Case Study: End-to-end and exploratory processing of a motor-bound nucleosome (EMPIAR-10739)

Cryo-EM Data Processing
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Hi CryoSPARC users! We are excited to share with you a new end-to-end processing case study of a nucleosome with a motor protein bound that you can follow along with!

Here we guide you all the way from downloading an EMPIAR dataset through to improving the quality of the particle stack, considering pseudosymmetry, classifying minor populations, and generating a map ready for model-building.

We walk through improving the particle stack by using:

  • Global CTF Estimation

  • Local CTF Estimation

  • Subset particles by Statistic on the basis of particle scale

We describe strategies to look for, and classify density for a low-population binding partner including:

  • 3DVA with a generous solvent mask

  • Creating a difference map from 3DVA volumes to aid mask generation

  • 3D Classification, and Particle Sets Tools to find minor classes

Our final sharpened maps, half maps and masks are downloadable to allow comparison with your results.

We hope this detailed guide is a useful resource for users old and new! If there are other types of targets, or particular single particle cryo-EM challenges that you would like to see a case study on in the future, let us know!

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Hello all,

I am one of the authors of the original study that generated EMPIAR-10739. If you explore this dataset and have any question, feel free to reach out! (If for some reason you prefer a different channel than this forum, my ORCiD always lists a current email address, and I am also active on Mastodon.)

As another example of the kind of things you might find in this dataset, you can check out our freshly released preprint about an activation intermediate of ALC1 loosely bound to the nucleosome. I just requested release of the PDB entry (9T4V), it should be online on the 19th.

This dataset is quite heterogeneous, and I am pretty sure there are other things to find in there. So if you find something that refines to domain or secondary structure resolution and does not look like any of the published structures (PDB 7OTQ or PDB 9T4V), please do tell me, I would love to know! And maybe we can even write a paper about it together.

I hope this will encourage more people to deposit their datasets to EMPIAR, especially if they are heterogeneous.

Happy processing!

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