Preferential orientation workflow

Hello There!
I have an elongated protein complex that exhibits preferred orientation. I am gearing to collect a new dataset using a tilted stage. My question is in regards to downstream processing: can I do all of my processing in cryoSparc, or will I need to incorporate Relion to process tilted data? I’ve been told I need Relion and I just wanted to confirm one way or the other (hopefully that I won’t need it!) Thanks!


You can do everything in CryoSPARC. :slight_smile: In my experience, highly tilted data are actually a little easier in CryoSPARC because Patch CTF Estimation can compensate for tilt across the image, while CTFFIND calculates a global (whole micrograph) CTF, which is significantly off for particles at the extremes of the tilt.


Possibly useful reference here (this is mixed csparc & relion, but definitely can all be done in CS):


Another option is GoCTF, which is CTFFIND modified to be able to do per-particle CTF estimation.

The Michigan site to download it is now dead, but the Google Site still works (at least the form is still there). If the form is broken (it was the last time I tried it a few months ago) I do have a previously downloaded copy of the source code in my archives (I’ll need a day or so to find it).

Warp is another option for defocus gradient estimation and subsequent per particle CTF interpolation, although updates have been hard to come by since Dimitry joined Genentech. Similar concept to Patch CTF. In our hands, it worked more robustly than GoCTF and GCTF for this purpose. Unfortunately, we hadn’t benchmarked it against Patch CTF, but I imagine the two would produce similar outcomes, at least up to 30-40deg stage tilt.


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