bearing nitrilotriacetic acid substituents at the end of a poly(ethylene glycol)- grafted surface that promotes specific capture of protein targets for single particle reconstruction analysis. The utilization of these grids for specific adsorption of the targeted protein onto the grid surface results in well- controlled surface concentration enhancements and a days-to-minutes reduc- tion in time required for the preparation of a purified sample for cryoEM anal- ysis from an E. coli expression system. The selective and reversible capture of his-tag T7 bacteriophage and GroEL from crude lysates, as well as purified nanodisc-solubilized his-malFGK2, on these NTA-modified grids with an exceptionally low level of adsorption by non-target proteins has been observed. Our data illustrates the utility of these grids for selective capture from complex mixtures, detergent-solubilized membrane protein isolates, and expression systems yielding low copy numbers of the desired target in a manner that is well-suited for single particle reconstruction analysis. 3116-Pos Board B546 Scanning Transmission Electron Tomography of Blood Platelets in Thick Sections Jake D. Hoyne 1 , Gina N. Calco 1 , Bryan C. Kuo 1 , Maria A. Aronova 1 , Alioscka A. Sousa 1 , Qianping He 1 , Guofeng Zhang 1 , Irina D. Pokrovskaya 2 , Laura MacDonald 2 , Andrew A. Prince 2 , Brian Storrie 2 , Richard D. Leapman 1 . 1 NIBIB, National Institutes of Health, Bethesda, MD, USA, 2 Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA. Electron tomography in the scanning transmission electron microscope (STEM) can be performed on sections of stained plastic-embedded tissues or cells of 1 to 2 micrometer thickness without effects of chromatic aberration because there are no imaging lenses after the specimen. By using a small STEM probe conver- gence angle of 1-2 mrad the geometrical broadening of the probe is restricted, which enables a spatial resolution of a few nanometers. Furthermore, by using an axial bright-field detector instead of the standard high-angle annular dark- field detector, image blurring due to multiple elastic scattering can be reduced in the lower part of the specimen. Here, we have applied STEM tomography to elucidate the 3D ultrastructure of human blood platelets, which are small anu- cleate blood cells that aggregate to seal leaks at sites of vascular injury and are important in the pathology of atherosclerosis and other diseases. Of particular interest are the morphological changes that occur in alpha-granules, which contain important proteins released when platelets are activated. Axial bright- field STEM electron tomographic tilt series were acquired at an accelerating voltage of 300 kV from 1.5-micrometer thick sections of platelets that had been prepared by rapid freezing and freeze-substitution; and the tomograms were reconstructed from dual-axis tilt series. The tomographic reconstructions revealed changes in ultrastructure that occurred on platelet activation including release of alpha granules through channels connecting to the plasma membrane. The research was supported by the intramural program of the National Institute of Biomedical Imaging and Bioengineering, and the research in the Storrie lab- oratory was supported in part by NIH grant R01 HL119393. 3117-Pos Board B547 Three-Dimensional Microstructural Visualization of Mitosis using Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) and 3Mv Ultra-High Voltage Electron Microscope (UHVEM) Tomography with Nanoscale Resolution at Whole Cell Level Atsuko H. Iwane 1,2 , Keisuke Ohta 2,3 . 1 Osaka University, Suita, Japan, 2 QBiC, RIKEN, Suita, Japan, 3 Kurume University, Kurume, Japan. To better understand fundamental cellular properties, such as differentiation and division, we are developing whole single cell 3D-structure analysis tech- nologies based on innovative electron microscopy. These new techniques are designed to reveal the dynamics and structure of intracellular material such as organelles and supramolecular proteins. Our main technologies include (1) Cryo-Tomography using Scanning-TEM and (2) FIB (Focused Ion Beam)- SEM and 3D-reconstruction. FIB-SEM is normally used to visualize metals and ceramics. We have modified it for the 3D reconstruction of an entire cell at a nanoscale resolution that lies between those of electron microscopy tomog- raphy and X-ray tomography. Last annual meeting, we described how FIB-SEM could visualize the basic 3D architecture of Cyanidioschyzon merolae (C. marolae). C. marolae is a primi- tive unicellular red algae whose cell division can be observed my manipulating the light/dark cycle. By synchronizing cells to a 6-h light/18-h dark cycle, we obtained > 75% S/M-phase cells at 89 hrs after synchronous culture start. Using these cells and FIB-SEM, we observed unique architectures of whole C. merolae cells during the mitotic cycle and successfully made 3D-models of individual double-membrane organelles such as the nucleus, chloroplast and mitochondria, and of single-membrane organelles such as the ER, lyso- some and peroxisome using ImageJ and Amira 3D software. 