Matching Items (8)
Description
With the advent of the X-ray free-electron laser (XFEL), an opportunity has arisen to break the nexus between radiation dose and spatial resolution in diffractive imaging, by outrunning radiation damage altogether when using single X-ray pulses so brief that they terminate before atomic motion commences. This dissertation concerns the application of XFELs to biomolecular imaging in an effort to overcome the severe challenges associated with radiation damage and macroscopic protein crystal growth. The method of femtosecond protein nanocrystallography (fsPNX) is investigated, and a new method for extracting crystallographic structure factors is demonstrated on simulated data and on the first experimental fsPNX data obtained at an XFEL. Errors are assessed based on standard metrics familiar to the crystallography community. It is shown that resulting structure factors match the quality of those measured conventionally, at least to 9 angstrom resolution. A new method for ab-initio phasing of coherently-illuminated nanocrystals is then demonstrated on simulated data. The method of correlated fluctuation small-angle X-ray scattering (CFSAXS) is also investigated as an alternative route to biomolecular structure determination, without the use of crystals. It is demonstrated that, for a constrained two-dimensional geometry, a projection image of a single particle can be formed, ab-initio and without modeling parameters, from measured diffracted intensity correlations arising from disordered ensembles of identical particles illuminated simultaneously. The method is demonstrated experimentally, based on soft X-ray diffraction from disordered but identical nanoparticles, providing the first experimental proof-of-principle result. Finally, the fundamental limitations of CFSAXS is investigated through both theory and simulations. It is found that the signal-to-noise ratio (SNR) for CFSAXS data is essentially independent of the number of particles exposed in each diffraction pattern. The dependence of SNR on particle size and resolution is considered, and realistic estimates are made (with the inclusion of solvent scatter) of the SNR for protein solution scattering experiments utilizing an XFEL source.
ContributorsKirian, Richard A (Author) / Spence, John C. H. (Committee member) / Doak, R. Bruce (Committee member) / Weierstall, Uwe (Committee member) / Bennett, Peter (Committee member) / Treacy, Michael M. J. (Committee member) / Arizona State University (Publisher)
Created2011
Description
One of the most important issues in femtosecond free electron laser X-ray diraction is to reconstruct the 3D charge density of molecule from a mass of diraction snapshots. In order to determine the orientation of single molecule from diraction patterns, we rst determine the moments and products of inertia of this from 2D experiment data (diraction patterns or EM images to obtain the elements of the inertia tensor. If diraction patterns from uniformly random orientations or some preferred orientations are collected, the principal axes of the molecule can be extracted, together with the Euler angles which relate the principal axes of the molecule to the laboratory frame axes. This is achieved by nding the maximum and minimum values for the measured moments from many single-molecule patterns. Simulations for GroEL protein indicates that the calculation of the autocorrelation help eliminate the Poisson noise in Cryo- EM images and can make correct orientation determination. The eect of water jacket surrounding the protein molecule is studied based on molecular dynamics simulation result. The intensities from water and interference is found to suppress those from protein itself. A method is proposed and applied to the simulation data to show the possibility for it to overcome the water background problem. The scattering between Bragg re ections from nanocrystals is used to aid solution of the phase problem. We describe a method for reconstructing the charge density of a typical molecule within a single unit cell, if suciently nely-sampled diraction data are available from many nanocrystals of dierent sizes lying in the same orientations without knowledge of the distribution of particle size or requiring atomic-resolution data. Triple correlation of the diraction patterns are made use of to reconiii
ContributorsWang, Xiaoyu (Author) / Spence, John C.H. (Thesis advisor) / Schmidt, Kevin (Committee member) / Doak, R. Bruce (Committee member) / Weierstall, Uwe (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2011
Description
A time-dependent semiclassical formalism is developed for the theory of incoherentdiffractive imaging (IDI), an atomically-precise imaging technique based on the principles of
intensity interferometry. The technique is applied to image inner-shell X-ray fluorescence
from heavy atoms excited by the femtosecond pulses of an X-ray free-electron laser (XFEL).
