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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 217 records · Page 12
All sourcesNASA NTRSDOE OSTI
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 OSTI2015

High-density grids for efficient data collection from multiple crystals

Higher throughput methods to mount and collect data from multiple small and radiation-sensitive crystals are important to support challenging structural investigations using microfocus synchrotron beamlines. Furthermore, efficient sample-delivery methods are essential to carry out productive femtosecond crystallography experiments at X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high-density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer-based sample delivery at synchrotron and XFEL sources. A single grid contains 75 mounting ports and fits inside an SSRL cassette or uni-puck storage container. The use of grids with an SSRL cassette expands the cassette capacity up to 7200 samples. Grids may also be covered with a polymer film or sleeve for efficient room-temperature data collection from multiple samples. New automated routines have been incorporated into the Blu-Ice / DCSS experimental control system to support grids, including semi-automated grid alignment, fully automated positioning of grid ports, rastering and automated data collection. Specialized tools have been developed to support crystallization experiments on grids, including a universal adaptor, which allows grids to be filled by commercial liquid-handling robots, as well as incubation chambers, which support vapor-diffusion and lipidic cubic phase crystallization experiments. Experiments in which crystals were loaded into grids or grown on grids using liquid-handling robots and incubation chambers are described. As a result, crystals were screened at LCLS-XPP and SSRL BL12-2 at room temperature and cryogenic temperatures.

59 BASIC BIOLOGICAL SCIENCES↗
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.↗
DOE OSTI2020

Beyond integration: modeling every pixel to obtain better structure factors from stills

Most crystallographic data processing methods use pixel integration. In serial femtosecond crystallography (SFX), the intricate interaction between the reciprocal lattice point and the Ewald sphere is integrated out by averaging symmetrically equivalent observations recorded across a large number (10 4 -10 6 ) of exposures. Although sufficient for generating biological insights, this approach converges slowly, and using it to accurately measure anomalous differences has proved difficult. This report presents a novel approach for increasing the accuracy of structure factors obtained from SFX data. A physical model describing all observed pixels is defined to a degree of complexity such that it can decouple the various contributions to the pixel intensities. Model dependencies include lattice orientation, unit-cell dimensions, mosaic structure, incident photon spectra and structure factor amplitudes. Maximum likelihood estimation is used to optimize all model parameters. The application of prior knowledge that structure factor amplitudes are positive quantities is included in the form of a reparameterization. The method is tested using a synthesized SFX dataset of ytterbium(III) lysozyme, where each X-ray laser pulse energy is centered at 9034 eV. This energy is 100 eV above the Yb 3+ L -III absorption edge, so the anomalous difference signal is stable at 10 electrons despite the inherent energy jitter of each femtosecond X-ray laser pulse. This work demonstrates that this approach allows the determination of anomalous structure factors with very high accuracy while requiring an order-of-magnitude fewer shots than conventional integration-based methods would require to achieve similar results.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Low- Z polymer sample supports for fixed-target serial femtosecond X-ray crystallography

X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introduction via a translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low- Z plastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. In conclusion, the benefits and limitations of these low- Z fixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature

Here, cytochrome c oxidase (C c O), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine C c O (bC c O) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe–C–O angle of ~142°. In contrast, in the synchrotron structure, the Fe–CO bond is cleaved; CO relocates to a new site near Cu B , which, in turn, moves closer to the heme a 3 iron by ~0.38 Å. Structural comparison reveals that ligand binding to the heme a 3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a 3 , thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2018

X-ray Emission Spectroscopy as an in Situ Diagnostic Tool for X-ray Crystallography of Metalloproteins Using an X-ray Free-Electron Laser

