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7 particle work, with built-in navigation for low-dose acquisition and advanced autotuning functions. This software package is installed on a wide range of TEMs which are equipped with remote control and computer-controlled goniometer, e. g. Mag Mag EM-TOMO: Automated Tilt Series Acquisition Software Two different views of a 3D model of IFT particles in fla14 flagella. The tilt series were acquired on aa CM200 using EM-TOMO (for further details please see Pigino G. et al. (2009): Electron-tomographic analysis of intraflagellar transport particle trains in situ, Journal of Cell Biology, 187: pp ). [1] Glaeser R. M. et al. (2011): Precise beam-tilt alignment and collimation are required to minimize the phase error associated with coma in high-resolution cryo-em, Journal of Structural Biology, 174: pp [2] Zemlin F. et al. (1978): Coma-free alignment of high resolution electron microscopes with the aid of optical diffractograms, Ultramicroscopy, 3: pp Zemlin tableau acquired on TemCam-F416, mounted on a JEM-3200FSC. Courtesy of O. Hofnagel, MPI of Molecular Physiology, Dortmund, Germany. Mag :1 digital zoom 5 x 5 tiled images Mag. 120 Schematic overview of the hierarchical navigator positions which can be utilized for tomographic and single particle data collection. Grid map: First a large scale image (e. g μm²) is recorded at low magnification. This image is composed of e. g. 5 5 tiles and provides an overview of the grid and is used to screen throughout the sample without unnecessarily exposing the specimen. Scan: At higher magnification (e. g ) several areas of interest can be localized more precisely. It is possible to define multiple scan positions. Search: Based on the previously defined scan positions the search position shows the area of interest at higher magnification (e. g ). Typically on this position the eucentric height will be corrected automatically (auto-eucentric). Focus: This position uses the same magnification as exposure position but is shifted e. g. along the tilt axis for the tomographic setup. On this position the defocus will be corrected automatically (auto-focus). To avoid illumination of adjacent areas the beam size and position is centered automatically (auto-beam-centering). Tracking: This position is mainly used for the tomographic setup and is shifted along the tilt axis of the stage. The tracking position is used to re-center the object for subsequent acquisitions. Exposure: This position records the final image of the area of interest. EM-SPC: Semi-Automated Data Collection Software for Single Particle Work Cryo-EM reconstructions of an archeal 70S ribosome at 6.6 Å resolution (average of 51 with the TemCam-F416 (for further details please see Becker T. et al. (2012): Structural basis of highly conserved ribosome recycling in eukaryotes and 14 15

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