Exploiting tertiary structure through local folds for crystallographic phasing.

We describe an algorithm for phasing protein crystal X-ray diffraction data that identifies, retrieves, refines and exploits general tertiary structural information from small fragments available in the Protein Data Bank. The algorithm successfully phased, through unspecific molecular replacement combined with density modification, all-helical, mixed alpha-beta, and all-beta protein structures. The method is available as a software implementation: Borges.

Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries.

Protein-DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein-DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite the fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein-DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein-DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.

Fundamental relation between molecular geometry and real-space topology. Combined AIM, ELI-D, and ASF analysis of hapticities and intramolecular hydrogen-hydrogen bonds in zincocene-related compounds.

Despite numerous advanced and widely distributed bonding theories such as MO, VB, NBO, AIM, and ELF/ELI-D, complex modes of bonding such as M-Cp*((R)) interactions (hapticities) in asymmetrical metallocenes or weak intramolecular interactions (e.g., hydrogen-hydrogen (H···H) bonds) still remain a challenge for these theories in terms of defining whether or not an atom-atom interaction line (a "chemical bond") should be drawn. In this work the intramolecular Zn-C(Cp*(R)) (R = Me, -(CH2)2NMe2, and -(CH2)3NMe2) and H···H connectivity of a systematic set of 12 zincocene-related compounds is analyzed in terms of AIM and ELI-D topology combined with the recently introduced aspherical stockholder fragment (ASF) surfaces. This computational analysis unravels a distinct dependency of the AIM and ELI-D topology against the molecular geometry for both types of interactions, which confirms and extends earlier findings on smaller sets of compounds. According to these results the complete real-space topology including strong, medium, and weak interactions of very large compounds such as proteins may be reliably predicted by sole inspection of accurately determined molecular geometries, which would on the one hand afford new applications (e.g., accurate estimation of numbers, types, and strengths of intra- and intermolecular interactions) and on the other hand have deep implications on the significance of the method.

Practical structure solution with ARCIMBOLDO.

Since its release in September 2009, the structure-solution program ARCIMBOLDO, based on the combination of locating small model fragments such as polyalanine α-helices with density modification with the program SHELXE in a multisolution frame, has evolved to incorporate other sources of stereochemical or experimental information. Fragments that are more sophisticated than the ubiquitous main-chain α-helix can be proposed by modelling side chains onto the main chain or extracted from low-homology models, as locally their structure may be similar enough to the unknown one even if the conventional molecular-replacement approach has been unsuccessful. In such cases, the program may test a set of alternative models in parallel against a specified figure of merit and proceed with the selected one(s). Experimental information can be incorporated in three ways: searching within ARCIMBOLDO for an anomalous fragment against anomalous differences or MAD data or finding model fragments when an anomalous substructure has been determined with another program such as SHELXD or is subsequently located in the anomalous Fourier map calculated from the partial fragment phases. Both sources of information may be combined in the expansion process. In all these cases the key is to control the workflow to maximize the chances of success whilst avoiding the creation of an intractable number of parallel processes. A GUI has been implemented to aid the setup of suitable strategies within the various typical scenarios. In the present work, the practical application of ARCIMBOLDO within each of these scenarios is described through the distributed test cases.

High-resolution crystal structure of MltE, an outer membrane-anchored endolytic peptidoglycan lytic transglycosylase from Escherichia coli.

The crystal structure of the first endolytic peptidoglycan lytic transglycosylase MltE from Escherichia coli is reported here. The degradative activity of this enzyme initiates the process of cell wall recycling, which is an integral event in the existence of bacteria. The structure sheds light on how MltE recognizes its substrate, the cell wall peptidoglycan. It also explains the ability of this endolytic enzyme to cleave in the middle of the peptidoglycan chains. Furthermore, the structure reveals how the enzyme is sequestered on the inner leaflet of the outer membrane.

Lewis-base-stabilized dichlorosilylene: a two-electron sigma-donor ligand.

The first structurally described cobalt(I) Lewis-base-stabilized silylene complex [Co(CO)(3){SiCl(2)(IPr)}(2)](+)[CoCl(3)(THF)](-) [1; IPr = 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene] was prepared by applying the two-electron sigma-donor ligand SiCl(2)(IPr) through coordination with Co(2)(CO)(8). The bonding situation between ligand SiCl(2)(IPr) and the cobalt(I) metal center in [Co(CO)(3){SiCl(2)(IPr)}(2)](+) of 1 was investigated by (1)H NMR and IR spectroscopy, single-crystal X-ray structural analysis, and density functional theoretical calculations.

SEQUENCE SLIDER: expanding polyalanine fragments for phasing with multiple side-chain hypotheses.

