DiSCaMB: a software library for aspherical atom model X-ray scattering factor calculations with CPUs and GPUs.

It has been recently established that the accuracy of structural parameters from X-ray refinement of crystal structures can be improved by using a bank of aspherical pseudoatoms instead of the classical spherical model of atomic form factors. This comes, however, at the cost of increased complexity of the underlying calculations. In order to facilitate the adoption of this more advanced electron density model by the broader community of crystallographers, a new software implementation called DiSCaMB, 'densities in structural chemistry and molecular biology', has been developed. It addresses the challenge of providing for high performance on modern computing architectures. With parallelization options for both multi-core processors and graphics processing units (using CUDA), the library features calculation of X-ray scattering factors and their derivatives with respect to structural parameters, gives access to intermediate steps of the scattering factor calculations (thus allowing for experimentation with modifications of the underlying electron density model), and provides tools for basic structural crystallographic operations. Permissively (MIT) licensed, DiSCaMB is an open-source C++ library that can be embedded in both academic and commercial tools for X-ray structure refinement.

Macromolecular X-ray structure determination using weak, single-wavelength anomalous data.

We describe a likelihood-based method for determining the substructure of anomalously scattering atoms in macromolecular crystals that allows successful structure determination by single-wavelength anomalous diffraction (SAD) X-ray analysis with weak anomalous signal. With the use of partial models and electron density maps in searches for anomalously scattering atoms, testing of alternative values of parameters and parallelized automated model-building, this method has the potential to extend the applicability of the SAD method in challenging cases.

Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers.

X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and without radiation damage. Results to date, however, have been limited by the challenge of deriving accurate Bragg intensities from a heterogeneous population of microcrystals, while at the same time modeling the X-ray spectrum and detector geometry. Here we present a computational approach designed to extract meaningful high-resolution signals from fewer diffraction measurements.

New Python-based methods for data processing.

Current pixel-array detectors produce diffraction images at extreme data rates (of up to 2 TB h(-1)) that make severe demands on computational resources. New multiprocessing frameworks are required to achieve rapid data analysis, as it is important to be able to inspect the data quickly in order to guide the experiment in real time. By utilizing readily available web-serving tools that interact with the Python scripting language, it was possible to implement a high-throughput Bragg-spot analyzer (cctbx.spotfinder) that is presently in use at numerous synchrotron-radiation beamlines. Similarly, Python interoperability enabled the production of a new data-reduction package (cctbx.xfel) for serial femtosecond crystallography experiments at the Linac Coherent Light Source (LCLS). Future data-reduction efforts will need to focus on specialized problems such as the treatment of diffraction spots on interleaved lattices arising from multi-crystal specimens. In these challenging cases, accurate modeling of close-lying Bragg spots could benefit from the high-performance computing capabilities of graphics-processing units.

Simultaneous femtosecond X-ray spectroscopy and diffraction of photosystem II at room temperature.

Intense femtosecond x-ray pulses produced at the Linac Coherent Light Source (LCLS) were used for simultaneous x-ray diffraction (XRD) and x-ray emission spectroscopy (XES) of microcrystals of photosystem II (PS II) at room temperature. This method probes the overall protein structure and the electronic structure of the Mn4CaO5 cluster in the oxygen-evolving complex of PS II. XRD data are presented from both the dark state (S1) and the first illuminated state (S2) of PS II. Our simultaneous XRD-XES study shows that the PS II crystals are intact during our measurements at the LCLS, not only with respect to the structure of PS II, but also with regard to the electronic structure of the highly radiation-sensitive Mn4CaO5 cluster, opening new directions for future dynamics studies.

Energy-dispersive X-ray emission spectroscopy using an X-ray free-electron laser in a shot-by-shot mode.

