Modes and model building in SHELXE.

Density modification is a standard step to provide a route for routine structure solution by any experimental phasing method, with single-wavelength or multi-wavelength anomalous diffraction being the most popular methods, as well as to extend fragments or incomplete models into a full solution. The effect of density modification on the starting maps from either source is illustrated in the case of SHELXE. The different modes in which the program can run are reviewed; these include less well known uses such as reading external phase values and weights or phase distributions encoded in Hendrickson-Lattman coefficients. Typically in SHELXE, initial phases are calculated from experimental data, from a partial model or map, or from a combination of both sources. The initial phase set is improved and extended by density modification and, if the resolution of the data and the type of structure permits, polyalanine tracing. As a feature to systematically eliminate model bias from phases derived from predicted models, the trace can be set to exclude the area occupied by the starting model. The trace now includes an extension into the gamma position or hydrophobic and aromatic side chains if a sequence is provided, which is performed in every tracing cycle. Once a correlation coefficient of over 30% between the structure factors calculated from such a trace and the native data indicates that the structure has been solved, the sequence is docked in all model-building cycles and side chains are fitted if the map supports it. The extensions to the tracing algorithm brought in to provide a complete model are discussed. The improvement in phasing performance is assessed using a set of tests.

Cln5 represents a new type of cysteine-based S-depalmitoylase linked to neurodegeneration.

Genetic CLN5 variants are associated with childhood neurodegeneration and Alzheimer's disease; however, the molecular function of ceroid lipofuscinosis neuronal protein 5 (Cln5) is unknown. We solved the Cln5 crystal structure and identified a region homologous to the catalytic domain of members of the N1pC/P60 superfamily of papain-like enzymes. However, we observed no protease activity for Cln5; and instead, we discovered that Cln5 and structurally related PPPDE1 and PPPDE2 have efficient cysteine palmitoyl thioesterase (S-depalmitoylation) activity using fluorescent substrates. Mutational analysis revealed that the predicted catalytic residues histidine-166 and cysteine-280 are critical for Cln5 thioesterase activity, uncovering a new cysteine-based catalytic mechanism for S-depalmitoylation enzymes. Last, we found that Cln5-deficient neuronal progenitor cells showed reduced thioesterase activity, confirming live cell function of Cln5 in setting S-depalmitoylation levels. Our results provide new insight into the function of Cln5, emphasize the importance of S-depalmitoylation in neuronal homeostasis, and disclose a new, unexpected enzymatic function for the N1pC/P60 superfamily of proteins.

Non-merohedral twinning: from minerals to proteins.

In contrast to twinning by merohedry, the reciprocal lattices of the different domains of non-merohedral twins do not overlap exactly. This leads to three kinds of reflections: reflections with no overlap, reflections with an exact overlap and reflections with a partial overlap of a reflection from a second domain. This complicates the unit-cell determination, indexing, data integration and scaling of X-ray diffraction data. However, with hindsight it is possible to detwin the data because there are reflections that are not affected by the twinning. In this article, the successful solution and refinement of one mineral, one organometallic and two protein non-merohedral twins using a common strategy are described. The unit-cell constants and the orientation matrices were determined by the program CELL_NOW. The data were then integrated with SAINT. TWINABS was used for scaling, empirical absorption corrections and the generation of two different data files, one with detwinned data for structure solution and refinement and a second one for (usually more accurate) structure refinement against total integrated intensities. The structures were solved by experimental phasing using SHELXT for the first two structures and SHELXC/D/E for the two protein structures; all models were refined with SHELXL.

PDB2INS: bridging the gap between small-molecule and macromolecular refinement.

The open-source Python program PDB2INS is designed to prepare a .ins file for refinement with SHELXL [Sheldrick (2015). Acta Cryst. C71, 3-8], taking atom coordinates and other information from a Protein Data Bank (PDB)-format file. If PDB2INS is provided with a four-character PDB code, both the PDB file and the accompanying mmCIF-format reflection data file (if available) are accessed via the internet from the PDB public archive [Read et al. (2011). Structure, 19, 1395-1412] or optionally from the PDB_REDO server [Joosten, Long, Murshudov & Perrakis (2014). IUCrJ, 1, 213-220]. The SHELX-format .ins (refinement instructions and atomic coordinates) and .hkl (reflection data) files can then be generated without further user intervention, appropriate restraints etc. being added automatically. PDB2INS was tested on the 23 974 X-ray structures deposited in the PDB between 2008 and 2018 that included reflection data to 1.7 Šor better resolution in a recognizable format. After creating the two input files for SHELXL without user intervention, ten cycles of conjugate-gradient least-squares refinement were performed. For 96% of these structures PDB2INS and SHELXL completed successfully without error messages.

