Diseases caused by pathogenic bacteria continue to be major health concerns. For example, it is estimated that in the year 2000 typhoid fever caused over 21,000,000 illnesses and ~200,000 deaths (Crump et al., 2004). The disease is caused by S. typhi, a closely-related serotype of S. typhiumurium, the salmonella strain in which BipA was first identified. The CDC estimated that in 2013, multidrug resistant bacteria caused over 2 million infections in the United States, ending in more than 23,000 deaths (CDC, 2013). This number is set to rise as more bacteria become resilient to the collection of conventional antibiotics. The increasing number of multidrug resistant bacterial strains necessitates the development of new antimicrobial drugs. BipA is an attractive target for drug research. As mentioned in Section 2.5.2, BipA is ubiquitous in eubacteria and lower eukaryotes such as protozoa, but is absent from higher-order eukaryotes such as humans. Because the protein is essential for bacterial survival, BipA presents a major vulnerability of pathogenic bacteria. A drug targeting the protein itself or its interactions to the ribosome will disable only the bacteria, but have no effect on the eukaryotic host. A comprehensive model of BipA bound to the 70S ribosome will provide unparalleled insight into BipA's binding site and its mechanism. Toward this goal, cryo-EM techniques were employed to visualize the binding site of BipA on the 70S ribosome, characterize its interactions with the ribosome, and elucidate its mechanism on the ribosome. An X-ray structure of isolated BipA-GMPPNP was elucidated, by collaborators, and used for further molecular modeling of the protein to reveal possible atomic interactions between BipA and 70S ribosome. Additional biochemical studies were performed to fully characterize the specific ribosomal complex that optimizes binding of the factor. Together, the cryo-EM reconstruction, the BipA X-ray structure, the subsequent molecular modeling, and the additional biochemical studies provide a comprehensive model for BipA binding. Over the last years, the introduction of new automated algorithms for particle selection (AutoPicker) and classification (RELION) for the cryo-EM technique has revolutionized the workflow of the entire imaging and reconstruction process. The BipA dataset was primed to be used as a test bed for these algorithms and classification technique, respectively. Using old and new techniques to process the dataset allows a discussion of how the single particle reconstruction process can be vastly improved, with greater automation and efficiency.

Translation is the process by which the cell produces new proteins on the ribosome, as directed by genetic instructions, in all living organisms. Structural studies of the ribosome have shed considerable lights on its mechanism and regulation. Cryogenic electron microscopy (cryo-EM) and single-particle reconstruction technique is one of the major approaches to studying ribosome structure. In this thesis, I report the use of cryo-EM and related new techniques to study the structure of ribosome complexes. This work is divided into three parts. First, in Chapter 3, I describe the development of a computational method in the classification of cryo-EM data. Recently developed classification methods have enabled resolving multiple structures/conformations of the molecules from cryo-EM data obtained on a heterogeneous biological sample. However, the classification methods all involve various amounts of arbitrary decisions made by researchers, which can limit the use of these methods by inexperienced users. As a step toward fully automated classification, I worked with colleagues to develop a "jumper analysis" to determine the number of distinguishable classes of 3D reconstruction, based on the statistics of cryo-EM particles. Second, in Chapter 4, I document the cryo-EM study of EttA-70S ribosome complex, which provided structural insights into the mechanism of EttA in translation regulation. Energy-dependent translation throttle A (EttA, previously named YjjK in Escherichia coli) is the most prevalent member of ATP-binding cassette F family proteins in eubacteria. Through a collaboration among the Hunt, Frank, and Gonzalez labs, we combined crystallography, biochemical, cryo-EM and single-molecule fluorescence energy transfer techniques to elucidate the function and mechanism of EttA. We demonstrated that EttA gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. We also showed that the ATP-bound form of EttA binds to the ribosomal tRNA-exit site, and restricts the ribosome and tRNA dynamics required for translation. Thirdly, in Chapter 5, I discuss the improvements to a new technique, time-resolved cryo-EM by mixing-spraying, and its application to ribosome studies. The mixing-spraying method can study processes involving two big biological molecules that are in the sub-second time scale. I worked with colleagues to apply this method to studying ribosome subunit association. By mixing the subunits and reacting for 60 ms and 140 ms, we were able to capture the association reaction in a pre-equilibrium state. We detected three 70S ribosome conformations in the system. Quantification of the proportions of particles assuming these conformations suggested that the 70S ribosome can undergo fast conformational changes upon formation, and reaches equilibrium among these conformations earlier than 60 ms. In addition, I present preliminary results of studying translation decoding using the mixing-spraying method. This study, performed before improving the mixing-spraying method, was inconclusive mainly due to the limited size of cryo-EM data. Now that we have demonstrated the capability of the mixing-spraying method to visualize multiple states of molecules in a sub-second reaction, the translation decoding process can be revisited and many other processes, such as translation initiation, can be studied.