The ultrabright femtosecond X-ray pulses provided by X-ray free-electron lasers open capabilities for studying the structure and dynamics of a wide variety of systems beyond what is possible with synchrotron sources. Recently, this "probe-before-destroy" approach has been demonstrated for atomic structure determination by serial X-ray diffraction of microcrystals. There has been the question whether a similar approach can be extended to probe the local electronic structure by X-ray spectroscopy. To address this, we have carried out femtosecond X-ray emission spectroscopy (XES) at the Linac Coherent Light Source using redox-active Mn complexes. XES probes the charge and spin states as well as the ligand environment, critical for understanding the functional role of redox-active metal sites. Kß(1,3) XES spectra of Mn(II) and Mn(2)(III,IV) complexes at room temperature were collected using a wavelength dispersive spectrometer and femtosecond X-ray pulses with an individual dose of up to >100 MGy. The spectra were found in agreement with undamaged spectra collected at low dose using synchrotron radiation. Our results demonstrate that the intact electronic structure of redox active transition metal compounds in different oxidation states can be characterized with this shot-by-shot method. This opens the door for studying the chemical dynamics of metal catalytic sites by following reactions under functional conditions. The technique can be combined with X-ray diffraction to simultaneously obtain the geometric structure of the overall protein and the local chemistry of active metal sites and is expected to prove valuable for understanding the mechanism of important metalloproteins, such as photosystem II.

Nanoflow electrospinning serial femtosecond crystallography.

An electrospun liquid microjet has been developed that delivers protein microcrystal suspensions at flow rates of 0.14-3.1 µl min(-1) to perform serial femtosecond crystallography (SFX) studies with X-ray lasers. Thermolysin microcrystals flowed at 0.17 µl min(-1) and diffracted to beyond 4 Å resolution, producing 14,000 indexable diffraction patterns, or four per second, from 140 µg of protein. Nanoflow electrospinning extends SFX to biological samples that necessitate minimal sample consumption.

Improved crystallographic models through iterated local density-guided model deformation and reciprocal-space refinement.

An approach is presented for addressing the challenge of model rebuilding after molecular replacement in cases where the placed template is very different from the structure to be determined. The approach takes advantage of the observation that a template and target structure may have local structures that can be superimposed much more closely than can their complete structures. A density-guided procedure for deformation of a properly placed template is introduced. A shift in the coordinates of each residue in the structure is calculated based on optimizing the match of model density within a 6 Šradius of the center of that residue with a prime-and-switch electron-density map. The shifts are smoothed and applied to the atoms in each residue, leading to local deformation of the template that improves the match of map and model. The model is then refined to improve the geometry and the fit of model to the structure-factor data. A new map is then calculated and the process is repeated until convergence. The procedure can extend the routine applicability of automated molecular replacement, model building and refinement to search models with over 2 Šr.m.s.d. representing 65-100% of the structure.

Graphical tools for macromolecular crystallography in PHENIX.

A new Python-based graphical user interface for the PHENIX suite of crystallography software is described. This interface unifies the command-line programs and their graphical displays, simplifying the development of new interfaces and avoiding duplication of function. With careful design, graphical interfaces can be displayed automatically, instead of being manually constructed. The resulting package is easily maintained and extended as new programs are added or modified.

Room temperature femtosecond X-ray diffraction of photosystem II microcrystals.

Most of the dioxygen on earth is generated by the oxidation of water by photosystem II (PS II) using light from the sun. This light-driven, four-photon reaction is catalyzed by the Mn(4)CaO(5) cluster located at the lumenal side of PS II. Various X-ray studies have been carried out at cryogenic temperatures to understand the intermediate steps involved in the water oxidation mechanism. However, the necessity for collecting data at room temperature, especially for studying the transient steps during the O-O bond formation, requires the development of new methodologies. In this paper we report room temperature X-ray diffraction data of PS II microcrystals obtained using ultrashort (< 50 fs) 9 keV X-ray pulses from a hard X-ray free electron laser, namely the Linac Coherent Light Source. The results presented here demonstrate that the "probe before destroy" approach using an X-ray free electron laser works even for the highly-sensitive Mn(4)CaO(5) cluster in PS II at room temperature. We show that these data are comparable to those obtained in synchrotron radiation studies as seen by the similarities in the overall structure of the helices, the protein subunits and the location of the various cofactors. This work is, therefore, an important step toward future studies for resolving the structure of the Mn(4)CaO(5) cluster without any damage at room temperature, and of the reaction intermediates of PS II during O-O bond formation.

