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The JUNGFRAU 4-megapixel (4M) charge-integrating pixel-array detector, when operated at a full 2â kHz frame rate, streams data at a rate of 17â GBâ s-1. To operate this detector for macromolecular crystallography beamlines, a data-acquisition system called Jungfraujoch was developed. The system, running on a single server with field-programmable gate arrays and general-purpose graphics processing units, is capable of handling data produced by the JUNGFRAU 4M detector, including conversion of raw pixel readout to photon counts, compression and on-the-fly spot finding. It was also demonstrated that 30â GBâ s-1 can be handled in performance tests, indicating that the operation of even larger and faster detectors will be achievable in the future. The source code is available from a public repository.
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Software , Síncrotrons , Raios X , Radiografia , Cristalografia por Raios XRESUMO
Formation of highly symmetric skeletal elements in demosponges, called spicules, follows a unique biomineralization mechanism in which polycondensation of an inherently disordered amorphous silica is guided by a highly ordered proteinaceous scaffold, the axial filament. The enzymatically active proteins, silicateins, are assembled into a slender hybrid silica/protein crystalline superstructure that directs the morphogenesis of the spicules. Furthermore, silicateins are known to catalyze the formation of a large variety of other technologically relevant organic and inorganic materials. However, despite the biological and biotechnological importance of this macromolecule, its tertiary structure was never determined. Here we report the atomic structure of silicatein and the entire mineral/organic hybrid assembly with a resolution of 2.4 Å. In this work, the serial X-ray crystallography method was successfully adopted to probe the 2-µm-thick filaments in situ, being embedded inside the skeletal elements. In combination with imaging and chemical analysis using high-resolution transmission electron microscopy, we provide detailed information on the enzymatic activity of silicatein, its crystallization, and the emergence of a functional three-dimensional silica/protein superstructure in vivo. Ultimately, we describe a naturally occurring mineral/protein crystalline assembly at atomic resolution.
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The accuracy of X-ray diffraction data is directly related to how the X-ray detector records photons. Here we describe the application of a direct-detection charge-integrating pixel-array detector (JUNGFRAU) in macromolecular crystallography (MX). JUNGFRAU features a uniform response on the subpixel level, linear behavior toward high photon rates, and low-noise performance across the whole dynamic range. We demonstrate that these features allow accurate MX data to be recorded at unprecedented speed. We also demonstrate improvements over previous-generation detectors in terms of data quality, using native single-wavelength anomalous diffraction (SAD) phasing, for thaumatin, lysozyme, and aminopeptidase N. Our results suggest that the JUNGFRAU detector will substantially improve the performance of synchrotron MX beamlines and equip them for future synchrotron light sources.
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Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/métodos , Coleta de Dados/métodos , Substâncias Macromoleculares/química , Síncrotrons/instrumentação , Antígenos CD13/química , Desenho de Equipamento , Humanos , Modelos Moleculares , Muramidase/químicaRESUMO
Precise knowledge of Mg2+ inner-sphere binding site properties is vital for understanding the structure and function of nucleic acid systems. Unfortunately, the PDB, which represents the main source of Mg2+ binding sites, contains a substantial number of assignment issues that blur our understanding of the functions of these ions. Here, following a previous study devoted to Mg2+ binding to nucleobase nitrogens, we surveyed nucleic acid X-ray structures from the PDB with resolutions ≤2.9 Å to classify the Mg2+ inner-sphere binding patterns to nucleotide carbonyl, ribose hydroxyl, cyclic ether, and phosphodiester oxygen atoms. From this classification, we derived a set of "prior-knowledge" nucleobase Mg2+ binding sites. We report that crystallographic examples of trustworthy nucleobase Mg2+ binding sites are fewer than expected since many of those are associated with misidentified Na+ or K+ We also emphasize that binding of Na+ and K+ to nucleic acids is much more frequent than anticipated. Overall, we provide evidence derived from X-ray structures that nucleobases are poor inner-sphere binders for Mg2+ but good binders for monovalent ions. Based on strict stereochemical criteria, we propose an extended set of guidelines designed to help in the assignment and validation of ions directly contacting nucleobase and ribose atoms. These guidelines should help in the interpretation of X-ray and cryo-EM solvent density maps. When borderline Mg2+ stereochemistry is observed, alternative placement of Na+, K+, or Ca2+ must be considered. We also critically examine the use of lanthanides (Yb3+, Tb3+) as Mg2+ substitutes in crystallography experiments.
