Detection of Zn Atoms on Ferritin by Annular Dark-Field cryo-STEM

Sunday, February 28, 2016 Jarvis, J.A., Haies, I.M., et al., An efficient NMR method for the characterisation of 14N sites through indirect 13C detection. Phys Chem Chem Phys, 2013. 15(20): p. 7613-20.

Electron Microscopy 776-Pos Board B556 3D Microstructural Visualization of the Simplest of Eukaryotic Cell (Cyanidioschyzon Merolae) during Mitosis Process using Several New Microscopic Techniques Atsuko H. Iwane1,2, Keisuke Ohta2,3. 1 Osaka University, Suita, Japan, 2QBiC, Riken, Osaka, Japan, 3Kurume University, Kurume, Japan. EM offers exceptional resolution of extremely small biological specimens, providing images of the minutest of organelles and molecules responsible for fundamental cellular phenomena such as division, differentiation and proliferation. The resulting fine structural information will be extraordinary in defining the relationship between cell morphology and cell function. FIB-SEM and 3view-SEM will provide us information of the specimen which will complement the 3D-structural information at whole cell level. We selected C. merolae cell, a primitive unicellular red algae, as an excellent model organism for cell mitotic cycle. Although the size of the cell is 2-5 micron, the body shape (3D-structure) has not well understood yet. Last annual meeting, we described how to analyze 3Dmicrostructural visualization of an entire cell at a nanoscale resolution using FIB-SEM and 3D reconstruction techniques, Image J and Amira 3D software. Forthermore, by using synchronizing cells to a 6-h light/18-h dark cycle and FIB-SEM, we observed unique architectures of whole C. merolae cells during the mitotic cycle and successfully made 3D-models of individual doublemembrane organelles and single-membrane organelles. In this meeting, in addition to analyze the shape and interaction between individual several organelles during mitosis cycle, we aim to reveal individual morphology details and compute the volume occupancy of each organelle. We will discuss you about the relationship between cell body mass and energy efficiency, also. Furthermore, using UHVEM tomography, we also observed the 3D-structure of phycobilisomes, which are essential supramolecular complexes on the surface of the thylakoid membrane in chloroplasts and the micro-compartments (cristae) in mitochondria. Although many reports have provided structural models, we offer the first 3D-structural model of the membrane surface from specimens that were not purified using specific detergents. 777-Pos Board B557 Three Dimensional Image Analysis Applying Various Serial Section Techniques on Study of Melanin Transfer in Human Skin Bo Ram Kim1, Hyo Sun Choi1, Il-Hwan Kim2, Ji Young Mun1. 1 Department of Biomedical Laboratory Science, Eulji University, Seongnam, Korea, Republic of, 2Department of Dermatology, Korea University Ansan Hospital, Ansan, Korea, Republic of. In the basal layer of the epidermis, there are melanocytes that produce melanin. Main function of melanin is to prevent UV-induced damage of keratinocytes by filtering out harmful UV radiation. Melanosomes containing melanin are organelle transferred from melanocytes to neighboring keratinocytes. However the mechanism of melanosome transfer is not fully understood. The study of the mechanism is necessary to understand the hyper or hypo-pigmentary disorders. Three possible mechanisms of melanosome transfer have been reported: (1) direct inoculation of melanosomes into keratinocytes via keratinocytemelanocyte membrane fusions through nanotubular filopodia, (2) release of individual melanosomes from melanocytes and their uptake by keratinocytes via phagocytosis, (3) partial cytophagocytosis of melanocyte dendrite tips containing melanosomes by keratinocytes, (4) coupled exocytosis of the melanin by melanocytes and subsequent endocytosis by keratinocytes. But those results were studied in different system such as cell culture, mouse skin and so on. In order to know which mechanism moves melanin from melanocyte to keratinocyte in human, we tried to use biopsy tissue of human skin. Structural studies using 2D images show limitations in understanding the structure of cellular organelle. To cope with the difficulty, over the last few years the 3D reconstruction techniques using electron microscopes have been developed at extremely high speed as the related equipments develop. In this study, 3D structure was reconstructed from newly developed serial section techniques. The serial section showed the detail of melanin transfer from melanocyte to keratinocyte in human skin. Our result will help to unravel the mechanisms behind a wide range of human pigmentary disease (This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning(2015R1C1A1A02037153).

