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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Robust error calibration for serial crystallography

Serial crystallography is an important technique with unique abilities to resolve enzymatic transition states, minimize radiation damage to sensitive metalloenzymes and perform de novo structure determination from micrometre-sized crystals. This technique requires the merging of data from thousands of crystals, making manual identification of errant crystals unfeasible. cctbx.xfel.merge uses filtering to remove problematic data. However, this process is imperfect, and data reduction must be robust to outliers. We add robustness to cctbx.xfel.merge at the step of uncertainty determination for reflection intensities. This step is a critical point for robustness because it is the first step where the data sets are considered as a whole, as opposed to individual lattices. Robustness is conferred by reformulating the error-calibration procedure to have fewer and less stringent statistical assumptions and incorporating the ability to down-weight low-quality lattices. We then apply this method to five macromolecular XFEL data sets and observe the improvements to each. The appropriateness of the intensity uncertainties is demonstrated through internal consistency. This is performed through theoretical CC 1/2 and I /σ relationships and by weighted second moments, which use Wilson's prior to connect intensity uncertainties with their expected distribution. This work presents new mathematical tools to analyze intensity statistics and demonstrates their effectiveness through the often underappreciated process of uncertainty analysis.

Mittan-Moreau, David W.↗
DOE OSTI2025

Real-time data processing for serial crystallography experiments

We report the use of streaming data interfaces to perform fully online data processing for serial crystallography experiments, without storing intermediate data on disk. The system produces Bragg reflection intensity measurements suitable for scaling and merging, with a latency of less than 1 s per frame. Our system uses the CrystFEL software in combination with the ASAP::O data framework. In a series of user experiments at PETRA III, frames from a 16 megapixel Dectris EIGER2 X detector were searched for peaks, indexed and integrated at the maximum full-frame readout speed of 133 frames per second. The computational resources required depend on various factors, most significantly the fraction of non-blank frames ('hits'). The average single-thread processing time per frame was 242 ms for blank frames and 455 ms for hits, meaning that a single 96-core computing node was sufficient to keep up with the data, with ample headroom for unexpected throughput reductions. Further significant improvements are expected, for example by binning pixel intensities together to reduce the pixel count. We discuss the implications of real-time data processing on the `data deluge' problem from recent and future photon-science experiments, in particular on calibration requirements, computing access patterns and the need for the preservation of raw data.

47 OTHER INSTRUMENTATION↗
DOE OSTI2024

Elucidating metal–organic framework structures using synchrotron serial crystallography

Metal organic frameworks (MOFs) are porous crystalline materials that display a wide variety of physical and chemical properties. Their single crystal structure determination is often challenging because in most cases micro- or nano-sized crystals spontaneously form upon MOF synthesis, which cannot be recrystallized. The production of larger single crystals for structure determination involves optimizing, and thus modifying, the conditions of synthesis, in which success cannot be guaranteed. Failure to produce crystals suitable for single-crystal X-ray diffraction leaves the 3D structure of the MOF compound unknown, and scientists must resort to more challenging structure solution methods based on X-ray powder or electron diffraction data. These laborious tasks can be avoided by using serial crystallography techniques which merge data collected on many micro-crystals. Here, we report the application of three synchrotron serial crystallography methods. We call these “mesh”, “grid” and “mesh&collect” scans. “Still” images (no rotation) are collected in the mesh scan approach, whereas small rotational wedges are collected in the grid scan method. The third protocol, mesh&collect, combines the acquisition of still images and rotational wedges. Using these means, we determine the ab initio structure of benchmark MOFs, MIL-100(Fe) and ZIF-8, that differ largely in unit cell size. These methods are expected to be widely applicable and facilitate structure determination of many MOF microcrystalline systems.

36 MATERIALS SCIENCE↗
DOE OSTI2024

In situ counter-diffusion crystallization and long-term crystal preservation in microfluidic fixed targets for serial crystallography