Interference between emission from different atoms is expected when the XFEL pulse duration
is shorter than the fluorescence lifetime. Simulations for atoms at the vertices of a simple
icosahedral virus capsid are used to generate mock IDI diffraction patterns. These are then
used to reconstruct the geometry by phase retrieval of the intensity correlation function between
photons emitted independently from many different atoms at two different detector
pixels. The dependence of the intensity correlation function on fluorescence lifetime relative
to XFEL pulse duration is computed, and a simple expression for the visibility (or contrast)
of IDI speckle as well as an upper bound on the IDI signal-to-noise ratio are obtained as a
function of XFEL flux and lifetime. This indicates that compact XFELs, with reduced flux
but attosecond pulses, should be ideally suited to 3D, atomic-resolution mapping of heavy
atoms in materials science, chemistry, and biology. As IDI is a new technique, not much has
yet been written about it in the literature. The current theoretical and experimental results
are reviewed, including a discussion of signal-to-noise issues that have been raised regarding
the idea that IDI is suitable for structural biology.
ContributorsShevchuk, Andrew Stewart Hegeman (Author) / Kirian, Richard A (Thesis advisor) / Schmidt, Kevin E (Committee member) / Weierstall, Uwe (Committee member) / Graves, William S (Committee member) / Arizona State University (Publisher)
Created2022
Description
X-ray free electron lasers (XFELs) provide several orders of magnitude brighter x-rays than 3rd generation sources. However, the electron beamlines and undulator magnets required are on the scale of kilometers, costing billions of dollars with only a half dozen or so currently operating worldwide. One way to overcome these limitations is to prebunch the electron beam on the scale of the x-ray wavelength. In this paper one such scheme is discussed, which uses a nanopatterned grating called a dynamical beam stop. This uses diffraction from crystal planes of the etched portion of a grating to impart a transverse modulation which becomes a temporal modulation via an emittance exchange (EEX). To expand upon this topic, dynamical electron diffraction intensities for a 200 nm thick Si(001) unpatterned membrane are simulated via the multislice method and compared to experiment for various crystallographic orientations at MeV energies. From this as well as an analysis of the experimental inelastic plasmon diffuse scattering, it is determined that the optimal transverse modulation would be formed from a bright field image of the beam stop, with the nanopattern being etched all the way through the membrane. A model quantifying the quality of the modulation - the bunching factor - as a function of contrast and duty factor is formulated and the optimal modulation is determined analytically. A prototype beam stop is then imaged in a transmission electron microscope (TEM) at 200 KeV, with the measured bunching factor of 0.5 agreeing with the model and approaching a saturated XFEL. Using the angular spectrum method, it is determined that the spatial coherence of the MeV energy electron beam is insufficient for significant self-imaging to occur for gratings with pitches of hundreds of nanometers. Finally, the first-order EEX input requirements for the electron beam are examined in the transverse dimension as are newly proposed longitudinal requirements to compensate for lingering correlations between the initial and final longitudinal phase spaces.
ContributorsMalin, Lucas Earle (Author) / Graves, William S (Thesis advisor) / Kirian, Richard A (Committee member) / Smith, David J. (Committee member) / Spence, John C. H. (Committee member) / Arizona State University (Publisher)
Created2020
Description
Optical trapping schemes that exploit radiation forces, such as optical tweezers, are well understood and widely used to manipulate microparticles; however, these are typically effective only on short (sub-millimeter) length scales. In the past decade, manipulating micron sized objects over large distances (∼0.5 meters) using photophoretic forces has been experimentally established. Photophoresis, discovered by Ehrenhaft in the early twentieth century, is the force a small particle feels when exposed to radiation while immersed in a gas. The anisotropic heating caused by the radiation results in a net momentum transfer on one side with the surrounding gas. To date, there is no theoretical evaluation of the photophoretic force in the case of an arbitrary illumination profile (i.e. not a plane wave) incident on a dielectric sphere, starting from Maxwell’s equations. Such a treatment is needed for the case of recently published photophoretic particle manipulation schemes that utilize complicated wavefronts such as diverging Laguerre-Gaussian-Bessel beams. Here the equations needed to determine the expansion coefficients for electromagnetic fields when represented as a superposition of spherical harmonics are derived. The algorithm of Driscoll and Healy for the efficient numerical integration of functions on the 2-sphere is applied and validated with the plane wave, whose analytic expansion is known. The expansion coefficients of the incident field are related to the field inside the sphere, from which the distribution of heat deposition can be evaluated. The incident beam is also related to the scattered field, from which the scattering forces may be evaluated through the Maxwell stress tensor. In future work, these results will be combined with heat diffusion/convection simulations within the sphere and a surrounding gas to predict the total forces on the sphere, which will be compared against experimental observations that so far remain unexplained.
ContributorsAlvarez, Roberto Carlos (Author) / Camacho, Erika T (Thesis advisor) / Kirian, Richard A (Thesis advisor) / Espanol, Malena I (Committee member) / Arizona State University (Publisher)
Created2021
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 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
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 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
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 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