Matching Items (8)
Description
Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.
ContributorsMunke, Anna (Author) / Andreasson, Jakob (Author) / Aquila, Andrew (Author) / Awel, Salah (Author) / Ayyer, Kartik (Author) / Barty, Anton (Author) / Bean, Richard J. (Author) / Berntsen, Peter (Author) / Bielecki, Johan (Author) / Boutet, Sebastien (Author) / Bucher, Maximilian (Author) / Chapman, Henry N. (Author) / Daurer, Benedikt J. (Author) / DeMirci, Hasan (Author) / Elser, Veit (Author) / Fromme, Petra (Author) / Hajdu, Janos (Author) / Hantke, Max F. (Author) / Higashiura, Akifumi (Author) / Hogue, Brenda (Author) / Hosseinizadeh, Ahmad (Author) / Kim, Yoonhee (Author) / Kirian, Richard (Author) / Reddy, Hemanth K. N. (Author) / Lan, Ti-Yen (Author) / Larsson, Daniel S. D. (Author) / Liu, Haiguang (Author) / Loh, N. Duane (Author) / Maia, Filipe R. N. C. (Author) / Mancuso, Adrian P. (Author) / Muhlig, Kerstin (Author) / Nakagawa, Atsushi (Author) / Nam, Daewoong (Author) / Nelson, Garrett (Author) / Nettelblad, Carl (Author) / Okamoto, Kenta (Author) / Ourmazd, Abbas (Author) / Rose, Max (Author) / van der Schot, Gijs (Author) / Schwander, Peter (Author) / Seibert, M. Marvin (Author) / Sellberg, Jonas A. (Author) / Sierra, Raymond G. (Author) / Song, Changyong (Author) / Svenda, Martin (Author) / Timneanu, Nicusor (Author) / Vartanyants, Ivan A. (Author) / Westphal, Daniel (Author) / Wiedom, Max O. (Author) / Williams, Garth J. (Author) / Xavier, Paulraj Lourdu (Author) / Soon, Chun Hong (Author) / Zook, James (Author) / College of Liberal Arts and Sciences (Contributor, Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / School of Life Sciences (Contributor) / Infectious Diseases and Vaccinology (Contributor) / Department of Physics (Contributor)
Created2016-08-01
Description
Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. In this study we report our high-resolution NMR results for a transmembrane protein, outer envelope protein of molar mass 16 kDa (OEP16), an amino acid transporter from the outer membrane of chloroplasts. Three-dimensional, high-resolution NMR experiments on the [superscript 13]C, [superscript 15]N, [superscript 2]H-triply-labeled protein were used to assign protein backbone resonances and to obtain secondary structure information. The results yield over 95% assignment of N, H[subscript N], CO, C[subscript α], and C[subscript β] chemical shifts, which is essential for obtaining a high resolution structure from NMR data. Chemical shift analysis from the assignment data reveals experimental evidence for the first time on the location of the secondary structure elements on a per residue basis. In addition T[subscript 1Z] and T[subscript 2] relaxation experiments were performed in order to better understand the protein dynamics. Arginine titration experiments yield an insight into the amino acid residues responsible for protein transporter function. The results provide the necessary basis for high-resolution structural determination of this important plant membrane protein.
ContributorsZook, James (Author) / Molugu, Trivikram R. (Author) / Jacobsen, Neil E. (Author) / Lin, Guangxin (Author) / Soll, Jurgen (Author) / Cherry, Brian (Author) / Brown, Michael F. (Author) / Fromme, Petra (Author) / Department of Chemistry and Biochemistry (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor)
Created2013-10-29
Description
Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.
ContributorsEdlund, Petra (Author) / Takala, Heikki (Author) / Claesson, Elin (Author) / Henry, Leocadie (Author) / Dods, Robert (Author) / Lehtivuori, Heli (Author) / Panman, Matthijs (Author) / Pande, Kanupriya (Author) / White, Thomas (Author) / Nakane, Takanori (Author) / Berntsson, Oskar (Author) / Gustavsson, Emil (Author) / Bath, Petra (Author) / Modi, Vaibhav (Author) / Roy Chowdhury, Shatabdi (Author) / Zook, James (Author) / Berntsen, Peter (Author) / Pandey, Suraj (Author) / Poudyal, Ishwor (Author) / Tenboer, Jason (Author) / Kupitz, Christopher (Author) / Barty, Anton (Author) / Fromme, Petra (Author) / Koralek, Jake D. (Author) / Tanaka, Tomoyuki (Author) / Spence, John (Author) / Liang, Mengning (Author) / Hunter, Mark S. (Author) / Boutet, Sebastien (Author) / Nango, Eriko (Author) / Moffat, Keith (Author) / Groenhof, Gerrit (Author) / Ihalainen, Janne (Author) / Stojkovic, Emina A. (Author) / Schmidt, Marius (Author) / Westenhoff, Sebastian (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2016-10-19
Description
The ASU Compact X-ray Free Electron Laser (CXFEL) is a first of its kind instrument that will illuminate the processes of life and allow scientists to create more effective treatments for disease. The dimensions of the linear accelerator (LINAC) cavities must remain stable during operation, for a change in the geometry alters the standing wave microwave energy resonance within the cavities and leads to reflected rather than coupled and useful microwave energy to electric field coupling. This disturbs the electron bunch acceleration dynamics critical to the ultimate generation of x-ray pulses. Cooling water must be supplied to the electron generating RF-GUN, and linear accelerator (LINAC) structures at unique flowrate and temperature setpoints that are specific to the operating mode of the CXFEL. Design specifications for the water supply to the RF-GUN and three LINACs and were made for the nominal operating mode, which adds a 3 kW heat load to the water. To maintain steady cavity dimensions, water must be supplied to each device under test at 30.0 ºC ± 0.06 ºC. The flowrate of water must be 3.5 GPM to the RF-GUN and 2.5 GPM to each of the three LINACs with ± 0.01 GPM flowrate resolution. The primary function of the Dedicated-Precision Thermal Trim Unit (D-PTTU) is to control the flowrate and temperature of water supply to each device under test. A simplified model of the system was developed to select valves that would meet our design specifications for flowrate and temperature control. After using this model for valve selection, a detailed system model was created to simulate relevant coupled-domain physics of the integrated system. The detailed system model was used to determine the critical sensitivities of the system and will be used to optimize the performance of the system in the future. Before the detailed system model can be verified and tuned with experiments, the sensors were calibrated in an ice-bath to ensure the sensors measure accurate and precise values. During initial testing, the D-PTTU was able to achieve ± 0.02 ºC temperature resolution, which exceeds the design specification by a factor of three.