Compared with batch and vapor diffusion methods, counter diffusion can generate larger and higher-quality protein crystals yielding improved diffraction data and higher-resolution structures. Typically, counter-diffusion experiments are conducted in elongated chambers, such as glass capillaries, and the crystals are either directly measured in the capillary or extracted and mounted at the X-ray beamline. Despite the advantages of counter-diffusion protein crystallization, there are few fixed-target devices that utilize counter diffusion for crystallization. In this article, different designs of user-friendly counter-diffusion chambers are presented which can be used to grow large protein crystals in a 2D polymer microfluidic fixed-target chip. Methods for rapid chip fabrication using commercially available thin-film materials such as Mylar, propylene and Kapton are also detailed. Rules of thumb are provided to tune the nucleation and crystal growth to meet users' needs while minimizing sample consumption. These designs provide a reliable approach to forming large crystals and maintaining their hydration for weeks and even months. This allows ample time to grow, select and preserve the best crystal batches before X-ray beam time. Importantly, the fixed-target microfluidic chip has a low background scatter and can be directly used at beamlines without any crystal handling, enabling crystal quality to be preserved. The approach is demonstrated with serial diffraction of photoactive yellow protein, yielding 1.32 Å resolution at room temperature. Fabrication of this standard microfluidic chip with commercially available thin films greatly simplifies fabrication and provides enhanced stability under vacuum. These advances will further broaden microfluidic fixed-target utilization by crystallographers.

Liu, Zhongrui↗
DOE OSTI2026

Detector alignment for X-ray crystallography using Millepede-II

I describe a method for accurately refining the geometrical parameters of segmented X-ray area detectors on the basis of serial crystallography data, using 'Millepede' – an algorithm created for a very similar problem in high-energy physics. The Millepede method for serial crystallography builds on the approach of Brewster et al. [Acta Cryst. (2018), D74, 877–894], in which the detector parameters are refined simultaneously with the parameters for each individual crystal. This accounts for the mutual dependency between the parameters and thereby avoids the bias and slow convergence problems that have afflicted older approaches in which the deviations between observed and calculated Bragg peak positions were taken directly as the updates for the detector panel positions. The Millepede method uses the special structure of the least-squares normal equations to reduce them to a much smaller form that can be solved very quickly, even compared with the sparse matrix methods used previously. This makes it practical to refine the detector geometry frequently and thereby maintain accurate calibration without specialized alignment campaigns. Tilts of detector panels out of the plane can be reliably refined, as can the overall distance of the detector in the beam direction. With a simulated test case, the new method produced panel shifts within 7% of the correct values with only one iteration, and produced almost exactly correct shifts after a second iteration. A simulated out-of-plane panel rotation was correctly determined to within 0.001°. Applied to experimental data from an X-ray free-electron laser, the method increased the indexable fraction of frames from 30% to 91% in a single iteration, and to 96% after two further iterations. Computing the geometry updates on the basis of 2060 crystals took only 0.819 s on desktop computing hardware, including the time taken to read the required data from disk. The scaling was found to be very close to linear for up to 100 980 sets of crystal parameters, which took only 78.2 s to process under the same conditions. The method has been applied as part of a real-time feedback system at a synchrotron radiation beamline, in which an out-of-plane detector tilt of 0.04° was detected and corrected. Possible further applications are also described here.

Millepede-II↗
DOE OSTI2025

Serial-femtosecond crystallography reveals how a phytochrome variant couples chromophore and protein structural changes

The photoreaction and commensurate structural changes of a chromophore within biological photoreceptors elicit conformational transitions of the protein promoting the switch between deactivated and activated states. We investigated how this coupling is achieved in a bacterial phytochrome variant, Agp2-PAiRFP2. Contrary to classical protein crystallography, which only allows probing (cryo-trapped) stable states, we have used time-resolved serial femtosecond x-ray crystallography (tr-SFX) and pump-probe techniques with various illumination and delay times with respect to photoexcitation of the parent Pfr state. Thus, structural data for seven time frames were sorted into groups of molecular events along the reaction coordinate. They range from chromophore isomerization to the formation of Meta-F, the intermediate that precedes the functional relevant secondary structure transition of the tongue. Structural data for the early events were used to calculate the photoisomerization pathway to complement the experimental data. Late events allow identifying the molecular switch that is linked to the intramolecular proton transfer as a prerequisite for the following structural transitions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Observation of early events in the photoactivation of Myxobacterial phytochrome using time-resolved serial femtosecond crystallography

Myxobacteria are non-photosynthetic, soil-dwelling bacteria distinguished by a multicellular stage in their life cycle known as fruiting bodies that are stimulated by light. Myxobacterial phytochromes are candidates for the perception of red-light. The mechanism how light is perceived and converted to a physiological response is unknown. Here, time-resolved serial femtosecond crystallographic (TR-SFX) experiments were conducted on microcrystals of the photosensory core module of the Stigmatella aurantiaca bacteriophytochrome 2 (SaBphP2). Initial events of the Z to E isomerization reaction of the covalently bound, open-chain tetrapyrrole biliverdin (BV) chromophore were determined. At 3 ps after light activation, the BV ring-D assumes a configuration needed for the isomerization. At 100 ps, a mixture of BV in the Z or E configuration is observed in subunit A, while in the other subunit the chromophore remains in the Z configuration. In conjunction with prior results, these structures reveal the molecular mechanism of phytochrome activation in the photomorphogenesis of the myxobacteria and provide the molecular foundation for physiological responses to red light in other bacteria.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Structural Mechanism of an Efficacy Photoswitch Targeting the β 2 ‐adrenergic Receptor

