Unlocking the secrets of doxepin hydrochloride's crystal structure: Insights from high-quality powder diffraction analysis.

Doxepin hydrochloride, a versatile pharmaceutical compound, has been the subject of extensive research aimed at elucidating its crystal structure and solid-state characteristics. In this manuscript, we explore the significance of high-quality powder diffraction data in unveiling the intricate details of doxepin hydrochloride's crystal lattice. By examining the refined atom coordinates, density functional theory (DFT) optimization, and intermolecular interactions, we gain valuable insights into its structural conformation. This knowledge highlights the importance of precise crystallographic data in advancing our understanding of complex compounds and their pharmaceutical applications.

Crystallographic raw data, education and refereeing.

This article provides an overview of the preservation of raw diffraction data, then addresses the impact on future plans in the education and training of our community with respect to raw diffraction data and its potential reuse, and, thirdly presents the issue of referee access to the underpinning diffraction data and coordinates, as well as the Protein Data Bank Validation Report, in the review process of structural biology articles submitted for publication. Overall I pay tribute to the scientific achievements of Alex Wlodawer, who is also an ardent advocate of the importance of experimental data.

PRIGo: a new multi-axis goniometer for macromolecular crystallography.

The Parallel Robotics Inspired Goniometer (PRIGo) is a novel compact and high-precision goniometer providing an alternative to (mini-)kappa, traditional three-circle goniometers and Eulerian cradles used for sample reorientation in macromolecular crystallography. Based on a combination of serial and parallel kinematics, PRIGo emulates an arc. It is mounted on an air-bearing stage for rotation around ω and consists of four linear positioners working synchronously to achieve x, y, z translations and χ rotation (0-90°), followed by a ϕ stage (0-360°) for rotation around the sample holder axis. Owing to the use of piezo linear positioners and active correction, PRIGo features spheres of confusion of <1 µm, <7 µm and <10 µm for ω, χ and ϕ, respectively, and is therefore very well suited for micro-crystallography. PRIGo enables optimal strategies for both native and experimental phasing crystallographic data collection. Herein, PRIGo hardware and software, its calibration, as well as applications in macromolecular crystallography are described.

Weak data do not make a free lunch, only a cheap meal.

Four data sets were processed at resolutions significantly exceeding the criteria traditionally used for estimating the diffraction data resolution limit. The analysis of these data and the corresponding model-quality indicators suggests that the criteria of resolution limits widely adopted in the past may be somewhat conservative. Various parameters, such as Rmerge and I/σ(I), optical resolution and the correlation coefficients CC1/2 and CC*, can be used for judging the internal data quality, whereas the reliability factors R and Rfree as well as the maximum-likelihood target values and real-space map correlation coefficients can be used to estimate the agreement between the data and the refined model. However, none of these criteria provide a reliable estimate of the data resolution cutoff limit. The analysis suggests that extension of the maximum resolution by about 0.2 Šbeyond the currently adopted limit where the I/σ(I) value drops to 2.0 does not degrade the quality of the refined structural models, but may sometimes be advantageous. Such an extension may be particularly beneficial for significantly anisotropic diffraction. Extension of the maximum resolution at the stage of data collection and structure refinement is cheap in terms of the required effort and is definitely more advisable than accepting a too conservative resolution cutoff, which is unfortunately quite frequent among the crystal structures deposited in the Protein Data Bank.

Data of crystal structure of the monomeric red fluorescent protein DsRed.

The monomeric red fluorescent protein DsRed (mDsRed) is an optical probe widely used in multicolor applications in flow cytometry and fluorescence microscopy. Although the crystal structure of monomeric DsRed has been determined, its molecular dynamics have not been fully elucidated. To better understand its molecular flexibility, the crystal structure of mDsRed was recently determined, and its structure and temperature factors were analyzed. Solvent-accessible hole connected with the mDsRed chromophore was observed on the mDsRed surface structure. Electron density map analysis showed the tyrosine-ring group of the mDsRed chromophore in a cis-conformation, exhibiting flexibility with a nonplanar configuration between the tyrosine and imidazoline rings of the chromophore. Temperature factor analysis indicated that the top and bottom of the ß-barrel are relatively flexible. These structural findings extended our understanding of the molecular flexibility of mDsRed. The detailed data collection and structure determination reported in this study can be used for future structural analyses.

Data of crystal structures of single-domain substrate-binding protein from Rhodothermus marinus.