ContributorsGardeck, Alex John (Author) / Holl, Mark (Thesis director) / Smith, Dean (Committee member) / Department of Physics (Contributor) / Mechanical and Aerospace Engineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
The Compact X-ray Light Source is an x-ray source at ASU that allows scientists to study the structures and dynamics of matter on an atomic scale. The radio frequency system that provides the power to accelerate electrons in the Compact X-ray Light Source must operate with a high degree of precision. This thesis measures the precision with which that system performs.
ContributorsBabic, Gregory (Author) / Graves, William (Thesis director) / Kitchen, Jennifer (Committee member) / Holl, Mark (Committee member) / Barrett, The Honors College (Contributor) / Electrical Engineering Program (Contributor) / Department of Physics (Contributor)
Created2022-05
Description
Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5–20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A[subscript 2A] adenosine receptor (A[subscript 2A]AR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A[subscript 2A]AR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A[subscript 2A]AR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5–20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.
ContributorsMartin Garcia, Jose Manuel (Author) / Conrad, Chelsie (Author) / Nelson, Garrett (Author) / Stander, Natasha (Author) / Zatsepin, Nadia (Author) / Zook, James (Author) / Zhu, Lan (Author) / Geiger, James (Author) / Chun, Eugene (Author) / Kissick, David (Author) / Hilgart, Mark C. (Author) / Ogata, Craig (Author) / Ishchenko, Andrii (Author) / Nagaratnam, Nirupa (Author) / Roy Chowdhury, Shatabdi (Author) / Coe, Jesse (Author) / Subramanian, Ganesh (Author) / Schaffer, Alexander (Author) / James, Daniel (Author) / Ketwala, Gihan (Author) / Venugopalan, Nagarajan (Author) / Xu, Shenglan (Author) / Corcoran, Stephen (Author) / Ferguson, Dale (Author) / Weierstall, Uwe (Author) / Spence, John (Author) / Cherezov, Vadim (Author) / Fromme, Petra (Author) / Fischetti, Robert F. (Author) / Liu, Wei (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2017-05-24
Description
Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.
ContributorsKupitz, Christopher (Author) / Olmos, Jose L. (Author) / Holl, Mark (Author) / Tremblay, Lee (Author) / Pande, Kanupriya (Author) / Pandey, Suraj (Author) / Oberthur, Dominik (Author) / Hunter, Mark (Author) / Liang, Mengning (Author) / Aquila, Andrew (Author) / Tenboer, Jason (Author) / Calvey, George (Author) / Katz, Andrea (Author) / Chen, Yujie (Author) / Wiedorn, Max O. (Author) / Knoska, Juraj (Author) / Meents, Alke (Author) / Majriani, Valerio (Author) / Norwood, Tyler (Author) / Poudyal, Ishwor (Author) / Grant, Thomas (Author) / Miller, Mitchell D. (Author) / Xu, Weijun (Author) / Tolstikova, Aleksandra (Author) / Morgan, Andrew (Author) / Metz, Markus (Author) / Martin Garcia, Jose Manuel (Author) / Zook, James (Author) / Roy Chowdhury, Shatabdi (Author) / Coe, Jesse (Author) / Nagaratnam, Nirupa (Author) / Meza-Aguilar, Domingo (Author) / Fromme, Raimund (Author) / Basu, Shibom (Author) / Frank, Matthias (Author) / White, Thomas (Author) / Barty, Anton (Author) / Bajt, Sasa (Author) / Yefanov, Oleksandr (Author) / Chapman, Henry N. (Author) / Zatsepin, Nadia (Author) / Nelson, Garrett (Author) / Weierstall, Uwe (Author) / Spence, John (Author) / Schwander, Peter (Author) / Pollack, Lois (Author) / Fromme, Petra (Author) / Ourmazd, Abbas (Author) / Phillips, George N. (Author) / Schmidt, Marius (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor)
Created2016-12-15
Description
Serial femtosecond crystallography (SFX) has opened a new era in crystallography by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.
ContributorsConrad, Chelsie (Author) / Basu, Shibom (Author) / James, Daniel (Author) / Wang, Dingjie (Author) / Schaffer, Alexander (Author) / Roy Chowdhury, Shatabdi (Author) / Zatsepin, Nadia (Author) / Aquila, Andrew (Author) / Coe, Jesse (Author) / Gati, Cornelius (Author) / Hunter, Mark S. (Author) / Koglin, Jason E. (Author) / Kupitz, Christopher (Author) / Nelson, Garrett (Author) / Subramanian, Ganesh (Author) / White, Thomas A. (Author) / Zhao, Yun (Author) / Zook, James (Author) / Boutet, Sebastien (Author) / Cherezov, Vadim (Author) / Spence, John (Author) / Fromme, Raimund (Author) / Weierstall, Uwe (Author) / Fromme, Petra (Author) / Department of Chemistry and Biochemistry (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor)
Created2015-06-30