The field of photopharmacology develops light-responsive drugs that can modulate protein activity, enabling precise and dynamic investigations of their roles in health and disease. Adrenergic receptors are prominent targets for this approach because they are prototypical G protein-coupled receptors with high clinical relevance in bronchial and cardiovascular diseases. Here, we employed the azobenzene-based compound photoazolol-1 in combination with time-resolved serial crystallography at X-ray free-electron lasers to resolve the molecular mechanisms by which photoswitchable β-blockers modulate activity of the β 2 -adrenoceptor (β 2 AR). Time-resolved structures of the receptor bound to trans-photoazolol-1 (pre-photoconversion), a strained intermediate in the nanosecond range, and the fully photoisomerized cis-photoazolol-1 reveal how isomerization of the azobenzene moiety induces distinct conformational changes within the orthosteric ligand binding pocket. Within seconds, light-excited photoazolol-1 adopts a new binding pose, altering interactions with extracellular loop 2 and shifting the positions of transmembrane helices 5, 6, and 7. Functional assays of β 2 AR in cellular membranes show that photoazolol-1 acts as an efficacy photoswitch, changing from an inverse agonist to a neutral antagonist upon isomerization without leaving the binding pocket. In combination, these findings suggest a molecular mechanism for activity modulation via efficacy photoswitches and provide a framework for designing ligands that exploit light-driven transitions within the binding pocket to achieve spatiotemporal control of receptor function.

G protein-coupled receptors↗
DOE OSTI2025

Scalable fabrication of an array-type fixed-target device for automated room temperature X-ray protein crystallography

X-ray crystallography is one of the leading tools to analyze the 3-D structure, and therefore, function of proteins and other biological macromolecules. Traditional methods of mounting individual crystals for X-ray diffraction analysis can be tedious and result in damage to fragile protein crystals. Furthermore, the advent of multi-crystal and serial crystallography methods explicitly require the mounting of larger numbers of crystals. To address this need, we have developed a device that facilitates the straightforward mounting of protein crystals for diffraction analysis, and that can be easily manufactured at scale. Inspired by grid-style devices that have been reported in the literature, we have developed an X-ray compatible microfluidic device that can be used to trap protein crystals in an array configuration, while also providing excellent optical transparency, a low X-ray background, and compatibility with the robotic sample handling and environmental controls used at synchrotron macromolecular crystallography beamlines. At the Stanford Synchrotron Radiation Lightsource (SSRL), these capabilities allow for fully remote-access data collection at controlled humidity conditions. Furthermore, we have demonstrated continuous manufacturing of these devices via roll-to-roll fabrication to enable cost-effective and efficient large-scale production.

chemical engineering↗
DOE OSTI2025

Probing substrate water access through the O1 channel of Photosystem II by single site mutations and membrane inlet mass spectrometry

Abstract Light-driven water oxidation by photosystem II sustains life on Earth by providing the electrons and protons for the reduction of CO 2 to carbohydrates and the molecular oxygen we breathe. The inorganic core of the oxygen evolving complex is made of the earth-abundant elements manganese, calcium and oxygen (Mn 4 CaO 5 cluster), and is situated in a binding pocket that is connected to the aqueous surrounding via water-filled channels that allow water intake and proton egress. Recent serial crystallography and infrared spectroscopy studies performed with PSII isolated fromThermosynechococcus vestitus(T. vestitus) support that one of these channels, the O1 channel, facilitates water access to the Mn 4 CaO 5 cluster during its S 2 →S 3 and S 3 →S 4 →S 0 state transitions, while a subsequent CryoEM study concluded that this channel is blocked in the cyanobacteriumSynechocystis sp.PCC 6803, questioning the role of the O1 channel in water delivery. Employing site-directed mutagenesis we modified the two O1 channel bottleneck residues D1-E329 and CP43-V410 (T. vestitusnumbering) and probed water access and substrate exchange via time resolved membrane inlet mass spectrometry. Our data demonstrates that water reaches the Mn 4 CaO 5 cluster via the O1 channel in both wildtype and mutant PSII. In addition, the detailed analysis provides functional insight into the intricate protein-water-cofactor network near the Mn 4 CaO 5 cluster that includes the pentameric, near planar ‘water wheel’ of the O1 channel.