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Bases de Dados de Proteínas , Magnésio/química , RNA Catalítico/química , Térbio/química , Itérbio/química , Sítios de Ligação/fisiologia , Cálcio/química , Domínio Catalítico , Cristalografia por Raios X , Potássio/química , Ribossomos/genética , Sódio/químicaRESUMO
Detection of heavy elements, such as metals, in macromolecular crystallography (MX) samples by X-ray fluorescence is a function traditionally covered at synchrotron MX beamlines by silicon drift detectors, which cannot be used at X-ray free-electron lasers because of the very short duration of the X-ray pulses. Here it is shown that the hybrid pixel charge-integrating detector JUNGFRAU can fulfill this function when operating in a low-flux regime. The feasibility of precise position determination of micrometre-sized metal marks is also demonstrated, to be used as fiducials for offline prelocation in serial crystallography experiments, based on the specific fluorescence signal measured with JUNGFRAU, both at the synchrotron and at SwissFEL. Finally, the measurement of elemental absorption edges at a synchrotron beamline using JUNGFRAU is also demonstrated.
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When thinking about RNA three-dimensional structures, coming across GNRA and UNCG tetraloops is perceived as a boon since their folds have been extensively described. Nevertheless, analyzing loop conformations within RNA and RNP structures led us to uncover several instances of GNRA and UNCG loops that do not fold as expected. We noticed that when a GNRA does not assume its "natural" fold, it adopts the one we typically associate with a UNCG sequence. The same folding interconversion may occur for loops with UNCG sequences, for instance within tRNA anticodon loops. Hence, we show that some structured tetranucleotide sequences starting with G or U can adopt either of these folds. The underlying structural basis that defines these two fold types is the mutually exclusive stacking of a backbone oxygen on either the first (in GNRA) or the last nucleobase (in UNCG), generating an oxygen-π contact. We thereby propose to refrain from using sequences to distinguish between loop conformations. Instead, we suggest using descriptors such as U-turn (for "GNRA-type" folds) and a newly described Z-turn (for "UNCG-type" folds). Because tetraloops adopt for the largest part only two (inter)convertible turns, we are better able to interpret from a structural perspective loop interchangeability occurring in ribosomes and viral RNA. In this respect, we propose a general view on the inclination for a given sequence to adopt (or not) a specific fold. We also suggest how long-noncoding RNAs may adopt discrete but transient structures, which are therefore hard to predict.
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Dobramento de RNA , RNA Viral/química , Ribossomos/química , Sequências Repetidas Invertidas , Modelos Moleculares , Motivos de Nucleotídeos , RNA Viral/metabolismo , Ribossomos/metabolismoRESUMO
Given the many roles proposed for Mg2+ in nucleic acids, it is essential to accurately determine their binding modes. Here, we surveyed the PDB to classify Mg2+ inner-sphere binding patterns to nucleobase imine N1/N3/N7 atoms. Among those, purine N7 atoms are considered to be the best nucleobase binding sites for divalent metals. Further, Mg2+ coordination to N7 has been implied in several ribozyme catalytic mechanisms. We report that Mg2+ assigned near imine nitrogens derive mostly from poor interpretations of electron density patterns and are most often misidentified Na+, K+, NH4+ ions, water molecules or spurious density peaks. Consequently, apart from few documented exceptions, Mg2+ ions do not bind to N7 atoms. Without much of a surprise, Mn2+, Zn2+ and Cd2+, which have a higher affinity for nitrogens, may contact N7 atoms when present in crystallization buffers. In this respect, we describe for the first time a potential Zn2+ ribosomal binding site involving two purine N7 atoms. Further, we provide a set of guidelines to help in the assignment of Mg2+ in crystallographic, cryo-EM, NMR and model building practices and discuss implications of our findings related to ion substitution experiments.
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Cátions Bivalentes/química , Magnésio/química , Nitrogênio/química , Ácidos Nucleicos/química , Cátions Monovalentes/química , Modelos Moleculares , Conformação Molecular , Ácidos Nucleicos/metabolismo , Oxigênio/química , SolventesRESUMO
Since the work of Alexander Rich, who solved the first Z-DNA crystal structure, we have known that d(CpG) steps can adopt a particular structure that leads to forming left-handed helices. However, it is still largely unrecognized that other sequences can adopt 'left-handed' conformations in DNA and RNA, in double as well as single stranded contexts. These 'Z-like' steps involve the coexistence of several rare structural features: a C2'-endo puckering, a syn nucleotide and a lone pair-π stacking between a ribose O4' atom and a nucleobase. This particular arrangement induces a conformational stress in the RNA backbone, which limits the occurrence of Z-like steps to ≈0.1% of all dinucleotide steps in the PDB. Here, we report over 600 instances of Z-like steps, which are located within r(UNCG) tetraloops but also in small and large RNAs including riboswitches, ribozymes and ribosomes. Given their complexity, Z-like steps are probably associated with slow folding kinetics and once formed could lock a fold through the formation of unique long-range contacts. Proteins involved in immunologic response also specifically recognize/induce these peculiar folds. Thus, characterizing the conformational features of these motifs could be a key to understanding the immune response at a structural level.