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778-Pos Board B558 Graphene-Enabled Electron Microscopy and Correlated Super-Resolution Microscopy of Wet Cells Michal Wojcik, Margaret Hauser, Wan Li, Seonah Moon, Ke Xu. Chemistry, UC Berkeley, Berkeley, CA, USA. The application of electron microscopy (EM) to hydrated biological samples has been limited by the harsh conditions found in EM chambers. Conventional methods require challenging and laborious sample dehydration procedures, often resulting in structural artifacts and causing difficulties in correlating results with those obtained from fluorescence microscopy (FM). We here present our recent results utilizing graphene, a single-atom-thick carbon sheet, as the thinnest possible impermeable and conductive membrane to protect whole cells from vacuum, thus enabling high-resolution EM of hydrated and untreated cells with unprecedented ease. Our approach further allows for facile correlative super-resolution FM and EM of wet, fixed cells directly on the culturing substrate. This simple, one-step procedure requires no special materials or equipment, and can be readily performed by biologists with basic experience using FM and SEM. Excellent results are obtained with conventional scanning electron microscopes. As an example of the power of this technique, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and are well correlated with super-resolution fluorescence results. Our approach thus offers an accessible method by which researchers can obtain multimodal information of biological specimens in their native, hydrated state. 779-Pos Board B559 Cryo-Electron Tomography and Nucleocapsid Protein Labeling by Tomo-Bubblegram Imaging Reveal a Role for HIV-1 Integrase in Viral Maturation Juan Fontana1, Kellie A. Jurado2, Naiqian Cheng1, Alan N. Engelman2, Alasdair C. Steven1. 1 Laboratory of Structural Biology Research, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD, USA, 2Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA, USA. HIV-1 maturation involves proteolysis of the precursor polyproteins, Gag and Gag-Pol, and assembly of a conical core containing the viral ribonucleoprotein (vRNP), comprised of the vRNA genome and associated proteins, notably nucleocapsid (NC). Allosteric integrase inhibitors (ALLINIs) are antiviral drugs initially thought to impede the viral integrase from inserting the DNA version of the genome into a host chromosome. However, recent studies indicate that their primary effects are on maturation. To investigate these effects, we imaged virions produced in the presence of ALLINIs by cryo-electron tomography (cryo-ET) and ‘‘tomo-bubblegram’’ imaging, a novel labeling technique that exploits the fact that vitrified protein subjected to relatively high levels of electron irradiation generates bubbles of hydrogen gas, which are readily visible. First, a regular cryo-tomogram is calculated. Then, after priming the specimen with additional exposures, a second tilt series is recorded, visualizing the bubbles. With HIV, we exploited the serendipitously discovered susceptibility of NC to bubbling. Most wild-type virions have conical cores with internal density, putatively the vRNP. In contrast, most ALLINI-treated virions have non-conical or conical cores with sparse internal contents but contain ‘‘eccentric condensates’’, outside the capsid. Tomo-bubblegrams showed that the bubbles specifically label the contents of filled cores and eccentric condensates. In immature virions, in which the domains of Gag and Gag-Pol are radially ordered in a spherical shell, the bubbles appear in the NC layer of that shell. Immature virions in which NC is replaced by a leucine zipper (another RNA binder) yielded no bubbles, confirming NC as the bubbling component. Taken together, these observations provide strong evidence that ALLINIs sabotage capsid assembly and vRNP encapsidation. They also illustrate the potential of bubblegram imaging for mapping components of large macromolecular complexes. 780-Pos Board B560 Detection of Zn Atoms on Ferritin by Annular Dark-Field cryo-STEM Nadav Elad1, Giuliano Bellapadrona2, Lothar Houben1, Irit Sagi3, Michael Elbaum2. 1 Chemical Research Support Department, Weizmann Institute of Science, Rehovot, Israel, 2Dept of Materials and Interfaces, Weizmann Institute of Science, Rehovot, Israel, 3Dept of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel. Metal ions play essential roles in many aspects of biological chemistry, including oxygen transport, enzyme catalysis, and maintenance of biopolymer integrity. Cryo-electron microscopy is in principle sensitive to metal ions due to the stronger Coulomb potential relative to surrounding light elements, but