Substrate-binding proteins (SBPs) are essential in ATP-binding cassette transporter systems to determine substrate specificity and delivery. A typical SBP comprises two domains that recognize ligands such as metal ions, amino acids, sugars, and peptides. Interestingly, single-domain SBPs are found in the genomic database, but the molecular function of single-domain SBPs is not fully elucidated. To better understand the molecular function of single-domain SBPs, the crystal structure of single-domain SBPs from Rhodothermus marinus (RmSBP) soaked with NaBr and HgCl2 were determined at 1.75 and 2.3 Å resolution, respectively. The molecular flexibility of RmSBP and Hg2+-bound RmSBP structure was determined. This structural information can be utilized to understand the molecular function of single-domain SBPs. This study reported the detailed process of data collection and structure determination.

Advanced exploitation of unmerged reflection data during processing and refinement with autoPROC and BUSTER.

The validation of structural models obtained by macromolecular X-ray crystallography against experimental diffraction data, whether before deposition into the PDB or after, is typically carried out exclusively against the merged data that are eventually archived along with the atomic coordinates. It is shown here that the availability of unmerged reflection data enables valuable additional analyses to be performed that yield improvements in the final models, and tools are presented to implement them, together with examples of the results to which they give access. The first example is the automatic identification and removal of image ranges affected by loss of crystal centering or by excessive decay of the diffraction pattern as a result of radiation damage. The second example is the `reflection-auditing' process, whereby individual merged data items showing especially poor agreement with model predictions during refinement are investigated thanks to the specific metadata (such as image number and detector position) that are available for the corresponding unmerged data, potentially revealing previously undiagnosed instrumental, experimental or processing problems. The third example is the calculation of so-called F(early) - F(late) maps from carefully selected subsets of unmerged amplitude data, which can not only highlight the location and extent of radiation damage but can also provide guidance towards suitable fine-grained parametrizations to model the localized effects of such damage.

TERSE/PROLIX (TRPX) - a new algorithm for fast and lossless compression and decompression of diffraction and cryo-EM data.

High-throughput data collection in crystallography poses significant challenges in handling massive amounts of data. Here, TERSE/PROLIX (or TRPX for short) is presented, a novel lossless compression algorithm specifically designed for diffraction data. The algorithm is compared with established lossless compression algorithms implemented in gzip, bzip2, CBF (crystallographic binary file), Zstandard(zstd), LZ4 and HDF5 with gzip, LZF and bitshuffle+LZ4 filters, in terms of compression efficiency and speed, using continuous-rotation electron diffraction data of an inorganic compound and raw cryo-EM data. The results show that TRPX significantly outperforms all these algorithms in terms of speed and compression rate. It was 60 times faster than bzip2 (which achieved a similar compression rate), and more than 3 times faster than LZ4, which was the runner-up in terms of speed, but had a much worse compression rate. TRPX files are byte-order independent and upon compilation the algorithm occupies very little memory. It can therefore be readily implemented in hardware. By providing a tailored solution for diffraction and raw cryo-EM data, TRPX facilitates more efficient data analysis and interpretation while mitigating storage and transmission concerns. The C++20 compression/decompression code, custom TIFF library and an ImageJ/Fiji Java plugin for reading TRPX files are open-sourced on GitHub under the permissive MIT license.

Raw diffraction data are our ground truth from which all subsequent workflows develop.

Defining best practice in science is challenging. International consensus is facilitated by the International Science Council via its members such as the International Union of Crystallography (IUCr). The crystallographic community has many decades of tradition linking articles with the underpinning data, and is admired across all sciences accordingly. Crystallography has always been at the forefront of harnessing new technology in the service of consensus. Technology has provided new vast data-archiving opportunities, allowing the preservation of raw diffraction data, along with article and database depositions of a model's coordinates and associated structure factors. The raw diffraction data, which can now be preserved, are the ground truth from which all subsequent workflows develop. Journal editorial boards provide a practical forum for setting the criteria to decide if a study's files are truly the version of record. Within that, reality involves a variance of reasonable workflows. But what is a reasonable variance? Workflows must be detailed carefully by authors in explaining what they have done. There is a great, and increasing, diversity of macromolecular crystallography analyses, and yet an increased constraint on how much can be written in an article about the workflow used. Raw data provide the ultimate reproducibility evidence. A part of reproducibility and replicability is using an agreed vocabulary; the meaning of words such as precision and accuracy and, more recently, the confidence of a protein structure prediction should feature in approaching `truth'.

A hybrid approach to study large conformational transitions of biomolecules from single particle XFEL diffraction data.

