Computational protocol: Molecular organization of the COG vesicle tethering complex

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Protocol publication

[…] Samples were prepared by conventional negative staining with 0.75% (w/v) uranyl formate as described previously. Images were collected with a Tecnai T12 electron microscope (FEI, Hillsboro, OR) equipped with a LaB6 filament and operated at an acceleration voltage of 120 kV. Images were recorded on imaging plates at a nominal magnification of 67,000× and a defocus value of −1.5 μm using low dose procedures. Imaging plates were read out with a scanner (DITABIS Digital Biomedical Imaging System AG, Pforzheim, Germany) using a step size of 15 μm, a gain setting of 20,000, and a laser power setting of 30% (ref. ); 2 × 2 pixels were averaged to yield a pixel size of 4.5 Å on the specimen level.A total of 11,336 Cog1-4 and 8,271 Cog2-4 particles were interactively selected from the raw images using BOXER, the display program associated with the EMAN software package. Using the SPIDER software package, these particles were windowed into 100 × 100 pixel images, rotationally and translationally aligned, and subjected to 10 cycles of multi-reference alignment. Each round of multi-reference alignment was followed by K-means classification specifying 200 output classes. The references used for the first multi-reference alignment were randomly chosen from the particle images. For GFP-labeled samples, a total of 7,904 particles were selected for GFP-Cog1/2/3/4, 8,698 particles for GFP-Cog2/1/3/4, 6,337 particles for GFP-Cog3/1/2/4, 10,014 particles for GFP-Cog4/1/2/3, 6,383 particles for Cog1-GFP/2/3/4, 5,923 particles for Cog2-GFP/1/3/4, 7,827 particles for Cog3-GFP/1/2/4, 7,790 particles for GFP-Cog4(1–553)/1/2/3, and 6,549 particles for GFP-Cog4/1/2/3/Cog8. The particle images were subjected to multi-reference alignment and K-means classification, specifying 200 output classes.Distance measurements of COG particles and averages were performed using the straight or segmented line distance measurement function in NIH ImageJ software version 1.41o (http://rsbweb.nih.gov/ij/). […]

Pipeline specifications

Software tools EMAN, SPIDER, ImageJ
Applications cryo-EM, Microscopic phenotype analysis