric state of P4 in the virus is a hexamer (with sixfold rotational symmetry) and it crystallizes in space group C2, with four hexamers in the crystallographic asymmetric unit. Current state-of-the-art ab initio phasing software yielded solutions consisting of 96 atoms arranged as sixfold symmetric clusters of Se atoms. However, although these solutions showed high correlation coefficients indicative that the substructure had been solved, the resulting phases produced uninterpretable electron-density maps. Only after further analysis were correct solutions found (also of 96 atoms), leading to the eventual identification of the positions of 120 Se atoms. Here, it is demonstrated how the difficulties in finding a correct phase solution arise from an intricate false-minima problem.