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conventional defocus phase contrast may not be the ideal approach to visualization. In tomography even small gold nanoparticles (<5 nm) are difficult to identify. We explore the alternative modality of scanning transmission EM (STEM), recently applied to cryo-microscopy and tomography. Annular dark-field STEM provides a quantitative image contrast based on atomic scattering, and has been exploited most effectively for mass measurements of macromolecules. Angular resolution of the scattering can provide further information to distinguish relatively heavy and light elements; in materials science this is known as Z contrast. We ask whether cryo-STEM can detect the presence and location of metal ions in protein complexes. Ferritin proteins forms a nearly spherical, highly symmetrical 3D shell structure by self-assembly of 24 polypeptide subunits. Ferritins bind iron as the primary physiological substrate, but also other metals. Zn binds to ferritin at precise ferroxidase sites within the structure. Taking advantage of these properties, we used cryo-STEM to identify Zn ions within the protein structure. Images were processed by single-particle alignment and averaging. Even in very small datasets, Zn stands out clearly at the predicted binding sites. The data provide an experimental measure of the signal contribution from single Zn atoms in cryo-STEM. This is key to understanding atomic detection of metals in 3D macromolecular and cellular contexts, as well as to use of synthetic metal tags as specific molecular labels. 781-Pos Board B561 Structure of the F-Actin-Tropomyosin Complex Revealed by Electron Cryomicroscopy Julian von der Ecken1, Mirco Mu¨ller2, William Lehman3, Dietmar Manstein2, Pawel Penczek4, Stefan Raunser1. 1 Structural Biochemistry, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany, 2Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany, 3Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA, USA, 4 Department of Biochemistry and Molecular Biology, The University of Texas, Houston, TX, USA. Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in nonmuscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional ˚ in complex with tropomyosin at structure of F-actin at a resolution of 3.7 A ˚ , determined by electron cryomicroscopy. The structure rea resolution of 6.5 A veals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg2þ and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin-tropomyosin with its position in our previously determined F-actin-tropomyosin-myosin structure reveals a myosininduced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs. 782-Pos Board B562 Using Electron Cryotomography and Coarse-Grained Molecular Dynamics to Study Contractile Mechanisms of Eukaryotic Cell Division Machinery Matthew T. Swulius1, Lam Nguyen1, Mark Ladinsky1, Mithilesh Mishra2, Grant Jensen1. 1 Biology, Caltech, Pasadena, CA, USA, 2Tata Institute, Mumbai, India. Division in eukaryotic cells depends on an actomyosin ring that forms at the midcell and then contracts throughout cytokinesis via interactions between actin filaments and myosin II motor proteins. While the protein players involved are well studied, the ultrastructure of the actomyosin division machinery has been elusive due to its highly dynamic nature, which makes it difficult to preserve without vitrification. Here we use cryo focused ion beam milling