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DNA Forma Z/química , RNA Catalítico/química , RNA/química , Ribossomos/química , Riboswitch/genética , Pareamento de Bases , DNA Forma Z/genética , DNA Forma Z/imunologia , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/imunologia , Humanos , Imunidade Inata , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , RNA/genética , RNA/imunologia , Dobramento de RNA , RNA Catalítico/genética , RNA Catalítico/imunologia , Ribossomos/genética , Ribossomos/imunologia , Riboswitch/imunologia , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/imunologiaRESUMO
Coarse-grained (CG) models in molecular dynamics (MD) are powerful tools to simulate the dynamics of large biomolecular systems on micro- to millisecond timescales. However, the CG model, potential energy terms, and parameters are typically not transferable between different molecules and problems. So parameterizing CG force fields, which is both tedious and time-consuming, is often necessary. We present RedMDStream, a software for developing, testing, and simulating biomolecules with CG MD models. Development includes an automatic procedure for the optimization of potential energy parameters based on metaheuristic methods. As an example we describe the parameterization of a simple CG MD model of an RNA hairpin.
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DNA/química , Simulação de Dinâmica Molecular , Software , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido NucleicoRESUMO
Serial crystallography (SX) involves combining observations from a very large number of diffraction patterns coming from crystals in random orientations. To compile a complete data set, these patterns must be indexed (i.e. their orientation determined), integrated and merged. Introduced here is TORO (Torch-powered robust optimization) Indexer, a robust and adaptable indexing algorithm developed using the PyTorch framework. TORO is capable of operating on graphics processing units (GPUs), central processing units (CPUs) and other hardware accelerators supported by PyTorch, ensuring compatibility with a wide variety of computational setups. In tests, TORO outpaces existing solutions, indexing thousands of frames per second when running on GPUs, which positions it as an attractive candidate to produce real-time indexing and user feedback. The algorithm streamlines some of the ideas introduced by previous indexers like DIALS real-space grid search [Gildea, Waterman, Parkhurst, Axford, Sutton, Stuart, Sauter, Evans & Winter (2014). Acta Cryst. D70, 2652-2666] and XGandalf [Gevorkov, Yefanov, Barty, White, Mariani, Brehm, Tolstikova, Grigat & Chapman (2019). Acta Cryst. A75, 694-704] and refines them using faster and principled robust optimization techniques which result in a concise code base consisting of less than 500 lines. On the basis of evaluations across four proteins, TORO consistently matches, and in certain instances outperforms, established algorithms such as XGandalf and MOSFLM [Powell (1999). Acta Cryst. D55, 1690-1695], occasionally amplifying the quality of the consolidated data while achieving superior indexing speed. The inherent modularity of TORO and the versatility of PyTorch code bases facilitate its deployment into a wide array of architectures, software platforms and bespoke applications, highlighting its prospective significance in SX.
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Serial and time-resolved macromolecular crystallography are on the rise. However, beam time at X-ray free-electron lasers is limited and most third-generation synchrotron-based macromolecular crystallography beamlines do not offer the necessary infrastructure yet. Here, a new setup is demonstrated, based on the JUNGFRAU detector and Jungfraujoch data-acquisition system, that enables collection of kilohertz serial crystallography data at fourth-generation synchrotrons. More importantly, it is shown that this setup is capable of collecting multiple-time-point time-resolved protein dynamics at kilohertz rates, allowing the probing of microsecond to second dynamics at synchrotrons in a fraction of the time needed previously. A high-quality complete X-ray dataset was obtained within 1â min from lysozyme microcrystals, and the dynamics of the light-driven sodium-pump membrane protein KR2 with a time resolution of 1â ms could be demonstrated. To make the setup more accessible for researchers, downstream data handling and analysis will be automated to allow on-the-fly spot finding and indexing, as well as data processing.
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Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15â Da fragment, undergoes binding-mode changes in endothiapepsin. A surprising fragment-binding discrepancy was observed between the cryo-cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure-based drug discovery.
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Proteínas , Ácido Aspártico Endopeptidases , Cristalografia por Raios X , Ligantes , Substâncias Macromoleculares , Proteínas/química , TemperaturaRESUMO
In this paper, we present a data workflow developed to operate the adJUstiNg Gain detector FoR the Aramis User station (JUNGFRAU) adaptive gain charge integrating pixel-array detectors at macromolecular crystallography beamlines. We summarize current achievements for operating at 9 GB/s data-rate a JUNGFRAU with 4 Mpixel at 1.1 kHz frame-rate and preparations to operate at 46 GB/s data-rate a JUNGFRAU with 10 Mpixel at 2.2 kHz in the future. In this context, we highlight the challenges for computer architecture and how these challenges can be addressed with innovative hardware including IBM POWER9 servers and field-programmable gate arrays. We discuss also data science challenges, showing the effect of rounding and lossy compression schemes on the MX JUNGFRAU detector images.