Serial femtosecond crystallography (SFX) using the ultrashort X-ray pulses from a X-ray free-electron laser (XFEL) provides a new way of collecting structural data at room temperature that allows for following the reaction in real time after initiation. XFEL experiments are conducted in a shot-by-shot mode as the sample is destroyed and replenished after each X-ray pulse, and therefore, monitoring and controlling the data quality by using in situ diagnostic tools is critical. To study metalloenzymes, we developed the use of simultaneous collection of X-ray diffraction of crystals along with X-ray emission spectroscopy (XES) data that is used as a diagnostic tool for crystallography, by monitoring the chemical state of the metal catalytic center. We have optimized data analysis methods and sample delivery techniques for fast and active feedback to ensure the quality of each batch of samples and the turnover of the catalytic reaction caused by reaction triggering methods. Here, we describe this active in situ feedback system using Photosystem II as an example that catalyzes the oxidation of H2O to O2at the Mn4CaO5active site. We used the first moments of the Mn Kβ1,3emission spectra, which are sensitive to the oxidation state of Mn, as the primary diagnostics. This approach is applicable to different metalloproteins to determine the integrity of samples and follow changes in the chemical states of the reaction that can be initiated by light or activated by substrates and offers a metric for determining the diffraction images that are used for the final data sets.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Radiation Damage in XFEL: Case study from the oxygen-evolving complex of Photosystem II

Structural changes induced by radiation damage in X-ray crystallography hinder the ability to understand the structure/function relationship in chemical reactions. Serial femtosecond crystallography overcomes this problem by exposing the sample to very short and intense laser pulse leading to measurement before destruction. Here we use molecular modeling to map the radiation damage during the 10–50 fs to the intensity, the energy and the time duration of the laser pulse on the oxygen-evolving complex (OEC) of photosystem II. In the model, the nuclei move classically in a fully quantum potential created by electron density under the effect of strong laser pulse in the Ehrenfest dynamics regime. The results show that the Mn-Mn and Mn-Ca distances are less affected by radiation damage due to the their heavy masses, while one μ-oxo bridge (O5) moves significantly. The radiation damage may induce conformational changes of the water ligands but only bond elongation for the amino acids ligands. These effects are relatively intensity independent from 10 16 to 10 17 W/cm 2 , but changes increase dramatically if the beam intensity is increased to 10 18 W/cm 2 . Finally, in addition, the self amplified spontaneous emission (SASE) nature of the laser beam does not affect the dynamics of the ions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography

Since early 2020, COVID-19 has grown to affect the lives of billions globally. A worldwide investigation has been ongoing for characterizing the virus and also for finding an effective drug and developing vaccines. As time has been of the essence, a crucial part of this research has been drug repurposing; therefore, confirmation of in silico drug screening studies have been carried out for this purpose. Here we demonstrated the possibility of screening a variety of drugs efficiently by leveraging a high data collection rate of 120 images/second with the new low-noise, high dynamic range ePix10k2M Pixel Array Detector installed at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS). The X-ray Free-Electron Laser (XFEL) is used for remote high-throughput data collection for drug repurposing of the main protease (Mpro) of SARS-CoV-2 at ambient temperature with mitigated X-ray radiation damage. We obtained multiple structures soaked with nine drug candidate molecules in two crystal forms. Although our drug binding attempts failed, we successfully established a high-throughput Serial Femtosecond X-ray crystallographic (SFX) data collection protocol.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Characterization and use of the spent beam for serial operation of LCLS

X-ray free-electron laser sources such as the Linac Coherent Light Source offer very exciting possibilities for unique research. However, beam time at such facilities is very limited and in high demand. This has led to significant efforts towards beam multiplexing of various forms. One such effort involves re-using the so-called spent beam that passes through the hole in an area detector after a weak interaction with a primary sample. This beam can be refocused into a secondary interaction region and used for a second, independent experiment operating in series. The beam profile of this refocused beam was characterized for a particular experimental geometry at the Coherent X-ray Imaging instrument at LCLS. A demonstration of this multiplexing capability was performed with two simultaneous serial femtosecond crystallography experiments, both yielding interpretable data of sufficient quality to produce electron density maps.

43 PARTICLE ACCELERATORS↗
DOE OSTI2020

Ultrafast structural response to charge redistribution within a photosinthetic reaction centre

This data set contains stream files, difference maps and SVD maps obtained from data processing of time-resolved serial femtosecond crystallography measurements performed at LCLS XFEL, used to construct the images and perform the analysis published in the paper "Ultrafast structural response to charge redistribution within a photosinthetic reaction centre". Two (2) time points plus a dark state from 2015 ('a' label) as well as five (5) time points plus a dark state from 2016 ('b' label) are provided.

