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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 145 records · Page 8
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Observations of phase changes in monoolein during high viscous injection

Serial crystallography of membrane proteins often employs high-viscosity injectors (HVIs) to deliver micrometre-sized crystals to the X-ray beam. Typically, the carrier medium is a lipidic cubic phase (LCP) media, which can also be used to nucleate and grow the crystals. However, despite the fact that the LCP is widely used with HVIs, the potential impact of the injection process on the LCP structure has not been reported and hence is not yet well understood. The self-assembled structure of the LCP can be affected by pressure, dehydration and temperature changes, all of which occur during continuous flow injection. These changes to the LCP structure may in turn impact the results of X-ray diffraction measurements from membrane protein crystals. To investigate the influence of HVIs on the structure of the LCP we conducted a study of the phase changes in monoolein/water and monoolein/buffer mixtures during continuous flow injection, at both atmospheric pressure and under vacuum. The reservoir pressure in the HVI was tracked to determine if there is any correlation with the phase behaviour of the LCP. The results indicated that, even though the reservoir pressure underwent (at times) significant variation, this did not appear to correlate with observed phase changes in the sample stream or correspond to shifts in the LCP lattice parameter. During vacuum injection, there was a three-way coexistence of the gyroid cubic phase, diamond cubic phase and lamellar phase. During injection at atmospheric pressure, the coexistence of a cubic phase and lamellar phase in the monoolein/water mixtures was also observed. The degree to which the lamellar phase is formed was found to be strongly dependent on the co-flowing gas conditions used to stabilize the LCP stream. A combination of laboratory-based optical polarization microscopy and simulation studies was used to investigate these observations.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗
DOE OSTI2021

The mechanism driving a solid–solid phase transition in a biomacromolecular crystal

A solid-solid phase transition (SSPT) occurs between distinguishable crystalline forms. Because of its importance in application and theory in material science and condensed matter physics, SSPT has been studied most extensively in metallic alloys, inorganic salt or small organic molecular crystals, but much less so in biomacromolecular crystals. In general, the mechanism of SSPT at atomic and molecular levels is not well understood. Here, we describe the ordered molecular rearrangements in biomacromolecular crystals of the adenine riboswitch (riboA) aptamer using real-time serial crystallography and solution atomic force microscopy (AFM). The large, ligand-induced conformational changes drive the initial phase transition from the apo unit cell (AUC) to the trans unit cell 1 (TUC1). During this transition, coaxial stacking of P1 duplexes becomes the dominant packing interface, whereas P2-P2 interactions are almost completely disrupted, resulting in “floating” layers of molecules. The coupling points in TUC1 and their local conformational flexibility allow the molecules to reorganize to achieve the more densely packed and energetically favorable bound unit cell (BUC). Our study thus reveals the interplay between the conformational changes and the crystal phases—the underlying mechanism that drives the phase transition. Using polarized video microscopy (PVM) to monitor the SSPT in small crystals at high ligand concentration, we have identified the time window during which the major conformational changes take place, and simulated the in crystallo kinetics. Together, these results provide the spatiotemporal information necessary for informing time-resolved crystallography (TRX) experiments. Moreover, this study illustrates a practical approach to characterize SSPT in transparent crystals.

36 MATERIALS SCIENCE↗
DOE OSTI2019

A space for lattice representation and clustering

Algorithms for quantifying the differences between two lattices are used for Bravais lattice determination, database lookup for unit cells to select candidates for molecular replacement, and recently for clustering to group together images from serial crystallography. It is particularly desirable for the differences between lattices to be computed as a perturbation-stable metric, i.e. as distances that satisfy the triangle inequality, so that standard tree-based nearest-neighbor algorithms can be used, and for which small changes in the lattices involved produce small changes in the distances computed. A perturbation-stable metric space related to the reduction algorithm of Selling and to the Bravais lattice determination methods of Delone is described. Two ways of representing the space, as six-dimensional real vectors or equivalently as three-dimensional complex vectors, are presented and applications of these metrics are discussed. (Note: in his later publications, Boris Delaunay used the Russian version of his surname, Delone.)

36 MATERIALS SCIENCE↗
DOE OSTI2023

An invertible seven-dimensional Dirichlet cell characterization of lattices

Characterization of crystallographic lattices is an important tool in structure solution, crystallographic database searches and clustering of diffraction images in serial crystallography. Characterization of lattices by Niggli-reduced cells (based on the three shortest non-coplanar lattice vectors) or by Delaunay-reduced cells (based on four non-coplanar vectors summing to zero and all meeting at obtuse or right angles) is commonly performed. The Niggli cell derives from Minkowski reduction. The Delaunay cell derives from Selling reduction. All are related to the Wigner–Seitz (or Dirichlet, or Voronoi) cell of the lattice, which consists of the points at least as close to a chosen lattice point as they are to any other lattice point. The three non-coplanar lattice vectors chosen are here called the Niggli-reduced cell edges. Starting from a Niggli-reduced cell, the Dirichlet cell is characterized by the planes determined by 13 lattice half-edges: the midpoints of the three Niggli cell edges, the six Niggli cell face-diagonals and the four body-diagonals, but seven of the lengths are sufficient: three edge lengths, the three shorter of each pair of face-diagonal lengths, and the shortest body-diagonal length. These seven are sufficient to recover the Niggli-reduced cell.

