Matching Items (4)
130320-Thumbnail Image.png
Description

X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a

X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.

ContributorsLi, Xuanxuan (Author) / Chiu, Chun-Ya (Author) / Wang, Hsiang-Ju (Author) / Kassemeyer, Stephan (Author) / Botha, Sabine (Author) / Shoeman, Robert L. (Author) / Lawrence, Robert (Author) / Kupitz, Christopher (Author) / Kirian, Richard (Author) / James, Daniel (Author) / Wang, Dingjie (Author) / Nelson, Garrett (Author) / Messerschmidt, Marc (Author) / Boutet, Sebastien (Author) / Williams, Garth J. (Author) / Hartman, Elisabeth (Author) / Jafarpour, Aliakbar (Author) / Foucar, Lutz M. (Author) / Barty, Anton (Author) / Chapman, Henry (Author) / Liang, Mengning (Author) / Menzel, Andreas (Author) / Wang, Fenglin (Author) / Basu, Shibom (Author) / Fromme, Raimund (Author) / Doak, R. Bruce (Author) / Fromme, Petra (Author) / Weierstall, Uwe (Author) / Huang, Michael H. (Author) / Spence, John (Author) / Schlichting, Ilme (Author) / Hogue, Brenda (Author) / Liu, Haiguang (Author) / ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Department of Physics (Contributor) / School of Life Sciences (Contributor)
Created2017-04-11
130284-Thumbnail Image.png
Description
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.
ContributorsLee, Ho-Hsien (Author) / Cherni, Irene (Author) / Yu, HongQi (Author) / Fromme, Raimund (Author) / Doran, Jeffrey (Author) / Grotjohann, Ingo (Author) / Mittman, Michele (Author) / Basu, Shibom (Author) / Deb, Arpan (Author) / Dorner, Katerina (Author) / Aquila, Andrew (Author) / Barty, Anton (Author) / Boutet, Sebastien (Author) / Chapman, Henry N. (Author) / Doak, R. Bruce (Author) / Hunter, Mark (Author) / James, Daniel (Author) / Kirian, Richard (Author) / Kupitz, Christopher (Author) / Lawrence, Robert (Author) / Liu, Haiguang (Author) / Nass, Karol (Author) / Schlichting, Ilme (Author) / Schmidt, Kevin (Author) / Seibert, M. Marvin (Author) / Shoeman, Robert L. (Author) / Spence, John (Author) / Stellato, Francesco (Author) / Weierstall, Uwe (Author) / Williams, Garth J. (Author) / Yoon, Chun Hong (Author) / Wang, Dingjie (Author) / Zatsepin, Nadia (Author) / Hogue, Brenda (Author) / Matoba, Nobuyuki (Author) / Fromme, Petra (Author) / Mor, Tsafrir (Author) / ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy (Contributor) / Department of Chemistry and Biochemistry (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor) / Biodesign Institute (Contributor) / Infectious Diseases and Vaccinology (Contributor) / Department of Physics (Contributor)
Created2014-08-20
130303-Thumbnail Image.png
Description
Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has produced high-resolution, room temperature, time-resolved protein structures. We report preliminary SFX of Sindbis virus, an enveloped icosahedral RNA virus with ∼700 Å diameter. Microcrystals delivered in viscous agarose medium diffracted to ∼40 Å resolution. Small-angle diffuse X-ray scattering overlaid Bragg peaks and analysis

Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has produced high-resolution, room temperature, time-resolved protein structures. We report preliminary SFX of Sindbis virus, an enveloped icosahedral RNA virus with ∼700 Å diameter. Microcrystals delivered in viscous agarose medium diffracted to ∼40 Å resolution. Small-angle diffuse X-ray scattering overlaid Bragg peaks and analysis suggests this results from molecular transforms of individual particles. Viral proteins undergo structural changes during entry and infection, which could, in principle, be studied with SFX. This is an important step toward determining room temperature structures from virus microcrystals that may enable time-resolved studies of enveloped viruses.
ContributorsLawrence, Robert (Author) / Conrad, Chelsie (Author) / Zatsepin, Nadia (Author) / Grant, Thomas D. (Author) / Liu, Haiguang (Author) / James, Daniel (Author) / Nelson, Garrett (Author) / Subramanian, Ganesh (Author) / Aquila, Andrew (Author) / Hunter, Mark S. (Author) / Liang, Mengning (Author) / Boutet, Sebastien (Author) / Coe, Jesse (Author) / Spence, John (Author) / Weierstall, Uwe (Author) / Liu, Wei (Author) / Fromme, Petra (Author) / Cherezov, Vadim (Author) / Hogue, Brenda (Author) / Biodesign Institute (Contributor) / Infectious Diseases and Vaccinology (Contributor) / Applied Structural Discovery (Contributor) / Department of Chemistry and Biochemistry (Contributor) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / School of Life Sciences (Contributor)
Created2015-08-20
129192-Thumbnail Image.png
Description

Membrane proteins are key players in biological systems, mediating signalling events and the specific transport of e.g. ions and metabolites. Consequently, membrane proteins are targeted by a large number of currently approved drugs. Understanding their functions and molecular mechanisms is greatly dependent on structural information, not least on complexes with

Membrane proteins are key players in biological systems, mediating signalling events and the specific transport of e.g. ions and metabolites. Consequently, membrane proteins are targeted by a large number of currently approved drugs. Understanding their functions and molecular mechanisms is greatly dependent on structural information, not least on complexes with functionally or medically important ligands. Structure determination, however, is hampered by the difficulty of obtaining well diffracting, macroscopic crystals. Here, the feasibility of X-ray free-electron-laser-based serial femtosecond crystallography (SFX) for the structure determination of membrane protein-ligand complexes using microcrystals of various native-source and recombinant P-type ATPase complexes is demonstrated. The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate. By analyzing the resolution dependence of ligand densities and overall model qualities, SFX data quality metrics as well as suitable refinement procedures are discussed. Even at relatively low resolution and multiplicity, the identification of ligands can be demonstrated. This makes SFX a useful tool for ligand screening and thus for unravelling the molecular mechanisms of biologically active proteins.

ContributorsBublitz, Maike (Author) / Nass, Karol (Author) / Drachmann, Nikolaj D. (Author) / Markvardsen, Anders J. (Author) / Gutmann, Matthias J. (Author) / Barends, Thomas R. M. (Author) / Mattle, Daniel (Author) / Shoeman, Robert L. (Author) / Doak, R. Bruce (Author) / Boutet, Sebastien (Author) / Messerschmidt, Marc (Author) / Seibert, M. Marvin (Author) / Williams, Garth J. (Author) / Foucar, Lutz (Author) / Reinhard, Linda (Author) / Sitsel, Oleg (Author) / Gregersen, Jonas L. (Author) / Clausen, Johannes D. (Author) / Boesen, Thomas (Author) / Gotfryd, Kamil (Author) / Wang, Kai-Tuo (Author) / Olesen, Claus (Author) / Moller, Jesper V. (Author) / Nissen, Poul (Author) / Schlichting, Ilme (Author) / College of Liberal Arts and Sciences (Contributor)
Created2015-07-01