Fragment-based molecular-replacement methods can solve a macromolecular structure quasi-ab initio. ARCIMBOLDO, using a common secondary-structure or tertiary-structure template or a library of folds, locates these with Phaser and reveals the rest of the structure by density modification and autotracing in SHELXE. The latter stage is challenging when dealing with diffraction data at lower resolution, low solvent content, high ß-sheet composition or situations in which the initial fragments represent a low fraction of the total scattering or where their accuracy is low. SEQUENCE SLIDER aims to overcome these complications by extending the initial polyalanine fragment with side chains in a multisolution framework. Its use is illustrated on test cases and previously unknown structures. The selection and order of fragments to be extended follows the decrease in log-likelihood gain (LLG) calculated with Phaser upon the omission of each single fragment. When the starting substructure is derived from a remote homolog, sequence assignment to fragments is restricted by the original alignment. Otherwise, the secondary-structure prediction is matched to that found in fragments and traces. Sequence hypotheses are trialled in a brute-force approach through side-chain building and refinement. Scoring the refined models through their LLG in Phaser may allow discrimination of the correct sequence or filter the best partial structures for further density modification and autotracing. The default limits for the number of models to pursue are hardware dependent. In its most economic implementation, suitable for a single laptop, the main-chain trace is extended as polyserine rather than trialling models with different sequence assignments, which requires a grid or multicore machine. SEQUENCE SLIDER has been instrumental in solving two novel structures: that of MltC from 2.7 Šresolution data and that of a pneumococcal lipoprotein with 638 residues and 35% solvent content.

Experimental charge density studies of disordered N-phenylpyrrole and N-(4-fluorophenyl)pyrrole.

The static electron densities of the title compounds were extracted from high-resolution X-ray diffraction data using the nucleus-centered finite multipole expansion technique. The interpretation of the data collected for the N-phenylpyrrole crystal revealed a static disorder that could be successfully resolved within the aspherical-atom formalism. The local and integrated topological properties of the density obtained via a constrained multipole refinement are in statistical agreement with those calculated at the B3LYP/cc-pVTZ level of theory for the isolated molecule and for those derived from the experimental density of the para-fluorinated derivative N-(4-fluorophenyl)pyrrole. The topological analysis of the densities indicates neither pyramidal character of the pyrrole N-atom nor a quinoidal structure of the phenyl rings in either molecule. The fluorine substitution appears to have only a minor effect on the density of the remaining constituents but it results in markedly different features of the electrostatic potential of the two compounds. The consistency of the multipole refinement is validated by residual density analysis.

6-Benzyl-3,4-dimeth-oxy-10-methyl-pyrido[2',1':2,3]imidazo[4,5-c]isoquinolin-5(6H)-one.

Pyrido[2',1':2,3]imidazo[4,5-c]isoquinolin-5(6H)-ones such as the title compound, C(24)H(21)N(3)O(3), can be obtained in a few minutes in a microwave-assisted three-component reaction from 2-amino-pyridines, isocyanides and 2-carboxy-benz-aldehydes. In the title compound, the pyrido[2',1':2,3]imidazo[4,5-c]isoquinolin-5(6H)-one ring system is almost planar (mean deviation 0.068 Å). The dihedral angle between the benzyl ring and the pyrido[2',1':2,3]imidazo[4,5-c]isoquinolin-5(6H)-one ring system is 78.2°. The crystal structure is stabilized by inter-molecular C-H⋯O and C-H⋯N hydrogen bonds.

Foundations of residual-density analysis.

New and concise descriptors of the residual density are presented, namely the gross residual electrons, the net residual electrons and the fractal dimension distribution. These descriptors indicate how much residual density is present and in what way it is distributed, i.e. the extent to which the distribution is featureless. The amount of residual density present accounts for noise in the experimental data as well as for modeling inadequacies. Therefore, the minimization of the gross residual electrons during refinement serves as a quality criterion. In the case where only Gaussian noise is present in the residual density, the fractal distribution is parabolic in shape. Deviations from this shape therefore serve as an indicator for systematic errors. The new measures have been applied to simulated and experimental data in order to study the effects of noise, model inadequacies and truncation in the experimental resolution. These measures, although designed and examined with particular regard to applications of space residual density, are very general and can in principle also be applied to space and momentum residual densities in a one-, two-, three- or higher-dimensional Euclidean space.

Conformations and tautomers of 5a,6-anhydrotetracycline.

Ab initio and density functional theory (DFT) calculations have been used to investigate the conformations and tautomeric forms of neutral anhydrotetracycline in aqueous solution.

Statistical tests against systematic errors in data sets based on the equality of residual means and variances from control samples: theory and applications.