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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.
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Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for de novo protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography (SFX) data. In this work, sensitive anomalous data-quality indicators and model proteins were used to quantify improvements in native-SAD at XFELs such as utilization of longer wavelengths, careful experimental geometry optimization, and better post-refinement and partiality correction. Compared with studies using shorter wavelengths at other XFELs and older software versions, up to one order of magnitude reduction in the required number of indexed images for native-SAD was achieved, hence lowering sample consumption and beam-time requirements significantly. Improved data quality and higher anomalous signal facilitate so-far underutilized de novo structure determination of challenging proteins at XFELs. Improvements presented in this work can be used in other types of SFX experiments that require accurate measurements of weak signals, for example time-resolved studies.
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Explicitly correlated Gaussian functions with floating centers have been employed to recalculate the ground state potential energy surface (PES) of the H(3) (+) ion with much higher accuracy than it was done before. The nonlinear parameters of the Gaussians (i.e., the exponents and the centers) have been variationally optimized with a procedure employing the analytical gradient of the energy with respect to these parameters. The basis sets for calculating new PES points were guessed from the points already calculated. This allowed us to considerably speed up the calculations and achieve very high accuracy of the results.
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Trítio/química , Sensibilidade e Especificidade , Propriedades de SuperfícieRESUMO
Very accurate variational calculations of all rotationless states (also called pure vibrational states) of the HD molecule have been performed within the framework that does not assume the Born-Oppenheimer (BO) approximation. The non-BO wave functions of the states describing the internal motion of the proton, the deuteron, and the two electrons were expanded in terms of one-center explicitly correlated Gaussian functions multiplied by even powers of the internuclear distance. Up to 6000 functions were used for each state. Both linear and nonlinear parameters of the wave functions of all 18 states were optimized with a procedure that employs the analytical gradient of the energy with respect to the nonlinear parameters of the Gaussians. These wave functions were used to calculate expectation values of the interparticle distances and some other related quantities. The results allow elucidation of the charge asymmetry in HD as a function of the vibrational excitation.
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Bacterial 5' untranslated regions of mRNA, termed thermal switches or thermometers, change their structure in response to temperature change. This structural change provides for the regulation of gene expression. One of such thermal switches, called fourU, is present in the Salmonella species. Mutations of fourU were found to abrogate its regulatory properties. We investigated the thermodynamics of the fourU fragment responsible for its structural changes. All-atom molecular dynamics simulations at various temperatures and spectroscopic experiments in solution were performed for the wild-type fourU and its mutants. We found that the U11C and A8C mutations stabilize the fourU structure in comparison to the wild-type fourU, and the double-point G14A/C25U mutant has the most destabilizing effect on the fourU hairpin 2 responsible for temperature sensing. The G14A/C25U mutant is also the easiest to strand-invade by a complementary oligonucleotide as indicated by fluorescence spectroscopy experiments.
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Simulação de Dinâmica Molecular , Dobramento de RNA , RNA Bacteriano/química , Riboswitch , Salmonella/químicaRESUMO
Native single-wavelength anomalous dispersion (SAD) is an attractive experimental phasing technique as it exploits weak anomalous signals from intrinsic light scatterers (Z < 20). The anomalous signal of sulfur in particular, is enhanced at long wavelengths, however the absorption of diffracted X-rays owing to the crystal, the sample support and air affects the recorded intensities. Thereby, the optimal measurable anomalous signals primarily depend on the counterplay of the absorption and the anomalous scattering factor at a given X-ray wavelength. Here, the benefit of using a wavelength of 2.7 over 1.9â Å is demonstrated for native-SAD phasing on a 266â kDa multiprotein-ligand tubulin complex (T2R-TTL) and is applied in the structure determination of an 86â kDa helicase Sen1 protein at beamline BL-1A of the KEK Photon Factory, Japan. Furthermore, X-ray absorption at long wavelengths was controlled by shaping a lysozyme crystal into spheres of defined thicknesses using a deep-UV laser, and a systematic comparison between wavelengths of 2.7 and 3.3â Å is reported for native SAD. The potential of laser-shaping technology and other challenges for an optimized native-SAD experiment at wavelengths >3â Å are discussed.
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A combined computational docking-molecular dynamics study has been performed on a system consisting of cytochrome c protein and alkanethiol self-assembled monolayers of various geometries. The results suggest that the orientation of the protein on the surface may be controlled or altered by means of designing specific structural motifs on the surface. The proposed computational approach may be used as a fast and reliable tool to complement other theoretical and experimental techniques of exploring other protein-surface interfaces.