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. 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. 3118-Pos Board B548 Regulation of Myosin VI Studied by Electron Microscopy Dario Saczko-Brack, Heike Ellrich, Christine Werner, Christopher Batters, Claudia Veigel. Department of Cellular Physiology, Ludwig-Maximilians-Universita ¨t Mu ¨nchen, Munich, Germany. Myosins are ATPase motor proteins that are activated by and traffic along actin filaments. This large protein family is divided into many classes with different functional properties and specializations for various roles, including membrane anchorage, longer range transport of cargo vesicles or cell signaling. Myosin class VI is unique due to its reversed directionality along actin fila- ments, moving towards the pointed end, in contrast to almost all other classes, which move towards the barbed end of F-actin. Whilst the directionality is well studied, other characteristics such as activation, cargo and lipid binding or dimerization are not fully understood. Using size exclusion chromatography, titration studies and gliding filament assays we investigated myosin VI back- folding, cargo binding and mechanical activity. Furthermore, we applied electron microscopy and single particle image pro- cessing to determine the structural properties of myosin VI in different ionic and nucleotide conditions. Two dimensional class averages based on various alignment and classification methods were made that allow for a detailed struc- tural analysis including a comparison with crystal structures. 3119-Pos Board B549 The Steric Fine Structure of Maurer’s Cleft in ‘‘Unroofed’’ Plasmodium Falciparum-Infected Erythrocytes Eri H. Hayakawa 1 , Fuyuki Tokumasu 2 , Jiro Usukura 3 , Hiroyuki Matsuoka 1 , Takafumi Tsuboi 4 , Thomas E. Wellems 5 . 1 Lab of Medical Zoology and Parasitology, Department of Infection and Immunity, Jichi Medical University, Shimotsuke, Japan, 2 Department of Lipidomics, Grad school of Medicine, The University of Tokyo, Tokyo, Japan, 3 Division of Integrated Project, EcoTopia Science Institute, Nagoya University, Nagoya, Japan, 4 Malaria Research Unit, Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Japan, 5 Lab of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases/NIH, Bethesda, MD, USA. Plasmodium falciparum produces additional membrane systems, Maurer’s cleft (MC) and tubulovesicular network (TVN), in the host erythrocytes. The para- sites use these membrane compartments to transport proteins to the surface of erythrocytes. Previous studies reported the structure of MCs by transmission electron microscopy (TEM) using ultra-thin layer specimen and suggested physical connections between MCs and erythrocyte membrane via an extension of MC membrane. However, fine structures of MC including filamentous exten- sions smaller than the thickness of diamond knives were likely missing in the TEM images. To obtain intact structural information of MCs, we used unroofing/rip-off technique for both normal- and parasitized-erythrocytes and successfully captured accurate oval/global shape of MCs with elongated-fine filamentous extensions (diameters <10 nm). We also treated parasitized eryth- rocyte with aluminum tetrafluoride, which are known to inhibit intracellular vesicle transport, to clarify if the oval/global structures are MCs. In the pres- ence of aluminum tetrafluoride, the vesicle was no longer observed in parasit- ized erythrocytes. This result was in agreement with the previous study (Trelka DP, et al., Mol Biochem Parasitol, 2000), demonstrating the oval/global struc- tures are MCs which extends filaments to host erythrocyte membrane. Our EM images demonstrated that MCs in P. falciparum-infected erythrocyte involve fine filaments reaching erythrocyte membrane which may provide a direct transport pathway for their proteins to the surface of erythrocytes. 3120-Pos Board B550 Towards Femtosecond Electron Diffraction of Proteins - Technical Challenges and Sample Preparation Strategies Henrike M. Mueller-Werkmeister 1,2 , Daniel Badali 3 , Oliver P. Ernst 2 , R.J. Dwayne Miller 1,3 . 1 Chemistry, University of Toronto, Toronto, ON, Canada, 2 Biochemistry, University of Toronto, Toronto, ON, Canada, 3 Max-Planck-Institute for Structure and Dynamics of Matter, Hamburg, Germany. To study protein dynamics in real-time with atomic resolution is one of the dream experiments in biophysics. Up to now experimental tools with full 618a Wednesday, February 11, 2015