Computational protocol: 2D hybrid analysis: Approach for building three-dimensional atomic model by electron microscopy image matching

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Protocol publication

[…] Approximately 10 μg of each purified integrin was subjected to an additional gel filtration process on a Superdex 200 HR column equibrated with 50 mM Tris, 150 mM NaCl, pH 7.5, containing 5 mM CaCl2 or 1 mM MnCl2. The samples after the gel filtration were immediately absorbed to glow-discharged carbon-coated copper grids. Samples were negatively stained with 2.5% (w/v) uranyl acetate and examined under an electron microscope (H9500SD, Hitachi, Japan) operated at 200 kV and a nominal magnification of ×80,000. Images were recorded on a 2,048 × 2,048 CCD camera (TVIPS, Gauting, Germany). Single-particle EM analysis, including particle selection and 2D classification and averaging, was performed using the EMAN suite and IMAGIC program. Particles were selected from individual frames (with an effective pixel size of 0.21 nm) by using the Boxer program in the EMAN suite. The particle images were rotationally and translationally aligned by a multireference alignment procedure and subjected to multivariate statistical analysis by specifying 20 classes using the IMAGIC program. […]

Pipeline specifications

Software tools EMAN, IMAGIC
Application cryo-EM