Aspherical scattering factors for SHELXL - model, implementation and application.

A new aspherical scattering factor formalism has been implemented in the crystallographic least-squares refinement program SHELXL. The formalism relies on Gaussian functions and can optionally complement the independent atom model to take into account the deformation of electron-density distribution due to chemical bonding and lone pairs. Asphericity contributions were derived from the electron density obtained from quantum-chemical density functional theory computations of suitable model compounds that contain particular chemical environments, as defined by the invariom formalism. Thanks to a new algorithm, invariom assignment for refinement in SHELXL is automated. A suitable parameterization for each chemical environment within the new model was achieved by metaheuristics. Figures of merit, precision and accuracy of crystallographic least-squares refinements improve significantly upon using the new model.

An introduction to experimental phasing of macromolecules illustrated by SHELX; new autotracing features.

For the purpose of this article, experimental phasing is understood to mean the determination of macromolecular structures by exploiting small intensity differences of Friedel opposites and possibly of reflections measured at different wavelengths or for heavy-atom derivatives, without the use of specific structural models. The SHELX programs provide a robust and efficient route for routine structure solution by the SAD, MAD and related methods, but involve a number of simplifying assumptions that may limit their applicability in borderline cases. The substructure atoms (i.e. those with significant anomalous scattering) are first located by direct methods, and the experimental data are then used to estimate phase shifts that are added to the substructure phases to obtain starting phases for the native reflections. These are then improved by density modification and, if the resolution of the data and the type of structure permit, polyalanine tracing. A number of extensions to the tracing algorithm are discussed; these are designed to improve its performance at low resolution. Given native data to 2.5 Šresolution or better, a correlation coefficient greater than 25% between the structure factors calculated from such a trace and the native data is usually a good indication that the structure has been solved.

Combining phase information in reciprocal space for molecular replacement with partial models.

ARCIMBOLDO allows ab initio phasing of macromolecular structures below atomic resolution by exploiting the location of small model fragments combined with density modification in a multisolution frame. The model fragments can be either secondary-structure elements predicted from the sequence or tertiary-structure fragments. The latter can be derived from libraries of typical local folds or from related structures, such as a low-homology model that is unsuccessful in molecular replacement. In all ARCIMBOLDO applications, fragments are searched for sequentially. Correct partial solutions obtained after each fragment-search stage but lacking the necessary phasing power can, if combined, succeed. Here, an analysis is presented of the clustering of partial solutions in reciprocal space and of its application to a set of different cases. In practice, the task of combining model fragments from an ARCIMBOLDO run requires their referral to a common origin and is complicated by the presence of correct and incorrect solutions as well as by their not being independent. The F-weighted mean phase difference has been used as a figure of merit. Clustering perfect, non-overlapping fragments dismembered from test structures in polar and nonpolar space groups shows that density modification before determining the relative origin shift enhances its discrimination. In the case of nonpolar space groups, clustering of ARCIMBOLDO solutions from secondary-structure models is feasible. The use of partially overlapping search fragments provides a more favourable circumstance and was assessed on a test case. Applying the devised strategy, a previously unknown structure was solved from clustered correct partial solutions.

Comparison of silver and molybdenum microfocus X-ray sources for single-crystal structure determination.

The quality of diffraction data obtained using silver and molybdenum microsources has been compared for six model compounds with a wide range of absorption factors. The experiments were performed on two 30 W air-cooled Incoatec IµS microfocus sources with multilayer optics mounted on a Bruker D8 goniometer with a SMART APEX II CCD detector. All data were analysed, processed and refined using standard Bruker software. The results show that Ag Kα radiation can be beneficial when heavy elements are involved. A numerical absorption correction based on the positions and indices of the crystal faces is shown to be of limited use for the highly focused microsource beams, presumably because the assumption that the crystal is completely bathed in a (top-hat profile) beam of uniform intensity is no longer valid. Fortunately the empirical corrections implemented in SADABS, although originally intended as a correction for absorption, also correct rather well for the variations in the effective volume of the crystal irradiated. In three of the cases studied (two Ag and one Mo) the final SHELXL R1 against all data after application of empirical corrections implemented in SADABS was below 1%. Since such corrections are designed to optimize the agreement of the intensities of equivalent reflections with different paths through the crystal but the same Bragg 2θ angles, a further correction is required for the 2θ dependence of the absorption. For this, SADABS uses the transmission factor of a spherical crystal with a user-defined value of µr (where µ is the linear absorption coefficient and r is the effective radius of the crystal); the best results are obtained when r is biased towards the smallest crystal dimension. The results presented here suggest that the IUCr publication requirement that a numerical absorption correction must be applied for strongly absorbing crystals is in need of revision.

