Hi all. I come from a crystallography background and am new to cryo-EM, so please bear with me.
I am facing processing issues with a multiprotein complex ~200 kDa. My 2D classes are decent and some of my ab initio classes look sensible, but I am lost in processing thereafter. I see what looks to be compositional heterogeneity in the sample and this is reflected in my 2D classes and ab initio classes (i.e. can see different binding stoichiometries). My main question is how do I sort through this heterogeneity? I’ve tried 3D classification and not sure how to interpret the results/where to go from there.
Thanks!
Hi @jeffamine7000, welcome to Cryo-EM and welcome to the forum!
Sorting through heterogeneity is one of the big challenges in Cryo-EM, especially coming from a crystallography background where that’s all done before you ever make the crystal 
If you haven’t already, I’d recommend checking out one (or more) of our case studies. They use known-good data to guide you through processing steps and explain why decisions are made at each stage. This way you can learn how to process data without worrying about whether the data itself is good, and then apply those lessons back to your own work. For your case, I’d recommend trying the DkTx-bound TRPV1 (EMPIAR-10059) case study, which lays out a nice particle curation workflow. There’s also a recording of a workshop lecture covering this same material, if you prefer a video.
If you still have questions after reading through a case study, go ahead and ask them here! In general, I think the community will have an easier time helping you if you can share a few example images – what do your 2D and Ab-Initio classes look like, etc.
Again, welcome to the forum! Please feel free to reply with questions you still have
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