No description available.

Thesis (Ph. D. in Molecular Physiology and Biophysics)--Vanderbilt University, Aug. 2009. / Title from title screen. Includes bibliographical references.

Single-particle reconstruction technique is one of the major approaches to studying ribosome structure and membrane proteins. In this thesis, I report the use of time-resolved cryo-EM technique to study the structure of short-lived ribosome complexes and conventional cryo-EM technique to study the structure of ribosome complexes and membrane proteins. The thesis consists three parts. The first part is the development of time-resolved cryo-EM technique. I document the protocol for how to capture short-lived states of the molecules with time-resolved cryo-EM technique using microfluidic chip. Working closely with Dr. Lin’s lab at Columbia University Engineering Department, I designed and tested a well-controlled and effective microspraying-plunging method to prepare cryo-grids. I demonstrated the performance of this device by a 3-Å reconstruction from about 4000 particles collected on grids sprayed with apoferritin suspension. The second part is the application of time-resolved cryo-EM technique for studying short-lived ribosome complexes in bacteria translation processes on the time-scale of 10-1000 ms. I document three applications on bacterial translation processes. The initiation project is collaborated with Dr. Gonzalez’s lab at Chemistry Department, Columbia University. The termination and recycling projects are collaborated with Dr. Ehrenberg’s lab at Department of Cell and Molecular Biology, Uppsala University. I captured and solved short-lived ribosome intermediates complexes in these processes. The results demonstrate the power of time-resolved cryo-EM to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology. The last part is the application of conventional cryo-EM technique to study ribosome complexes and membrane proteins. This part includes five collaboration projects. Human GABA(B) receptor project is the collaboration with Dr. Fan at Department of Pharmacology, Columbia University. Cyclic nucleotide-gated (CNG) channels project is the collaboration with Dr. Yang at Department of Biological Sciences, Columbia University. The cryo-EM study of Ybit-70S ribosome complex and Cystic fibrosis transmembrane conductance regulator (CFTR) project are the collaboration with Dr. Hunt at Department of Biological Sciences, Columbia University. The cryo-EM study of native lipid bilayer in membrane protein transporter is the collaboration with Dr. Hendrickson at Department of Biochemistry and Molecular Biophysics, Columbia University and Dr. Guo at Department of Medicinal Chemistry, Virginia Commonwealth University.

Adenosine triphosphate (ATP) is the molecular currency of intracellular energy transfer in living organisms. The enzyme ATP synthase is primarily responsible for ATP production in eukaryotes. In archaea and some bacteria, ATP is synthesized by V-ATPase that is related to ATP synthase both in structure and function. Both of these enzymes are reversible rotary motors capable of catalyzing ATP synthesis or hydrolysis. The rotation of the central rotor, which is powered by the flow of proton (or sometimes sodium ion) down the electrochemical gradient through the membrane-bound Fo/Vo region, leads to the chemical synthesis of ATP in F1/V1 region. The F1/V1 region, on the other hand, can catalyze ATP hydrolysis, which in turn leads to proton (or sodium) pumping across the membrane through rotation of the central rotor in the opposite direction. This thesis describes structure determination of both the intact F-type and V-type enzymes using single particle electron cryomicroscopy (cryo-EM), with the aim of better understanding their overall architecture, subunit organization and the mechanism of proton translocation. Our cryo-EM structural analysis on the F-type ATP synthase from Saccharomyces cerevisiae uncovered the arrangement of subunits a, b, c, and the two dimer-specific subunits e and g within the membrane-bound region of Fo. A model of oligomerization of the ATP synthase involving two distinct dimerization interfaces was proposed.The rotor-stator interaction within the membrane-bound region of both enzymes is responsible for proton translocation. Our cryo-EM structures of the V-ATPase from Thermus thermophilus reveal that the interaction between the rotary ring (rotor) and the I-subunit (stator) is surprisingly small, with only two subunits from the ring making contact with the I-subunit near the middle of the membrane. Furthermore, the spatial arrangement of transmembrane helices resolved in subunit I can form two passageways that could provide proton access through the membrane-bound region and is consistent with a two-channel model of proton translocation.

