Computational protocol: Human T-Lymphotropic Virus-1 Visualized at the Virological Synapse by Electron Tomography

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Protocol publication

[…] Electron microscopic data were collected with an FEI Tecnai F30 FEG transmission electron microscope (FEG-TEM) (FEI Company, Eindhoven, The Netherlands) at an electron voltage of 300 kV. The TEM was equipped with an energy filter (Gatan imaging filter (GIF), Gatan, Inc., Abingdon, UK). Electron micrographs were recorded using the software Digital Micrograph (Gatan, Inc., Abingdon, UK) with a Gatan 2k×2k slow-scan CCD camera. Most tomographic data sets were recorded at a few microns under focus using the GIF with a slit width of 20–25 eV to accentuate contrast. Tilt series for tomography were recorded in steps of 1° over a tilt range of typically −65° to +65° using the FEI software. Most tomographic data sets were recorded using a flip-flop sample holder (Gatan) that allows one to turn the sample by 90° inside the EM column. A few data sets were recorded in the Laboratory for 3-Dimensional Electron Microscopy of Cells in Boulder, Colorado with a FEI Tecnai F30 FEG-TEM (without energy filter). These data were recorded using the software SerialEM (Boulder, Colorado) with a Gatan CCD camera. Tomograms were processed with the tomography software IMOD and surface representations were generated using both IMOD and the software package AMIRA (Mercury Computer Systems, Inc.). Data analysis was performed using IMOD, AMIRA and Matlab (The MathWorks). Membrane-membrane spacings were measured using a program that calculates the distance between each voxel of the digitized membrane model (surface representation) and its nearest neighbour in the opposite cell membrane. The size of individual HTLV-1 particles was determined by measuring an averaged diameter in the section (through the 3D object) of largest appearance of the particle. […]

Pipeline specifications

Software tools SerialEM, IMOD
Application cryo-EM
Organisms Human T-cell leukemia virus type I