X-ray free-electron laser (XFEL) is the latest generation of the X-ray source that could become an invaluable technique in structural biology. XFEL has ultrashort pulse duration, extreme peak brilliance, and high spatial coherence, which could enable the observation of the biological molecules in near nature state at room temperature without crystallization. However, for biological systems, due to their low diffraction power and complexity of sample delivery, experiments and data analysis are not straightforward, making it extremely challenging to reconstruct three-dimensional (3D) structures from single particle XFEL data. Given the current limitations to the amount and resolution of the data from such XFEL experiments, we propose a new hybrid approach for characterizing biomolecular conformational transitions by using a single 2D low-resolution XFEL diffraction pattern in combination with another known conformation. In our method, we represent the molecular structure with a coarse-grained model, the Gaussian mixture model, to describe large conformational transitions from low-resolution XFEL data. We obtain plausible 3D structural models that are consistent with the XFEL diffraction pattern by deforming an initial structural model to maximize the similarity between the target pattern and the simulated diffraction patterns from the candidate models. We tested the proposed algorithm on two biomolecules of different sizes with different complexities of conformational transitions, adenylate kinase, and elongation factor 2, using synthetic XFEL data. The results show that, with the proposed algorithm, we can successfully describe the conformational transitions by flexibly fitting the coarse-grained model of one conformation to become consistent with an XFEL diffraction pattern simulated from another conformation. In addition, we showed that the incident beam orientation has some effect on the accuracy of the 3D structure modeling and discussed the reasons for the inaccuracies for certain orientations. The proposed method could serve as an alternative approach for retrieving information on 3D conformational transitions from the XFEL diffraction patterns to interpret experimental data. Since the molecules are represented by Gaussian kernels and no atomic structure is needed in principle, such a method could also be used as a tool to seek initial models for 3D reconstruction algorithms.

Structure determination from unindexed powder data from scratch by a global optimization approach using pattern comparison based on cross-correlation functions.

A method of ab initio crystal structure determination from powder diffraction data for organic and metal-organic compounds, which does not require prior indexing of the powder pattern, has been developed. Only a reasonable molecular geometry is required, needing knowledge of neither unit-cell parameters nor space group. The structures are solved from scratch by a global fit to the powder data using the new program FIDEL-GO (`FIt with DEviating Lattice parameters - Global Optimization'). FIDEL-GO uses a similarity measure based on cross-correlation functions, which allows the comparison of simulated and experimental powder data even if the unit-cell parameters deviate strongly. The optimization starts from large sets of random structures in various space groups. The unit-cell parameters, molecular position and orientation, and selected internal degrees of freedom are fitted simultaneously to the powder pattern. The optimization proceeds in an elaborate multi-step procedure with built-in clustering of duplicate structures and iterative adaptation of parameter ranges. The best structures are selected for an automatic Rietveld refinement. Finally, a user-controlled Rietveld refinement is performed. The procedure aims for the analysis of a wide range of `problematic' powder patterns, in particular powders of low crystallinity. The method can also be used for the clustering and screening of a large number of possible structure candidates and other application scenarios. Examples are presented for structure determination from unindexed powder data of the previously unknown structures of the nanocrystalline phases of 4,11-difluoro-, 2,9-dichloro- and 2,9-dichloro-6,13-dihydro-quinacridone, which were solved from powder patterns with 14-20 peaks only, and of the coordination polymer dichloro-bis(pyridine-N)copper(II).

EDDIDAT: a graphical user interface for the analysis of energy-dispersive diffraction data.

EDDIDAT is a MATLAB-based graphical user interface for the convenient and versatile analysis of energy-dispersive diffraction data obtained at laboratory and synchrotron sources. The main focus of EDDIDAT up to now has been on the analysis of residual stresses, but it can also be used to prepare measurement data for subsequent phase analysis or analysis of preferred orientation. The program provides access to the depth-resolved analysis of residual stresses at different levels of approximation. Furthermore, the graphic representation of the results also serves for the consideration of microstructural and texture-related properties. The included material database allows for the quick analysis of the most common materials and is easily extendable. The plots and results produced with EDDIDAT can be exported to graphics and text files. EDDIDAT is designed to analyze diffraction data from various energy-dispersive X-ray sources. Hence it is possible to add new sources and implement the device-specific properties into EDDIDAT. The program is freely available to academic users.

Application of X-Ray Powder Diffraction for Analysis of Selected Dietary Supplements Containing Magnesium and Calcium.

It is estimated that ~50% of medications and dietary supplements offered in the Internet are counterfeit. X-ray diffraction is one of the techniques which may be successfully applied to identify various chemical compounds in polycrystalline mixtures such as dietary supplements, but also medications, narcotics or designer drugs. X-ray diffraction enables the understanding of compositions of such mixtures. For the tests, 22 dietary supplements which should contain magnesium and calcium compounds, available in pharmacies, groceries, Internet shops, as well as in shops for sportspersons, were selected. Identification of crystalline substances present in the tested sample consists in determination of inter-planar distances d hkl of investigated substances and determination of intensity of the obtained diffraction lines, and then in comparing them with values contained in diffraction databases. In this study, the ICDD-PDF2 database was used. The most important criterion in qualitative analysis, confirming the presence of a given phase, is the conformity of positions of diffraction lines in the recorded diffraction image with those in the reference image. Reflection shifts for the individual 2θ angles compared with the data from the database should not exceed 0.2°. In most cases, X-ray analysis of the investigated dietary supplements proved the presence of magnesium and calcium compounds declared by the manufacturer, as well as allowing the identification of auxiliary substances present in the tested products. In the case of two magnesium-containing dietary supplements, the magnesium compounds declared by the manufacturer were not found. Our studies confirmed the effectiveness of X-ray structural analysis and proved the possibility of distinguishing counterfeit preparations from authentic products, as well as to use this method for the quality control of such pharmaceutical preparations.