CXI↗
DOE OSTI2020

Mechanism and dynamics of fatty acid photodecarboxylase

The deposition contains diffraction patterns after hit-finding of time-resolved serial femtosecond crystallography measurements on fatty acid photodecarboxylase performed at LCLS XFEL (proposal ID: LT59). Five datasets are available. One dataset without pump laser excitation, labeled as "dark" and four time-resolved datasets with pump laser excitation at four different time-points labeled as "20 ps", "900 ps", "300 ns" and "2 μs". Please check the publication for more information about the datasets.

CXI↗
DOE OSTI2022

Structural basis for receptor selectivity and inverse agonism in S1P5 receptors

This dataset contains a subset of raw diffraction images for the serial femtosecond crystallography data of S1P5-ONO-5430608 complex. The diffraction data contain a strong systematic background as described in Lyapina et al. No additional background subtraction was applied to the data. In order to avoid its effects, the background subtraction implemented in CrystFEL, was applied (--rings-grad subtraction). The final stream, supplemented with this dataset, was obtained with: indexamajig 0.8.0 Respectively, the hkl file was obtained with: partialator 0.8.0

Human sphingosine-1-phosphate receptor 5 (S1P5) in↗
DOE OSTI2019

Toward G protein-coupled receptor structure-based drug design using X-ray lasers

Rational structure-based drug design (SBDD) relies on the availability of a large number of co-crystal structures to map the ligand-binding pocket of the target protein and use this information for lead-compound optimization via an iterative process. While SBDD has proven successful for many drug-discovery projects, its application to G protein-coupled receptors (GPCRs) has been limited owing to extreme difficulties with their crystallization. Here, a method is presented for the rapid determination of multiple co-crystal structures for a target GPCR in complex with various ligands, taking advantage of the serial femtosecond crystallography approach, which obviates the need for large crystals and requires only submilligram quantities of purified protein. The method was applied to the human β 2 -adrenergic receptor, resulting in eight room-temperature co-crystal structures with six different ligands, including previously unreported structures with carvedilol and propranolol. The generality of the proposed method was tested with three other receptors. This approach has the potential to enable SBDD for GPCRs and other difficult-to-crystallize membrane proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Towards phasing using high X-ray intensity

X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography. The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential `bleaching' of heavy atoms. This paper investigates the effects of electronic damage on experimental data collected from a Gd derivative of lysozyme microcrystals at different X-ray intensities, and the degree of ionization of Gd atoms is quantified from phased difference Fourier maps. In conclusion, a pattern sorting scheme is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed.

74 ATOMIC AND MOLECULAR PHYSICS↗
DOE OSTI2016

Protein structure determination by single-wavelength anomalous diffraction phasing of X-ray free-electron laser data

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems that are prone to radiation damage. However, phasing XFEL data de novo is complicated by the inherent inaccuracy of SFX data, and only a few successful examples, mostly based on exceedingly strong anomalous or isomorphous difference signals, have been reported. Here, it is shown that SFX data from thaumatin microcrystals can be successfully phased using only the weak anomalous scattering from the endogenous S atoms. Furthermore, a step-by-step investigation is presented of the particular problems of SAD phasing of SFX data, analysing data from a derivative with a strong anomalous signal as well as the weak signal from endogenous S atoms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

On the release of cppxfel for processing X-ray free-electron laser images

As serial femtosecond crystallography expands towards a variety of delivery methods, including chip-based methods, and smaller collected data sets, the requirement to optimize the data analysis to produce maximum structure quality is becoming increasingly pressing. Here cppxfel , a software package primarily written in C++, which showcases several data analysis techniques, is released. This software package presently indexes images using DIALS (diffraction integration for advanced light sources) and performs an initial orientation matrix refinement, followed by post-refinement of individual images against a reference data set. Cppxfel is released with the hope that the unique and useful elements of this package can be repurposed for existing software packages. However, as released, it produces high-quality crystal structures and is therefore likely to be also useful to experienced users of X-ray free-electron laser (XFEL) software who wish to maximize the information extracted from a limited number of XFEL images.

97 MATHEMATICS AND COMPUTING↗