Graphene and other two-dimensional materials can be stacked together to form vander Waals heterostructures: synthetic crystals composed of different atomically thin layers with a bespoke electronic band structure. Structural characterisation of vander Waals heterostructures is difficult using conventional methods as the properties are almost entirely defined by the nature of the buried interfaces between dissimilar crystals. These methods also fall short of resolving the atomic structure of buried defects in van der Waals materials such as graphite. This work demonstrates the refinement and successful application of ion beam specimen preparation to produce cross sectional slices through these unique crystals so that they can be characterised by high resolution scanning transmission electron microscopy (STEM). Cross sectional specimen were prepared using in situ lift-out in a focused ion beam (FIB) dual-beam instrument. The fine polishing steps were optimised to prevent damage to the core of the specimen. High resolution STEM imaging of twin defects in graphene, hexagonal boron ni-tride and MoSe2 revealed that the boundaries are not atomically sharp but extended across many atoms. Advanced processing and analysis of these images uncovered fundamental mechanics which govern their geometry. This technique was further applied to complex transition metal dichalcogenide heterostructures to quantitatively determine the properties of buried interfaces between atomically thin crystals.

Multicellular development in fungi is fundamentally different from that of animals or plants. In filamentous fungi, multicellular structures are formed by aggregation and adhesion of hyphae, followed by septation and specialisation of hyphal compartments within the aggregate. The perithecium, a flask-shaped sexual fruitbody produced by both Neurospora crassa and Sordaria macrospora, provides a model system in which to study fungal multicellular development. This study presents a detailed description of the morphological stages of perithecial morphogenesis in N. crassa and S. macrospora and its early stages, the ascogonial and protoperithecial stages, using a range of microscopical techniques. Details of the development of several mutants impaired in perithecial development are described, including: gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; and three mutants pro22, pro40 and pro41 of S. macrospora, and their corresponding gene-deletions in N. crassa. The results confirm that all three MAP kinase cascades are required for sexual development. However, only the pheromone response and cell-wall integrity MAP kinase pathways, but not the osmoregulatory MAP kinase pathway, are essential for hyphal cell fusion. Evidence of cell fusion-related processes, regulated through MAP kinase signalling, have been identified as novel features important for the construction of fertilisable protoperithecia. These cell-fusion related processes include extracellular matrix deposition, hyphal attachment and envelopment. A novel phenotype of S. macrospora with defective ascogonial septation is presented. This pro22 mutant also has impaired hyphal cell fusion and produces only small, defective protoperithecia. The pro22 gene encodes a protein that is highly conserved throughout eukaryotes. Live-cell imaging revealed that this PRO22 protein is localised in the dynamic tubular and vesicular vacuolar-network of the colony periphery and in ascogonia. PRO22 is absent from the large spherical vacuoles in the vegetative hyphae of the sub-peripheral region of the colony. This points to a specific role of PRO22 in the tubular and vesicular vacuolar-network. Furthermore, the loss of intercalary septation in ascogonia suggests that PRO22 functions during the initiation of sexual development.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a transmembrane protein that functions as an ion channel. Mutations in this protein cause Cystic Fibrosis. For this reason, it is important to study the structure and function of CFTR. In this study, constructs of CFTR (C-terminii), a CFTR-interacting protein and full-length CFTR were cloned, expressed and purified for structural and functional studies. The purified C-terminal polypeptides of CFTR were soluble and shown to interact with NHERF1 PDZ 1 (a CFTR-interacting protein). The CFTR C-terminus and NHERF1 PDZ 1 domain were co-expressed and co-purified. The purified complex showed a strong interaction that might induces a conformational change. Site-directed mutation of the C-terminus of CFTR was performed in order to examine the effect of removing a potentially flexible amino acid (Arginine) on protein crystallization. Pull-down assay experiments with full-length CFTR demonstrated an interaction between CFTR (in DDM detergent) and NHERF1 PDZ 1(+). No interaction was observed for CFTR in LPG (a relatively denaturing detergent) and NHERF1, implying that the interaction between the PDZ motive of CFTR and NHERF1 requires a stable folded structure for both proteins. In addition, full-length CFTR in DDM has been studied by electron microscopy and Single Particle Analysis in the presence of NHERF1 PDZ 1(+). A 3D structure was generated for the CFTR-NHERF1 PDZ 1(+) complex at a resolution of ~ 18 A. This 3D structure showed a new open conformation of CFTR (V shape). In comparable studies with CFTR alone, a 3D structure was generated at a resolution of 27 A and this structure showed a closed state as previously reported. This new data suggest a possible role for NHERF1 in terms of CFTR channel gating or activation.