Gold Standard for macromolecular crystallography diffraction data.

Macromolecular crystallography (MX) is the dominant means of determining the three-dimensional structures of biological macromolecules. Over the last few decades, most MX data have been collected at synchrotron beamlines using a large number of different detectors produced by various manufacturers and taking advantage of various protocols and goniometries. These data came in their own formats: sometimes proprietary, sometimes open. The associated metadata rarely reached the degree of completeness required for data management according to Findability, Accessibility, Interoperability and Reusability (FAIR) principles. Efforts to reuse old data by other investigators or even by the original investigators some time later were often frustrated. In the culmination of an effort dating back more than two decades, a large portion of the research community concerned with high data-rate macromolecular crystallography (HDRMX) has now agreed to an updated specification of data and metadata for diffraction images produced at synchrotron light sources and X-ray free-electron lasers (XFELs). This 'Gold Standard' will facilitate the processing of data sets independent of the facility at which they were collected and enable data archiving according to FAIR principles, with a particular focus on interoperability and reusability. This agreed standard builds on the NeXus/HDF5 NXmx application definition and the International Union of Crystallo-graphy (IUCr) imgCIF/CBF dictionary, and it is compatible with major data-processing programs and pipelines. Just as with the IUCr CBF/imgCIF standard from which it arose and to which it is tied, the NeXus/HDF5 NXmx Gold Standard application definition is intended to be applicable to all detectors used for crystallography, and all hardware and software developers in the field are encouraged to adopt and contribute to the standard.

Findable Accessible Interoperable Re-usable (FAIR) diffraction data are coming to protein crystallography.

The policy of IUCr Journals on diffraction data is defined.

Findable Accessible Interoperable Re-usable (FAIR) diffraction data are coming to protein crystallography.

The policy of IUCr Journals on diffraction data is defined.

Interactive visualization of multi-data-set Rietveld analyses using Cinema:Debye-Scherrer.

A tool named Cinema:Debye-Scherrer to visualize the results of a series of Rietveld analyses is presented. The multi-axis visualization of the high-dimensional data sets resulting from powder diffraction analyses allows identification of analysis problems, prediction of suitable starting values, identification of gaps in the experimental parameter space and acceleration of scientific insight from the experimental data. The tool is demonstrated with analysis results from 59 U-Nb alloy samples with different compositions, annealing times and annealing temperatures as well as with a high-temperature study of the crystal structure of CsPbBr3. A script to extract parameters from a series of Rietveld analyses employing the widely used GSAS Rietveld software is also described. Both software tools are available for download.

Against Method: Table 1-Cui Bono?

The almost universally required "Table 1," summarizing data-collection and data-processing statistics, has in its present form outlived its usefulness in almost all publications of biomolecular crystal structure reports. Information contained in "Table 1" is insufficient to evaluate or repeat the experiment; is redundant with information extractable from deposited diffraction data; and includes data items whose meaning is under increased scrutiny in the crystallographic community. Direct and consistent extraction and analysis of data quality metrics from preferably unmerged intensity data with graphical presentation of reciprocal space features, including impact on map and model features, should replace "Table 1."

Status of the neutron time-of-flight single-crystal diffraction data-processing software STARGazer.

The STARGazer data-processing software is used for neutron time-of-flight (TOF) single-crystal diffraction data collected using the IBARAKI Biological Crystal Diffractometer (iBIX) at the Japan Proton Accelerator Research Complex (J-PARC). This software creates hkl intensity data from three-dimensional (x, y, TOF) diffraction data. STARGazer is composed of a data-processing component and a data-visualization component. The former is used to calculate the hkl intensity data. The latter displays the three-dimensional diffraction data with searched or predicted peak positions and is used to determine and confirm integration regions. STARGazer has been developed to make it easier to use and to obtain more accurate intensity data. For example, a profile-fitting method for peak integration was developed and the data statistics were improved. STARGazer and its manual, containing installation and data-processing components, have been prepared and provided to iBIX users. This article describes the status of the STARGazer data-processing software and its data-processing algorithms.