Computational protocol: Three-dimensional structure of recombinant type 1 inositol 1,4,5-trisphosphate receptor

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Protocol publication

[…] Purified IP3R1 was loaded on to glow-discharged carbon-coated copper grids and negatively stained with 2% uranyl acetate. Micrographs were collected on a Tecnai T-12 electron microscope in low-dose mode (20 electrons/Å2) at a calibrated magnification of 41125, at 120 kV with ~700 nm defocus. The quality of the micrographs was assessed using an optical diffractometer. Only micrographs with circular and isotropic diffraction rings (Thon rings) consistent with a resolution of at least 20 Å within the first ring were used for further processing. Micrographs were digitized using a Nikon Coolscan 9000ED at a step size of 6.35 μm. Scanned micrographs were then converted for processing using IMAGIC programs [] and 3-pixel×3-pixel areas were averaged, resulting in a final pixel size of 0.45 nm. The particles were selected with a box size of 100 pixels×100 pixels using the BOXER tool of the EMAN package []. IMAGIC [] was used for all other image processing, except for the multi-reference alignment routine for which SPIDER [] was used. In the first multi-reference alignment and first angular assignment, our previously published IP3R structure [], filtered to 54 Å, was used as a reference. The resolution of the three-dimensional reconstruction was determined using the half-bit criterion. […]

Pipeline specifications

Software tools IMAGIC, EMAN, SPIDER
Application cryo-EM
Chemicals Inositol 1,4,5-Trisphosphate