Automatic Fortran to C++ conversion with FABLE.

BACKGROUND: In scientific computing, Fortran was the dominant implementation language throughout most of the second part of the 20th century. The many tools accumulated during this time have been difficult to integrate with modern software, which is now dominated by object-oriented languages. RESULTS: Driven by the requirements of a large-scale scientific software project, we have developed a Fortran to C++ source-to-source conversion tool named FABLE. This enables the continued development of new methods even while switching languages. We report the application of FABLE in three major projects and present detailed comparisons of Fortran and C++ runtime performances. CONCLUSIONS: Our experience suggests that most Fortran 77 codes can be converted with an effort that is minor (measured in days) compared to the original development time (often measured in years). With FABLE it is possible to reuse and evolve legacy work in modern object-oriented environments, in a portable and maintainable way. FABLE is available under a nonrestrictive open source license. In FABLE the analysis of the Fortran sources is separated from the generation of the C++ sources. Therefore parts of FABLE could be reused for other target languages.

Towards automated crystallographic structure refinement with phenix.refine.

phenix.refine is a program within the PHENIX package that supports crystallographic structure refinement against experimental data with a wide range of upper resolution limits using a large repertoire of model parameterizations. It has several automation features and is also highly flexible. Several hundred parameters enable extensive customizations for complex use cases. Multiple user-defined refinement strategies can be applied to specific parts of the model in a single refinement run. An intuitive graphical user interface is available to guide novice users and to assist advanced users in managing refinement projects. X-ray or neutron diffraction data can be used separately or jointly in refinement. phenix.refine is tightly integrated into the PHENIX suite, where it serves as a critical component in automated model building, final structure refinement, structure validation and deposition to the wwPDB. This paper presents an overview of the major phenix.refine features, with extensive literature references for readers interested in more detailed discussions of the methods.

Use of knowledge-based restraints in phenix.refine to improve macromolecular refinement at low resolution.

Traditional methods for macromolecular refinement often have limited success at low resolution (3.0-3.5 Šor worse), producing models that score poorly on crystallographic and geometric validation criteria. To improve low-resolution refinement, knowledge from macromolecular chemistry and homology was used to add three new coordinate-restraint functions to the refinement program phenix.refine. Firstly, a `reference-model' method uses an identical or homologous higher resolution model to add restraints on torsion angles to the geometric target function. Secondly, automatic restraints for common secondary-structure elements in proteins and nucleic acids were implemented that can help to preserve the secondary-structure geometry, which is often distorted at low resolution. Lastly, we have implemented Ramachandran-based restraints on the backbone torsion angles. In this method, a ϕ,ψ term is added to the geometric target function to minimize a modified Ramachandran landscape that smoothly combines favorable peaks identified from nonredundant high-quality data with unfavorable peaks calculated using a clash-based pseudo-energy function. All three methods show improved MolProbity validation statistics, typically complemented by a lowered R(free) and a decreased gap between R(work) and R(free).

phenix.mr_rosetta: molecular replacement and model rebuilding with Phenix and Rosetta.

The combination of algorithms from the structure-modeling field with those of crystallographic structure determination can broaden the range of templates that are useful for structure determination by the method of molecular replacement. Automated tools in phenix.mr_rosetta simplify the application of these combined approaches by integrating Phenix crystallographic algorithms and Rosetta structure-modeling algorithms and by systematically generating and evaluating models with a combination of these methods. The phenix.mr_rosetta algorithms can be used to automatically determine challenging structures. The approaches used in phenix.mr_rosetta are described along with examples that show roles that structure-modeling can play in molecular replacement.

iotbx.cif: a comprehensive CIF toolbox.

iotbx.cif is a new software module for the development of applications that make use of the CIF format. Comprehensive tools are provided for input, output and validation of CIFs, as well as for interconversion with high-level cctbx [Grosse-Kunstleve, Sauter, Moriarty & Adams (2002). J. Appl. Cryst.35, 126-136] crystallographic objects. The interface to the library is written in Python, whilst parsing is carried out using a compiled parser, combining the performance of a compiled language (C++) with the benefits of using an interpreted language.