Crystal structure refinement with SHELXL.

The improvements in the crystal structure refinement program SHELXL have been closely coupled with the development and increasing importance of the CIF (Crystallographic Information Framework) format for validating and archiving crystal structures. An important simplification is that now only one file in CIF format (for convenience, referred to simply as `a CIF') containing embedded reflection data and SHELXL instructions is needed for a complete structure archive; the program SHREDCIF can be used to extract the .hkl and .ins files required for further refinement with SHELXL. Recent developments in SHELXL facilitate refinement against neutron diffraction data, the treatment of H atoms, the determination of absolute structure, the input of partial structure factors and the refinement of twinned and disordered structures. SHELXL is available free to academics for the Windows, Linux and Mac OS X operating systems, and is particularly suitable for multiple-core processors.

SHELXT - integrated space-group and crystal-structure determination.

The new computer program SHELXT employs a novel dual-space algorithm to solve the phase problem for single-crystal reflection data expanded to the space group P1. Missing data are taken into account and the resolution extended if necessary. All space groups in the specified Laue group are tested to find which are consistent with the P1 phases. After applying the resulting origin shifts and space-group symmetry, the solutions are subject to further dual-space recycling followed by a peak search and summation of the electron density around each peak. Elements are assigned to give the best fit to the integrated peak densities and if necessary additional elements are considered. An isotropic refinement is followed for non-centrosymmetric space groups by the calculation of a Flack parameter and, if appropriate, inversion of the structure. The structure is assembled to maximize its connectivity and centred optimally in the unit cell. SHELXT has already solved many thousand structures with a high success rate, and is optimized for multiprocessor computers. It is, however, unsuitable for severely disordered and twinned structures because it is based on the assumption that the structure consists of atoms.

Triostin a derived cyclopeptide as architectural template for the alignment of four recognition units.

The DNA bisintercalator triostin A is structurally based on a disulfide-bridged depsipeptide scaffold that provides preorganization of two quinoxaline units in 10.5 Å distance. Triostin A analogues are synthesized with nucleobase recognition units replacing the quinoxalines and containing two additional recognition units in between. Thus, four nucleobase recognition units are organized on a rigid template, well suited for DNA double strand interactions. The new tetra-nucleobase binders are synthesized as aza-TANDEM derivatives lacking the N-methylation of triostin A and based on a cyclopeptide backbone. Synthesis of two tetra-nucleobase aza-TANDEM derivatives is established, DNA interaction analyzed by microscale thermophoresis, cytotoxic activity studied and a nucleobase sequence dependent self-aggregation investigated by mass spectrometry.

Ion permeation in K⁺ channels occurs by direct Coulomb knock-on.

Potassium channels selectively conduct K(+) ions across cellular membranes with extraordinary efficiency. Their selectivity filter exhibits four binding sites with approximately equal electron density in crystal structures with high K(+) concentrations, previously thought to reflect a superposition of alternating ion- and water-occupied states. Consequently, cotranslocation of ions with water has become a widely accepted ion conduction mechanism for potassium channels. By analyzing more than 1300 permeation events from molecular dynamics simulations at physiological voltages, we observed instead that permeation occurs via ion-ion contacts between neighboring K(+) ions. Coulomb repulsion between adjacent ions is found to be the key to high-efficiency K(+) conduction. Crystallographic data are consistent with directly neighboring K(+) ions in the selectivity filter, and our model offers an intuitive explanation for the high throughput rates of K(+) channels.

Molecular architecture with carbohydrate functionalized ß-peptides adopting 314-helical conformation.

Carbohydrate recognition is essential in cellular interactions and biological processes. It is characterized by structural diversity, multivalency and cooperative effects. To evaluate carbohydrate interaction and recognition, the structurally defined attachment of sugar units to a rigid template is highly desired. ß-Peptide helices offer conformationally stable templates for the linear presentation of sugar units in defined distances. The synthesis and ß-peptide incorporation of sugar-ß-amino acids are described providing the saccharide units as amino acid side chain. The respective sugar-ß-amino acids are accessible by Michael addition of ammonia to sugar units derivatized as α,ß-unsaturated esters. Three sugar units were incorporated in ß-peptide oligomers varying the sugar (glucose, galactose, xylose) and sugar protecting groups. The influence of sugar units and the configuration of sugar-ß-amino acids on ß-peptide secondary structure were investigated by CD spectroscopy.

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.

Structure of sulfamidase provides insight into the molecular pathology of mucopolysaccharidosis IIIA.