Chemistry↗
DOE OSTI2023

Determining biomolecular structures near room temperature using X-ray crystallography: concepts, methods and future optimization

For roughly two decades, cryocrystallography has been the overwhelmingly dominant method for determining high-resolution biomolecular structures. Competition from single-particle cryo-electron microscopy and micro-electron diffraction, increased interest in functionally relevant information that may be missing or corrupted in structures determined at cryogenic temperature, and interest in time-resolved studies of the biomolecular response to chemical and optical stimuli have driven renewed interest in data collection at room temperature and, more generally, at temperatures from the protein–solvent glass transition near 200 K to ∼350 K. Fischer has recently reviewed practical methods for room-temperature data collection and analysis [Fischer (2021), Q. Rev. Biophys. 54 , e1]. Here, the key advantages and physical principles of, and methods for, crystallographic data collection at noncryogenic temperatures and some factors relevant to interpreting the resulting data are discussed. For room-temperature data collection to realize its potential within the structural biology toolkit, streamlined and standardized methods for delivering crystals prepared in the home laboratory to the synchrotron and for automated handling and data collection, similar to those for cryocrystallography, should be implemented.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Deep residual networks for crystallography trained on synthetic data

The use of artificial intelligence to process diffraction images is challenged by the need to assemble large and precisely designed training data sets. To address this, a codebase called Resonet was developed for synthesizing diffraction data and training residual neural networks on these data. Here, two per-pattern capabilities of Resonet are demonstrated: (i) interpretation of crystal resolution and (ii) identification of overlapping lattices. Resonet was tested across a compilation of diffraction images from synchrotron experiments and X-ray free-electron laser experiments. Crucially, these models readily execute on graphics processing units and can thus significantly outperform conventional algorithms. While Resonet is currently utilized to provide real-time feedback for macromolecular crystallography users at the Stanford Synchrotron Radiation Lightsource, its simple Python-based interface makes it easy to embed in other processing frameworks. This work highlights the utility of physics-based simulation for training deep neural networks and lays the groundwork for the development of additional models to enhance diffraction collection and analysis.

36 MATERIALS SCIENCE↗
DOE OSTI2019

Structural isomers of the S 2 state in photosystem II: do they exist at room temperature and are they important for function?

In nature, an oxo‐bridged Mn 4 CaO 5 cluster embedded in photosystem II (PSII), a membrane‐bound multi‐subunit pigment protein complex, catalyzes the water oxidation reaction that is driven by light‐induced charge separations in the reaction center of PSII. The Mn 4 CaO 5 cluster accumulates four oxidizing equivalents to enable the four‐electron four‐proton catalysis of two water molecules to one dioxygen molecule and cycles through five intermediate S‐states, S 0 – S 4 in the Kok cycle. One important question related to the catalytic mechanism of the oxygen‐evolving complex (OEC) that remains is, whether structural isomers are present in some of the intermediate S‐states and if such equilibria are essential for the mechanism of the O‐O bond formation. Here we compare results from electron paramagnetic resonance (EPR) and X‐ray absorption spectroscopy (XAS) obtained at cryogenic temperatures for the S 2 state of PSII with structural data collected of the S 1 , S 2 and S 3 states by serial crystallography at neutral pH (∼6.5) using an X‐ray free electron laser at room temperature. While the cryogenic data show the presence of at least two structural forms of the S 2 state, the room temperature crystallography data can be well‐described by just one S 2 structure. We discuss the deviating results and outline experimental strategies for clarifying this mechanistically important question.

Chatterjee, Ruchira↗
DOE OSTI2020

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.

36 MATERIALS SCIENCE↗
DOE OSTI2018

Demonstration of femtosecond X-ray pump X-ray probe diffraction on protein crystals

The development of X-ray free-electron lasers (XFELs) has opened the possibility to investigate the ultrafast dynamics of biomacromolecules using X-ray diffraction. Whereas an increasing number of structures solved by means of serial femtosecond crystallography at XFELs is available, the effect of radiation damage on protein crystals during ultrafast exposures has remained an open question. We used a split-and-delay line based on diffractive X-ray optics at the Linac Coherent Light Source XFEL to investigate the time dependence of X-ray radiation damage to lysozyme crystals. For these tests, crystals were delivered to the X-ray beam using a fixed-target approach. The presented experiments provide probe signals at eight different delay times between 19 and 213 femtoseconds after a single pump event, thereby covering the time-scales relevant for femtosecond serial crystallography. Even though significant impact on the crystals was observed at long time scales after exposure with a single X-ray pulse, the collected diffraction data did not show significant signal reduction that could be assigned to beam damage on the crystals in the sampled time window and resolution range. This observation is in agreement with estimations of the applied radiation dose, which in our experiment was clearly below the values expected to cause damage on the femtosecond time scale. The experiments presented here demonstrate the feasibility of time-resolved pump-multiprobe X-ray diffraction experiments on protein crystals.

