Appion

h2. Trimer processing workflow

h1. reconstruct an ab initio model using common lines

# select a set of ~20 good class averages in the image viewer, and upload them as a template stack (call it, e.g. "ts_initial_models")

# select as many of the class averages that have reliable features which you think are real and correspond to the Fab-bound complex (could be 50, could be 100, etc …), and upload that as another template stack (call it, e.g. "ts_refinement")

# You can access the common lines method under the menu "ab initio reconstruction" and "automated common lines" in Appion. Go there. 

# Under "iterative model creation" select the first template stack, "ts_initial_models"

# Under "refinement of aligned and clustered model" select the second template stack, "ts_refinement"

# keep all parameters as default, and try EMAN for iterative model creation. Then fill out the number of images per volume. This should be ~50% of the number of images in the "ts_2g12_initial_models" stack. If you have, e.g. 30 images, this value should be ~15. You can also try IMAGIC for iterative model creation by switching between the two options. 

# Fill out the bottom radii for the particles and give a mass. For example, for 2g12, these should probably be: "radius of mask"=200 ; "inner alignment radius" = 0 ; "outer alignment radius" = 140 ; "particle mass" = 750

# Launch the job

# repeat 1-8 with an identical job, EXCEPT that uncheck "iteratively align class averages to each other", and give the run a different name (for example, you can have two runs, titled "acl1" and "acl1_noiteralign" …).