Plant Sciences↗
DOE OSTI2025

Structural and Mechanistic Advances in the Chemistry of Methyl-Coenzyme M Reductase (MCR)

Methane represents 34% of U.S. energy consumption and is a major greenhouse gas related to the global carbon cycle and energy production. However, current industrial practices significantly increase atmospheric methane levels, necessitating a deeper understanding of its biosynthesis and oxidation. Methyl-coenzyme M reductase (MCR) is central to biological methane metabolism, catalyzing the final step of methanogenesis and the first step in anaerobic methane oxidation. It is also a key target for strategies to capture and transform methane into value-added chemicals.The active site of MCR is a buried Ni-based cofactor only accessible by the substrates via a 50 Å long tunnel. Although the Ni(I) state is required to initiate catalysis, capturing this state remains a challenge for the current structural techniques. Recent advances in structural biology using X-ray Free-Electron Laser serial crystallography have provided insights into MCR's inactive Ni(II) state at room temperature and show promise for capturing its active Ni(I) form.Our team has established several critical aspects of the MCR mechanism using a combination of experimental and computational studies. MCR uses CH 3 -SCoM and CoBSH as substrates, producing methane and a disulfide product CoMSSCoB. Kinetic analysis showed that productive substrate binding requires CH 3 -SCoM to bind first, inducing conformational changes that optimize the active site for subsequent CoBSH binding. Following substrate binding, four proposed methane production/oxidation mechanisms were examined, establishing whether the reaction proceeds through an organometallic methyl-nickel(III), methyl anion ion, or methyl radical intermediate. Experimental measurements using CoBSH analogs successfully slowed the reaction, allowing for mechanistic insight that demonstrated the methyl radical pathway, where the initial interactions involve homolytic cleavage of the methyl-sulfur bond, generating a methyl radical that quickly abstracts the thiol hydrogen atom of CoBSH to form methane. Computational studies further confirmed that, compared to other mechanisms, the methyl radical mechanism is thermodynamically more favorable and accessible under physiological conditions.Spectroscopic and computational studies challenged the conventional understanding of substrate binding in MCR by proposing an alternative positioning of CH 3 -SCoM and CoMSSCoB in the active site pocket. The research suggested that CH 3 -SCoM (substrate) and CoMSSCoB (product) bind via their sulfonate groups to the Ni(I) center of cofactor F 430 . This binding allows for the reaction without substrate reorganization in the pocket but would require a long-range electron transfer. Altogether, the work summarized in this review reflects our current understanding of the enzyme's catalytic mechanism and structural dynamics. This is essential for developing efficient methane conversion technologies that could mitigate its environmental impact while harnessing energy-storage properties.

03 NATURAL GAS↗
DOE OSTI2026

Structural basis of the promiscuity of the unusual Fe(II) and 2-oxoglutarate dependent human aspartate/asparagine-β-hydroxylase

Protein-hydroxylation catalysed by Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases is an important regulatory mechanism in human biology. Such oxygenases typically coordinate their Fe(II) cofactor via a conserved triad of an aspartate- or glutamate- and two histidine-residues. By contrast, aspartate/asparagine β-hydroxylase (AspH), which catalyses asparagine/aspartate-residue oxidation in epidermal growth factor-like domains (EGFDs), has only two histidine-residues (H679, H725), with a water occupying the site normally occupied by an aspartate- or glutamate-residue. We describe mechanistic studies with catalytically active AspH crystals. Turnover studies with single crystals under cryogenic conditions give (3 R)-hydroxylated EGFDs with the product alcohol coordinating Fe(II) trans to H725. Time-resolved serial crystallography of microcrystals using an acoustic droplet ejection system, coupled to X-ray emission analyses, demonstrate turnover within 1.5 s, giving a product complex in which Fe(II) is regenerated. Solution and crystallographic studies with the O 2 surrogate nitric oxide imply O 2 binds to Fe(II) trans to H725. The additional Fe-chelating water is maintained throughout AspH catalysis and is not directly involved in substrate hydroxylation, because O 2 is the sole oxygen source in alcohol products, as shown by 18 O labelling studies. The results reveal how AspH accommodates both aspartate- and asparagine-substrates and will assist in efforts targeting AspH for cancer treatment.

de Munnik, Mariska↗