Statistical tests are applied for the detection of systematic errors in data sets from least-squares refinements or other residual-based reconstruction processes. Samples of the residuals of the data are tested against the hypothesis that they belong to the same distribution. For this it is necessary that they show the same mean values and variances within the limits given by statistical fluctuations. When the samples differ significantly from each other, they are not from the same distribution within the limits set by the significance level. Therefore they cannot originate from a single Gaussian function in this case. It is shown that a significance cutoff results in exactly this case. Significance cutoffs are still frequently used in charge-density studies. The tests are applied to artificial data with and without systematic errors and to experimental data from the literature.

About systematic errors in charge-density studies.

The formerly introduced theoretical R values [Henn & Schönleber (2013). Acta Cryst. A69, 549-558] are used to develop a relative indicator of systematic errors in model refinements, R(meta), and applied to published charge-density data. The counter of R(meta) gives an absolute measure of systematic errors in percentage points. The residuals (Io - Ic)/σ(Io) of published data are examined. It is found that most published models correspond to residual distributions that are not consistent with the assumption of a Gaussian distribution. The consistency with a Gaussian distribution, however, is important, as the model parameter estimates and their standard uncertainties from a least-squares procedure are valid only under this assumption. The effect of correlations introduced by the structure model is briefly discussed with the help of artificial data and discarded as a source of serious correlations in the examined example. Intensity and significance cutoffs applied in the refinement procedure are found to be mechanisms preventing residual distributions from becoming Gaussian. Model refinements against artificial data yield zero or close-to-zero values for R(meta) when the data are not truncated and small negative values in the case of application of a moderate cutoff Io > 0. It is well known from the literature that the application of cutoff values leads to model bias [Hirshfeld & Rabinovich (1973). Acta Cryst. A29, 510-513].

More about systematic errors in charge-density studies.

In order to detect and graphically visualize the absence or presence of systematic errors in fit data, conditional probabilities are employed to analyze the statistical independence or dependence of fit residuals. This concept is completely general and applicable to all scientific fields in which model parameters are fitted to experimental data. The applications presented in this work refer to published charge-density data.

Structure solution with ARCIMBOLDO using fragments derived from distant homology models.

Molecular replacement, one of the general methods used to solve the crystallographic phase problem, relies on the availability of suitable models for placement in the unit cell of the unknown structure in order to provide initial phases. ARCIMBOLDO, originally conceived for ab initio phasing, operates at the limit of this approach, using small, very accurate fragments such as polyalanine α-helices. A distant homolog may contain accurate building blocks, but it may not be evident which sub-structure is the most suitable purely from the degree of conservation. Trying out all alternative possibilities in a systematic way is computationally expensive, even if effective. In the present study, the solution of the previously unknown structure of MltE, an outer membrane-anchored endolytic peptidoglycan lytic transglycosylase from Escherichia coli, is described. The asymmetric unit contains a dimer of this 194 amino acid protein. The closest available homolog was the catalytic domain of Slt70 (PDB code 1QTE). Originally, this template was used omitting contiguous spans of aminoacids and setting as many ARCIMBOLDO runs as models, each aiming to locate two copies sequentially with PHASER. Fragment trimming against the correlation coefficient prior to expansion through density modification and autotracing in SHELXE was essential. Analysis of the figures of merit led to the strategy to optimize the search model against the experimental data now implemented within ARCIMBOLDO-SHREDDER (http://chango.ibmb.csic.es/SHREDDER). In this strategy, the initial template is systematically shredded, and fragments are scored against each unique solution of the rotation function. Results are combined into a score per residue and the template is trimmed accordingly.

Structure and cell wall cleavage by modular lytic transglycosylase MltC of Escherichia coli.

The lytic transglycosylases are essential bacterial enzymes that catalyze the nonhydrolytic cleavage of the glycan strands of the bacterial cell wall. We describe here the structural and catalytic properties of MltC, one of the seven lytic transglycosylases found in the genome of the Gram-negative bacterium Escherichia coli. The 2.3 Å resolution X-ray structure of a soluble construct of MltC shows a unique, compared to known lytic transglycosylase structures, two-domain structure characterized by an expansive active site of 53 Å length extending through an interface between the domains. The structures of three complexes of MltC with cell wall analogues suggest the positioning of the peptidoglycan in the active site both as a substrate and as a product. One complex is suggested to correspond to an intermediate in the course of sequential and exolytic cleavage of the peptidoglycan. Moreover, MltC partitioned its reactive oxocarbenium-like intermediate between trapping by the C6-hydroxyl of the muramyl moiety (lytic transglycosylase activity, the major path) and by water (muramidase activity). Genomic analysis identifies the presence of an MltC homologue in no less than 791 bacterial genomes. While the role of MltC in cell wall assembly and maturation remains uncertain, we propose a functional role for this enzyme as befits the uniqueness of its two-domain structure.