43 PARTICLE ACCELERATORS↗
DOE OSTI2019

The Macromolecular Femtosecond Crystallography Instrument at the Linac Coherent Light Source

The Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS) is the seventh and newest instrument at the world's first hard X-ray free-electron laser. It was designed with a primary focus on structural biology, employing the ultrafast pulses of X-rays from LCLS at atmospheric conditions to overcome radiation damage limitations in biological measurements. It is also capable of performing various time-resolved measurements. The MFX design consists of a versatile base system capable of supporting multiple methods, techniques and experimental endstations. The primary techniques supported are forward scattering and crystallography, with capabilities for various spectroscopic methods and time-resolved measurements. The location of the MFX instrument allows for utilization of multiplexing methods, increasing user access to LCLS by running multiple experiments simultaneously.

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗
DOE OSTI2025

Macromolecular crystallography and biology at the Linac Coherent Light Source

The Linac Coherent Light Source (LCLS) has significantly impacted the field of biology by providing advanced capabilities for probing the structure and dynamics of biological molecules with high precision. The ultrashort coherent X-ray pulses from the LCLS have enabled ultrafast, time-resolved, serial femtosecond crystallography that is inaccessible at conventional synchrotron light sources. Since the facility's founding, scientists have captured detailed insights into biological processes at atomic resolution and fundamental timescales. The ability to observe these processes in real time and under conditions closely resembling their natural state is transforming our approach to studying biochemical mechanisms and developing new medical and energy applications. This work recounts some of the history of the LCLS, advances in biological research enabled by the LCLS, key biological areas that have been impacted and how the LCLS has helped to unravel complex biological phenomena in these fields.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Filling data analysis gaps in time-resolved crystallography by machine learning

There is a growing understanding of the structural dynamics of biological molecules fueled by x-ray crystallography experiments. Time-resolved serial femtosecond crystallography (TR-SFX) with x-ray Free Electron Lasers allows the measurement of ultrafast structural changes in proteins. Nevertheless, this technique comes with some limitations. One major challenge is the quality of data from TR-SFX measurements, which often faces issues like data sparsity, partial recording of Bragg reflections, timing errors, and pixel noise. To overcome these difficulties, conventionally, large volumes of data are collected and grouped into a few temporal bins. The data in each bin are then averaged and paired with the mean of their corresponding jittered timestamps. This procedure provides one structure per bin, resulting in a limited number of averaged structures for the entire time interval spanned by the experiment. Therefore, the information on ultrafast structural dynamics at high temporal resolution is lost. This has initiated research for advanced methods of analyzing experimental TR-SFX data beyond the standard binning and averaging method. To address this problem, we use a machine learning algorithm called Nonlinear Laplacian Spectral Analysis (NLSA), which has emerged as a promising technique for studying the dynamics of complex systems. In this work, we demonstrate the power of this algorithm using synthetic x-ray diffraction snapshots from a protein with significant data incompleteness, timing uncertainties, and noise. Our study confirms that NLSA is a suitable approach that effectively mitigates the effects of these artifacts in TR-SFX data and recovers accurate structural dynamics information hidden in such data.

Trujillo, Justin (ORCID:0000000285505360)↗
DOE OSTI2022

Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature

Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser

NendoU from SARS-CoV-2 is responsible for the virus’s ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

The crystal structure of dGTPase reveals the molecular basis of dGTP selectivity

Deoxynucleotide triphosphohydrolases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools. While the vast majority of these enzymes display broad activity toward canonical dNTPs, such as the dNTPase SAMHD1 that blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, Escherichia coli ( Ec )-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric Ec- dGTPase, including an X-ray free-electron laser structure of the free Ec- -dGTPase enzyme to 3.2 Å. To obtain this structure, we developed a method that applied UV-fluorescence microscopy, video analysis, and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly located on fixed target holders, resulting in the highest indexing rates observed for a serial femtosecond crystallography experiment. Our structures show a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo- form. Moreover, despite no sequence homology, Ec- dGTPase and SAMHD1 share similar active-site and HD motif architectures; however, Ec- dGTPase residues at the end of the substrate-binding pocket mimic Watson–Crick interactions providing guanine base specificity, while a 7-Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures shed light on the mechanism by which long distance binding (25 Å) of single-stranded DNA in an allosteric site primes the active site by conformationally “opening” a tyrosine gate allowing enhanced substrate binding.

Barnes, Christopher O.↗