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.

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.

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 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.

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 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.

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].

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.

The structure of the oligomerization domain of Lsr2 from Mycobacterium tuberculosis reveals a mechanism for chromosome organization and protection.

Lsr2 is a small DNA-binding protein present in mycobacteria and related actinobacteria that regulates gene expression and influences the organization of bacterial chromatin. Lsr2 is a dimer that binds to AT-rich regions of chromosomal DNA and physically protects DNA from damage by reactive oxygen intermediates (ROI). A recent structure of the C-terminal DNA-binding domain of Lsr2 provides a rationale for its interaction with the minor groove of DNA, its preference for AT-rich tracts, and its similarity to other bacterial nucleoid-associated DNA-binding domains. In contrast, the details of Lsr2 dimerization (and oligomerization) via its N-terminal domain, and the mechanism of Lsr2-mediated chromosomal cross-linking and protection is unknown. We have solved the structure of the N-terminal domain of Lsr2 (N-Lsr2) at 1.73 Å resolution using crystallographic ab initio approaches. The structure shows an intimate dimer of two ß-ß-a motifs with no close homologues in the structural databases. The organization of individual N-Lsr2 dimers in the crystal also reveals a mechanism for oligomerization. Proteolytic removal of three N-terminal residues from Lsr2 results in the formation of an anti-parallel ß-sheet between neighboring molecules and the formation of linear chains of N-Lsr2. Oligomerization can be artificially induced using low concentrations of trypsin and the arrangement of N-Lsr2 into long chains is observed in both monoclinic and hexagonal crystallographic space groups. In solution, oligomerization of N-Lsr2 is also observed following treatment with trypsin. A change in chromosomal topology after the addition of trypsin to full-length Lsr2-DNA complexes and protection of DNA towards DNAse digestion can be observed using electron microscopy and electrophoresis. These results suggest a mechanism for oligomerization of Lsr2 via protease-activation leading to chromosome compaction and protection, and concomitant down-regulation of large numbers of genes. This mechanism is likely to be relevant under conditions of stress where cellular proteases are known to be upregulated.

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.

Is there a fundamental upper limit for the significance I/σ(I) of observations from X-ray and neutron diffraction experiments?

The answer is yes. A fundamental limit exists, which is not strongly applicable to individual reflections but to a sufficiently large set of reflections such as any set for structure determination. The limit originates from Poisson statistics which gives a minimum (average) error. The proposed limit of significance and a proposed decrease in significance due to data processing are also tested by monitoring W = / for raw and for Bragg data. Since Poisson statistics are the lower limit for the experimental standard uncertainties, it is expected that W < 1 for raw and Bragg data, and that W decreases upon data processing. W also gives a measure of systematic errors in the experimental data as W ≈ 1 characterizes pure Poisson data, W >> 1 is physically impossible for sufficiently large data sets of unmerged reflections and W < 1 describes the contamination with systematic errors. Systematic differences depending on the software used to process the data were found. Also, the frequency distributions in particular of σ(I) values change considerably depending on the data-processing software used. We have no explanation for these differences in the distributions of σ(I), which lead to distinct changes in the frequency distribution of the significances I/σ(I) compared with the raw data. Another consequence of Poisson statistics is that lower limits also exist for the agreement factors, the internal agreement factor and the goodness of fit. These limits depend on the moments <1/I(o)>, , and of the observed set of intensities I(o) about the origin. These agreement factors are theoretically attainable when no systematic sources of error apply. They may be used in future to construct further measures of systematic error in experimental data.

Synthesis, structure, and theoretical investigation of amidinato supported 1,4-disilabenzene.

Reaction of LSi-SiL (1) (L = PhC(NtBu)(2)) with diphenyl alkyne gave the first example of a room temperature stable 1,4-disilabenzene. The compound was characterized by means of single crystal X-ray diffraction studies. NICS (nucleus independent chemical shift) calculations showed that the compound has some aromatic character.

The first asymmetric organolithium tetramers with simple ether donor bases.

The donor-free trimethylsilylmethyllithium [LiCH(2)SiMe(3)](6) hexameric aggregate is for the first time broken up by simple ether donors such as diethyl ether (Et(2)O) and tert-butylmethyl ether ((t)BuOMe) to give the unprecedented asymmetric tetramers while the chelating dimethoxyethane (DME) gives the expected dimer.

On the effect of neglecting anharmonic nuclear motion in charge density studies.

The effect of neglecting anharmonic nuclear motion when it is definitely present is studied. To ensure the presence of anharmonic nuclear motion a model was used that was previously refined against experimental data including anharmonic nuclear motion, and these calculated structure factors were used as observed data for a multipole refinement. It was then studied how the neglect of anharmonic nuclear motion and noise in the data affects the usual crystallographic quality measure R, the density parameters and the residual density distribution. It is demonstrated that the neglect of anharmonic nuclear motion leads to a characteristic imprint onto the residual density distribution in terms of residual density peaks and holes, in terms of the whole residual density distribution and in terms of the number, location and strength of valence shell charge concentrations (VSCCs). These VSCCs differ from that of the input model in a way which heavily influences and misleads the chemical interpretation of the charge density. This imprint vanishes after taking anharmonic nuclear motion into account. Also the input model VSCCs are restored. The importance of modeling anharmonic nuclear motion is furthermore shown by the characteristic imprint on the residual density distribution, even in the case of a numerically almost unaffected R value.