Mucopolysaccharidosis type IIIA (Sanfilippo A syndrome), a fatal childhood-onset neurodegenerative disease with mild facial, visceral and skeletal abnormalities, is caused by an inherited deficiency of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH; sulfamidase). More than 100 mutations in the SGSH gene have been found to reduce or eliminate its enzymatic activity. However, the molecular understanding of the effect of these mutations has been confined by a lack of structural data for this enzyme. Here, the crystal structure of glycosylated SGSH is presented at 2 Å resolution. Despite the low sequence identity between this unique N-sulfatase and the group of O-sulfatases, they share a similar overall fold and active-site architecture, including a catalytic formylglycine, a divalent metal-binding site and a sulfate-binding site. However, a highly conserved lysine in O-sulfatases is replaced in SGSH by an arginine (Arg282) that is positioned to bind the N-linked sulfate substrate. The structure also provides insight into the diverse effects of pathogenic mutations on SGSH function in mucopolysaccharidosis type IIIA and convincing evidence for the molecular consequences of many missense mutations. Further, the molecular characterization of SGSH mutations will lay the groundwork for the development of structure-based drug design for this devastating neurodegenerative disorder.

Refinement of macromolecular structures against neutron data with SHELXL2013.

Some of the improvements in SHELX2013 make SHELXL convenient to use for refinement of macromolecular structures against neutron data without the support of X-ray data. The new NEUT instruction adjusts the behaviour of the SFAC instruction as well as the default bond lengths of the AFIX instructions. This work presents a protocol on how to use SHELXL for refinement of protein structures against neutron data. It includes restraints extending the Engh & Huber [Acta Cryst. (1991), A47, 392-400] restraints to H atoms and discusses several of the features of SHELXL that make the program particularly useful for the investigation of H atoms with neutron diffraction. SHELXL2013 is already adequate for the refinement of small molecules against neutron data, but there is still room for improvement, like the introduction of chain IDs for the refinement of macromolecular structures.

On the temperature dependence of H-U(iso) in the riding hydrogen model.

The temperature dependence of H-U(iso) in N-acetyl-L-4-hydroxyproline monohydrate is investigated. Imposing a constant temperature-independent multiplier of 1.2 or 1.5 for the riding hydrogen model is found to be inaccurate, and severely underestimates H-U(iso) below 100 K. Neutron diffraction data at temperatures of 9, 150, 200 and 250 K provide benchmark results for this study. X-ray diffraction data to high resolution, collected at temperatures of 9, 30, 50, 75, 100, 150, 200 and 250 K (synchrotron and home source), reproduce neutron results only when evaluated by aspherical-atom refinement models, since these take into account bonding and lone-pair electron density; both invariom and Hirshfeld-atom refinement models enable a more precise determination of the magnitude of H-atom displacements than independent-atom model refinements. Experimental efforts are complemented by computing displacement parameters following the TLS+ONIOM approach. A satisfactory agreement between all approaches is found.

Validation of metal-binding sites in macromolecular structures with the CheckMyMetal web server.

Metals have vital roles in both the mechanism and architecture of biological macromolecules. Yet structures of metal-containing macromolecules in which metals are misidentified and/or suboptimally modeled are abundant in the Protein Data Bank (PDB). This shows the need for a diagnostic tool to identify and correct such modeling problems with metal-binding environments. The CheckMyMetal (CMM) web server (http://csgid.org/csgid/metal_sites/) is a sophisticated, user-friendly web-based method to evaluate metal-binding sites in macromolecular structures using parameters derived from 7,350 metal-binding sites observed in a benchmark data set of 2,304 high-resolution crystal structures. The protocol outlines how the CMM server can be used to detect geometric and other irregularities in the structures of metal-binding sites, as well as how it can alert researchers to potential errors in metal assignment. The protocol also gives practical guidelines for correcting problematic sites by modifying the metal-binding environment and/or redefining metal identity in the PDB file. Several examples where this has led to meaningful results are described in the ANTICIPATED RESULTS section. CMM was designed for a broad audience--biomedical researchers studying metal-containing proteins and nucleic acids--but it is equally well suited for structural biologists validating new structures during modeling or refinement. The CMM server takes the coordinates of a metal-containing macromolecule structure in the PDB format as input and responds within a few seconds for a typical protein structure with 2-5 metal sites and a few hundred amino acids.

Extending molecular-replacement solutions with SHELXE.

Although the program SHELXE was originally intended for the experimental phasing of macromolecules, it can also prove useful for expanding a small protein fragment to an almost complete polyalanine trace of the structure, given a favourable combination of native data resolution (better than about 2.1 Å) and solvent content. A correlation coefficient (CC) of more than 25% between the native structure factors and those calculated from the polyalanine trace appears to be a reliable indicator of success and has already been exploited in a number of pipelines. Here, a more detailed account of this usage of SHELXE for molecular-replacement solutions is given.

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.