Trimer processing workflow¶

reconstruct an ab initio model using common lines¶

1. select a set of ~20 good class averages, and upload them as a template stack (call it, e.g. "ts_2g12_initial_models")
2. 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_2g12_refinement")
3. You can access the common lines method under the menu "ab initio reconstruction" and "automated common lines" in Appion. Go there.
4. Under "iterative model creation" select the first template stack, "ts_2g12_initial_models"
5. Under "refinement of aligned and clustered model" select the second template stack, "ts_2g12_refinement"
6. keep all parameters as default, and use 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.
7. 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

1. Launch the job on guppy (you can also try garibaldi, but I know that guppy works)
2. 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" …).