The Phenix software for automated determination of macromolecular structures.

X-ray crystallography is a critical tool in the study of biological systems. It is able to provide information that has been a prerequisite to understanding the fundamentals of life. It is also a method that is central to the development of new therapeutics for human disease. Significant time and effort are required to determine and optimize many macromolecular structures because of the need for manual interpretation of complex numerical data, often using many different software packages, and the repeated use of interactive three-dimensional graphics. The Phenix software package has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on automation. This has required the development of new algorithms that minimize or eliminate subjective input in favor of built-in expert-systems knowledge, the automation of procedures that are traditionally performed by hand, and the development of a computational framework that allows a tight integration between the algorithms. The application of automated methods is particularly appropriate in the field of structural proteomics, where high throughput is desired. Features in Phenix for the automation of experimental phasing with subsequent model building, molecular replacement, structure refinement and validation are described and examples given of running Phenix from both the command line and graphical user interface.

Exact direct-space asymmetric units for the 230 crystallographic space groups.

It is well known that the direct-space asymmetric unit definitions found in the International Tables for Crystallography, Volume A, are inexact at the borders. Face- and edge-specific sub-conditions have to be added to remove parts redundant under symmetry. This paper introduces a concise geometric notation for asymmetric unit conditions. The notation is the foundation for a reference table of exact direct-space asymmetric unit definitions for the 230 crystallographic space-group types. The change-of-basis transformation law for the conditions is derived, which allows the information from the reference table to be used for any space-group setting. We also show how the vertices of an asymmetric unit can easily be computed from the information in the reference table.

Joint X-ray and neutron refinement with phenix.refine.

Approximately 85% of the structures deposited in the Protein Data Bank have been solved using X-ray crystallography, making it the leading method for three-dimensional structure determination of macromolecules. One of the limitations of the method is that the typical data quality (resolution) does not allow the direct determination of H-atom positions. Most hydrogen positions can be inferred from the positions of other atoms and therefore can be readily included into the structure model as a priori knowledge. However, this may not be the case in biologically active sites of macromolecules, where the presence and position of hydrogen is crucial to the enzymatic mechanism. This makes the application of neutron crystallography in biology particularly important, as H atoms can be clearly located in experimental neutron scattering density maps. Without exception, when a neutron structure is determined the corresponding X-ray structure is also known, making it possible to derive the complete structure using both data sets. Here, the implementation of crystallographic structure-refinement procedures that include both X-ray and neutron data (separate or jointly) in the PHENIX system is described.

phenix.model_vs_data: a high-level tool for the calculation of crystallographic model and data statistics.

phenix.model_vs_data is a high-level command-line tool for the computation of crystallographic model and data statistics, and the evaluation of the fit of the model to data. Analysis of all Protein Data Bank structures that have experimental data available shows that in most cases the reported statistics, in particular R factors, can be reproduced within a few percentage points. However, there are a number of outliers where the recomputed R values are significantly different from those originally reported. The reasons for these discrepancies are discussed.

Detection and correction of underassigned rotational symmetry prior to structure deposition.

Up to 2% of X-ray structures in the Protein Data Bank (PDB) potentially fit into a higher symmetry space group. Redundant protein chains in these structures can be made compatible with exact crystallographic symmetry with minimal atomic movements that are smaller than the expected range of coordinate uncertainty. The incidence of problem cases is somewhat difficult to define precisely, as there is no clear line between underassigned symmetry, in which the subunit differences are unsupported by the data, and pseudosymmetry, in which the subunit differences rest on small but significant intensity differences in the diffraction pattern. To help catch symmetry-assignment problems in the future, it is useful to add a validation step that operates on the refined coordinates just prior to structure deposition. If redundant symmetry-related chains can be removed at this stage, the resulting model (in a higher symmetry space group) can readily serve as an isomorphous replacement starting point for re-refinement using re-indexed and re-integrated raw data. These ideas are implemented in new software tools available at http://cci.lbl.gov/labelit.