and cryosectioning to examine the actomyosin machinery of fission yeast (S. pombe) for the first time in its native state, at different stages of contraction. Due to the machinery’s complex and dynamic nature, and because EM images are static, we used structural data from our tomographic reconstructions to inform and guide 3D coarse-grained molecular dynamics simulations of the actomyosin ring contracting. To make our simulations as realistic as possible, for the first time, we have modeled the myosin ATPase cycle step-by-step and a coarse-grained membrane was used to test the ring’s ability to constrict the membrane. By simulating all current models (and many variations) of actomyosin architecture and by comparing the results to tomographic data, we propose a working model for the contractile machinery, including how the actin filaments are arranged, how they might be tethered to the membrane and the structure of myosin II in the ring. 783-Pos Board B563 Mitochondrial Networks in Beta Cells of Pancreatic Islet of Langerhans Investigated by Serial Block Face Scanning Electron Microscopy Gina N. Calco, Bryan C. Kuo, Jake D. Hoyne, Maria A. Aronova, Guofeng Zhang, Richard D. Leapman. NIBIB, National Institutes of Health, Bethesda, MD, USA. The process whereby mitochondrial tubules fuse to form extended networks within cells has been associated with metabolic activity, and may provide a defense against generation of reactive oxygen species, whereas fission of such mitochondrial networks has been associated with apoptosis. We have used serial block-face scanning electron microscopy (SBF-SEM) to analyze distributions of mitochondrial network lengths in metabolically active insulin secreting beta cells of mouse pancreatic islets of Langerhans, with the aim of quantifying differences between wild type and diabetic disease models (Pfeifer et al., 2015). This technique provides three-dimensional ultrastructure of beta cells at a spatial resolution of about 10 nm in the x-y plane and 25 nm in the z-direction, from which we have been able to obtain cross sectional areas, volumes and network lengths of mitochondrial tubules in a population of beta cells. Blocks of isolated pancreatic islets were prepared by fixation, staining with heavy atoms, and embedding in Epon-Araldite resin, and were imaged at 1.5-keV primary beam energy with the backscattered electron signal. Our results showed that approximately one-third of the total mitochondrial volume originated from short networks less than 10 mm in length, one-third came from tubules of length 10 to 45 mm, and one-third from networks of length 45 to 150 mm. We conclude that SBF-SEM can provide a quantitative characterization of mitochondrial networks in wild type and disease models of endocrine tissues. This research was funded by the intramural program of NIBIB, NIH. C.R. Pfeifer, A. Shomorony, M.A. Aronova, G. Zhang, T. Cai, H. Xu. A.L. Notkins, R.D. Leapman. Quantitative analysis of mouse pancreatic islet architecture by serial block-face SEM. J Struct Biol, 189 (2015) 44-52. 784-Pos Board B564 From CRYO-EM Densities to Atom Coordinates and Ensembles with Bayes Approach Christian Blau1, Nicolas Lenner2, Carsten Kutzner2, Helmut Grubmuller2, Erik Lindahl1. 1 Theoretical and computational Biophysics, Science for Life Laboratory, Solna, Sweden, 2Theoretical and computational Biophysics, Max Planck Institute for biophysical Chemistry, Go¨ttingen, Germany. Near-atom resolution in 3D cryo-electron microscopy brings two new challenges for refining atom coordinates to densities. Because their underlying potentials become more rugged with increased resolution, established refinement methods are easily trapped in local minima. Second, single structures no longer well describe the inherent structural diversity that can be resolved with cryoEM. Here, we developed a method to derive cryo-EM refinement potentials from a Bayesian approach. Our refinement potential takes a physical model of the cryo-EM measuring and reconstruction process into account using Bayesian statistics. The result is a potential that statistically correctly reflects the given EM-data and is smooth even at high resolution. Previously developed algorithms are contained as limiting cases. With our method we are able to represent the configurational dynamics that is captured in cryo-EM density maps through a series of features. The smoothness of our refinement energy landscape allows efficient sampling and refinement while also taking into account thermal fluctuations. We provide an refinement force constant and potential from the Bayes approach that truthfully represents the complete distribution of atom configurations underlying the given EM maps. Thus, in combination with traditional molecular dynamics simulation we create refined ensembles that - as a whole - represent a given cryo-EM map. We further use the advantage of the Bayes approach to generate molecular dynamics ensembles that represent the simultaneous input from multiple cryo-EM