Adenosine triphosphate (ATP) is the molecular currency of intracellular energy transfer in living organisms. The enzyme ATP synthase is primarily responsible for ATP production in eukaryotes. In archaea and some bacteria, ATP is synthesized by V-ATPase that is related to ATP synthase both in structure and function. Both of these enzymes are reversible rotary motors capable of catalyzing ATP synthesis or hydrolysis. The rotation of the central rotor, which is powered by the flow of proton (or sometimes sodium ion) down the electrochemical gradient through the membrane-bound Fo/Vo region, leads to the chemical synthesis of ATP in F1/V1 region. The F1/V1 region, on the other hand, can catalyze ATP hydrolysis, which in turn leads to proton (or sodium) pumping across the membrane through rotation of the central rotor in the opposite direction. This thesis describes structure determination of both the intact F-type and V-type enzymes using single particle electron cryomicroscopy (cryo-EM), with the aim of better understanding their overall architecture, subunit organization and the mechanism of proton translocation. Our cryo-EM structural analysis on the F-type ATP synthase from Saccharomyces cerevisiae uncovered the arrangement of subunits a, b, c, and the two dimer-specific subunits e and g within the membrane-bound region of Fo. A model of oligomerization of the ATP synthase involving two distinct dimerization interfaces was proposed.The rotor-stator interaction within the membrane-bound region of both enzymes is responsible for proton translocation. Our cryo-EM structures of the V-ATPase from Thermus thermophilus reveal that the interaction between the rotary ring (rotor) and the I-subunit (stator) is surprisingly small, with only two subunits from the ring making contact with the I-subunit near the middle of the membrane. Furthermore, the spatial arrangement of transmembrane helices resolved in subunit I can form two passageways that could provide proton access through the membrane-bound region and is consistent with a two-channel model of proton translocation.

Intracellular ice formation (IIF), a major cause of cryoinjury in biological cells, is significantly more pronounced during freezing of tissue than during freezing of suspended cells. While extensive studies of IIF have been conducted for single cells in suspension, few have investigated IIF in tissue. Due to the increased complexity that arises from both cell-substrate and cell-cell interactions in tissue, knowledge of cryobiology of isolated cells cannot simply be extrapolated to tissue. Different theories have been hypothesized for the mechanisms of IIF in tissue, but none have been conclusively proven. Towards the goal of developing mathematical models to accurately predict the probability of IIF in tissues of one or more cell types, we have developed a novel high-speed video cryomicroscopy system capable of image acquisition at sampling rates up to 32,000 Hz. Specifically, the effects of cell adhesion to the substrate and cell-cell interactions were investigated with experimental (micropatterned endothelial cell constructs) and mathematical models (Monte Carlo simulations). We have reported the first direct observations of the IIF process recorded at unprecedented sub-millisecond and sub-micron resolution. For the majority of our experiments, IIF nucleation was determined to occur preferentially at the cell perimeter. This observation was not consistent with the commonly accepted hypotheses of ice nucleation in suspended cells and suggests that an alternative mechanism of IIF initiation is dominant in adherent cells. In addition, the kinetics of ice nucleation were shown to be influenced by time in culture, attached cell perimeter, fibronectin coating density, and degree of cell-cell contact. Moreover, an additional phenomenon, paracellular ice penetration was identified, and the frequency of formation was correlated with focal adhesion formation. The data and mathematical models presented in this thesis bring closer the goal of elucidating the primary mechanisms contributing to IIF in tissue; providing important contributions to both the fields of cryopreservation (minimizing IIF) and cryosurgery (maximizing IIF).

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 203-215). Available also in a digital version from Dissertation Abstracts.