Xing, Xiaochen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references. / Abstract also in Chinese. / Abstract (Chinese) --- p.i / Abstract --- p.iii / Contents --- p.v / Acknowledgement --- p.ix / Chapter Chapter1 --- Introduction and background / Chapter 1.1 --- Overview of Studies in Colloid-Polymer mixture --- p.1 / Chapter 1.2 --- Depletion Force in Colloid-Polymer Mixture --- p.1 / Chapter 1.2.1. --- Depletion Interaction in Monodisperse and Neutral Polymer-Colloid Mixtures: Theory --- p.3 / Chapter 1.2.1.1. --- An Exact Result: the Interaction between Parallel Plates due to Ideal Polymer Chains --- p.3 / Chapter 1.2.1.2. --- Penetrable Hard Sphere (PHS) Approach --- p.4 / Chapter 1.2.2. --- Early Experimental Findings of Depletion Interaction --- p.6 / Chapter 1.3 --- References and Notes --- p.8 / Chapter Chapter2 --- Principle of Total Internal Reflection Microscopy (TIRM) and Instrumentation / Chapter 2.1 --- Introduction of Total Internal Reflection Microscopy (TIRM) --- p.10 / Chapter 2.2 --- The Principle of TIRM Technique --- p.11 / Chapter 2.2.1 --- Total Internal Reflection --- p.11 / Chapter 2.2.2 --- Details on Scattering of the Evanescent Wave --- p.13 / Chapter 2.2.3 --- Data Analysis --- p.16 / Chapter 2.3 --- Instrumentation --- p.20 / Chapter 2.3.1 --- Apparatus --- p.20 / Chapter 2.3.2 --- Optical Tweezer --- p.23 / Chapter 2.3.3 --- Cleaning of the Slide Surface --- p.24 / Chapter 2.3.4 --- A Typical Potential Energy Profile --- p.25 / Chapter 2.4 --- Laser Light Scattering (LLS) --- p.26 / Chapter 2.5 --- Zeta-potential Measurements --- p.27 / Chapter 2.6 --- References and Notes --- p.28 / Chapter Chapter3 --- Depletion Attraction between a Polystyrene Sphere and a Hydrophilic Surface in a Pluronic Aqueous Solution / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Experimental Section --- p.34 / Chapter 3.2.1 --- Sample Preparation --- p.34 / Chapter 3.2.2 --- Total Internal Reflection Microscopy --- p.35 / Chapter 3.2.3 --- Laser Light Scattering --- p.36 / Chapter 3.3 --- Results and Discussion --- p.37 / Chapter 3.4 --- Conclusion --- p.48 / Chapter 3.5 --- References and Notes --- p.50 / Chapter Chapter4 --- pH-Controllable Depletion Attraction Induced by Microgel Particles / Chapter 4.1 --- Introduction --- p.53 / Chapter 4.2 --- Experimental Section --- p.54 / Chapter 4.2.1 --- Sample Preparation --- p.54 / Chapter 4.2.2 --- Total Internal Reflection Microscopy --- p.56 / Chapter 4.3 --- Results and Discussion --- p.58 / Chapter 4.4 --- Conclusion --- p.63 / Chapter 4.5 --- References and Notes --- p.64 / Publication List --- p.65

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.

With the development of ultra-fast laser technology, several new imaging techniques have pushed optical resolution past the diffraction limit for traditional light-based optics. Advancements in lithography have enabled the straightforward creation of micron- and nanometer-sized optical devices. Exposing metal-dielectric structures to light can result in surface plasmon excitation and propagation along the transition interface, creating a surface plasmon polariton (SPP) response. Varying the materials or geometry of the structures, the plasmonic response can be tailored for a wide range of applications. Photoemission electron microscopy (PEEM) has been used to image excitations in micron-sized plasmonic devices. With PEEM, optical responses can be characterized in detail, aiding in the development of new types of plasmonic structures and their applications. We show here that in thin, triangular gold platelets SPPs can be excited and concentrated within specific regions of the material (thickness ~50 nm); resulting in localized photoemission in areas of high electric field intensity. In this regard, the platelets behave as receiver antennas by converting the incident light into localized excitations in specific regions of the gold platelets. The excited areas can be significantly smaller than the wavelength of the incident light (λ≤1µ). By varying the wavelength of the light, the brightness of the excited spots can be changed and by varying the polarization of the light, the brightness and position can be changed, effectively switching the photoemission on or off for a specific region within the triangular gold structure. In this work, the spatial distribution of surface plasmons and the imaging results from photoemission electron microscopy are reproduced in simulation using finite element analysis (FEA). In addition, we show that electromagnetic theory and simulation enable a detailed and quantitative analysis of the excited SPP modes, an explanation of the overall optical responses seen in PEEM images, and prediction of new results.

Typewritten copy Includes bibliographical references (leaves 141-144) Pt. 1. Historical review -- pt. 2. Techniques used in electron microscopy -- pt. 3. Experiments on cilia from Hydrdella australis -- pt. 4. Electron microscope experiments on cilia from the rat trachea -- pt. 5. Electron microscope experiments on cilia from paramecium -- pt. 6. Electron microscope experiments on the trichocysts of paramecium -- pt. 7. Discussion An account of experimental investigations carried out from January 1952 to September 1954.

Thesis (Ph.D.)--University of Hong Kong, 1994. / Includes bibliographical references. Also available in print.