This dissertation reports on data acquisition method and the application of world’s first cryogenic apertureless near-field microscope designed for terahertz frequencies. The dissertation briefly summarizes the commonly used data acquisition methods and the existing challenges in applying near-field technology using broadband terahertz sources. We devised, implemented, and validated a novel measurement technique to resolve the challenges. The novel method improves the traditional method by providing the information of the carrier-envelop-phase of the terahertz pulse. The physical properties of WTe₂ microcrystals depend sensitively on the layer number. By applying both the traditional and the novel techniques, we systematically explored the layer-dependent electromagnetic response of mono-layer and few-layer tungsten ditelluride (WTe₂ microcrystals. On tri-layer WTe₂, we discovered the plasmonic response and imaged the real-space pattern of the terahertz plasmon using the novel measurement technique. On bi-layer WTe₂, our measurements support that the band alignment is semi-metallic instead of semi-conducting. Near-field technology at terahertz frequency is sensitive to the Drude behavior of condensed matters. We imaged the electromagnetic response of the transition of cadmium osmate (Cd₂Os₂O₇) crystals from a high temperature metal to a low temperature magnetic insulator. The result is consistent with the temperature dependence in the direct-current conductivity. In the end, the dissertation discusses the theory and simulation of imaging hydrodynamic flow of materials with viscous electron systems via nano-photocurrent technique. In anisotropic material, nano-photocurrent measures the geometrical properties of the Shockley-Ramo auxiliary field or flux. As a result, the nano-photocurrent is a good candidate to detect the boundary layer and vortex flow pattern of a viscous electron system.

In an effort to minimize the harmful effects of intracellular ice formation (IIF) during cryopreservation of confluent tissues, computer simulations based on Monte Carlo methods were performed to predict the probability of IIF in confluent monolayers during various freezing procedures. To overcome the prohibitive computational costs of such simulations for large tissues, the well-known Johnson-Mehl-Avrami (JMA) model of crystallization kinetics was implemented as a continuum approximation of IIF in tissues. This model, which describes nucleation, growth, and impingement of crystals in a supercooled melt, is analogous to the process of intracellular ice formation and propagation in biological tissues. Based on the work of Weinberg and Kapral (1989), the JMA model was modified to account for finite-size effects, and was shown to predict accurately the results of freezing simulations in 1-D tissue constructs, for various propagation rates and tissue sizes. An initial analysis of IIF kinetics in 2-D tissues is also presented. The probability of IIF in 2-D liver tissue was measured experimentally during freezing of HepG2 cells cultured in monolayers, and compared to Monte Carlo simulations and predictions of the continuum model. The Avrami coefficient and exponent for IIF in HepG2 tissue were estimated to be k = 0.19 and n = 0.45.

Translation is the process by which proteins are synthesized from the instructions in the genetic code. Translation is mediated by the ribosome, a large ribonucleoprotein complex, in concert with messenger RNA (mRNA), transfer RNA (tRNA), and a variety of proteins. The canonical mechanism of translation, introduced in Part I of my thesis, is divided into four distinct phases: initiation, elongation, termination, and recycling. Under unusual circumstances, each phase of translation can also proceed via a number of noncanonical mechanisms, many of which are vitally important for cellular growth or viral infectivity. My thesis describes structural insights into two such noncanonical mechanisms. The aim of the first project, described in Part II, was to structurally characterize a noncanonical mechanism of translational termination in bacteria. In the absence of a stop codon, ribosomes arrest at the 3′ end of an mRNA and are unable to terminate. In bacteria, the primary mechanism for rescuing such nonstop complexes is known as trans-translation. In the absence of a functional trans-translation system, however, the small protein ArfA recognizes the empty mRNA channel and recruits the release factor RF2 to the ribosome, enabling termination to occur. Using single-particle electron cryomicroscopy (cryo-EM), I obtained four high-resolution structures of nonstop complexes that reveal the mechanism of ArfA-mediated ribosome rescue and have wider implications for understanding canonical termination in bacteria. The aim of the second project, described in Part III, was to gain structural insights into a noncanonical mechanism of translational initiation in eukaryotes known as internal ribosome entry. Instead of a 5′ cap, many viruses contain intricately structured, cis-acting internal-ribosome-entry sites (IRESs) within their genomes that direct end-independent initiation. The IRES of hepatitis-C virus (HCV), for example, interacts directly with the mammalian ribosome and functionally replaces many of the canonical initiation factors. However, the mechanism by which the HCV IRES coordinates assembly of an initiation complex and progresses through the initiation phase remains poorly understood. I developed a method for purifying native ribosomal complexes from cell lysate that enabled me to obtain multiple cryo-EM maps of the HCV IRES in complex with the 80S ribosome, including a previously unseen conformation of the IRES induced by rotation of the ribosomal small subunit, and to make progress towards capturing earlier steps in the initiation pathway.