We did screen through other surfactants but found these four to be the most reliable in our hands. With the number of options for surfactants and their concentrations, using a low concentration combo hopes to take out the guess work of which surfactant to use and the associated cost of screening.
Hey Sean, with both HA and MoFeP, there have been single surfactants used and we find that SurfACT matches or succeeds the resolution and particle distributions. Especially for HA, we had minimal optimization and screening, the data shown was from one single freezing session and collection.
9) This was a fantastic team effort led by Suzanne Enos with a fun and insightful collaboration between the Herzik lab at @ucsandiego.bsky.social and Grotjahn lab at @scripps.edu. #cryoET #cryoEM #HerzikLab @sptlabtech.bsky.social @structurabio.bsky.social @thermofishersci.bsky.social
8) While we explore SurfACT in our preprint: tinyurl.com/SurfACT-prep..., we show that this cocktail can be a generalizable solution to enable many difficult proteins adopt more ideal behaviors during freezing.
7) Using cryoET and STA we find that aldolase particles stuck at the AWI not only adopt a single orientation, but these particles are also partially denatured at the AWI! Only when aldolase particles were pushed into bulk ice with SurfACT did they remain intact.
6) Importantly, we demonstrate that SurfACT can be used with any of your favorite grid prep devices! SPT Labtech chameleon ✅ TFS Vitrobot ✅ Custom manually-operated plunger ✅.
5) We first sought to test SurfACT with a pathologically preferred specimen, hemagglutinin. We find that without SurfACT, the particles adopt a “top-down” view and the density is incomplete. Addition of SurfACT drastically reduces PO, flipping particles and leading to a complete ~2.1 Å density.
4) We teamed up with @nanigrotjahn.bsky.social and @hamid13r.bsky.social to do a through examination of SurfACT with four known proteins that suffer from PO. Combining #SPA and #cryoET paired with #STA, we show that SurfACT overcomes PO for these proteins and provides insights into this problem.
3) Our surfactant cocktail, SurfACT, is designed with minimal sample-dependent screening in mind and can be used across protein classes and EM sample preparation devices.
2) ) PO, where particles adopt non-random orientations at the AWI, is a leading causes of failure in single-particle cryoEM. We rationally designed a cocktail of four synergistic surfactants that disrupt particle interactions with the AWI to improve reconstruction quality and particle integrity.
1) The Herzik lab is excited to announce our newest preprint in collaboration with the Grotjahn lab! We have developed a surfactant cocktail that seeks to overcome one of the more nefarious problems in #cryoEM, Air-water interface (AWI)-induced damage and preferred orientation (PO).
Unfortunately, im not finding a great review of current SPA data processing bu the cryoSPARC case studies can give a good idea of where things are: guide.cryosparc.com/processing-d...
For a pretty good (although detailed) book: iopscience.iop.org/book/edit/97...
And pretty good review of sample prep and imaging: www.sciencedirect.com/science/arti...
Subtomogram Averaging of a mitochondrially co-translating ribosome
Happy to share that the final version of record for our (
@nanigrotjahn.bsky.social) #teamtomo mitochondrial cotranslation story is online at @jcb.org! We combined STA with surface morphometrics to learn how ribosomes bind to and alter mitochondrial membranes in yeast.
rupress.org/jcb/article/...
Nature research paper: Structural basis for the conformational protection of nitrogenase from O2
https://go.nature.com/4aiD344
Awesome work from Sarah, @bcryo.bsky.social and friends in the Herzik Lab! It was cool to see this come together in such a short time.
7) This was an exciting study led by Sarah Narehood in collaboration with Suppachai Srisantitham, Vanessa Eng, Angela Shiau, Kelly McGuire, David Britt. #cryoEM #cryoSPARC #Nitrogenase #HerizkLab @nature.com @sptlabtech.bsky.social @structurabio.bsky.social #UCSDChemBioChem #anaerobicEM
6) We were able to achieve these structures using our newly developed (an)aerobic freezing protocol using the SPT chameleon. This allowed us to generate high quality girds with thin ice without having to be in an anaerobic chamber!
www.biorxiv.org/content/10.1...
5) Our structures combined with our analytical ultracentrifugation and EPR experiments allow us to propose a mechanism by which these oxygen protecting complexes and filaments form in oxidizing conditions, protecting MoFeP and FeP, and disassemble in reducing conditions.
4) Surprisingly, the binding of Shethna occurs at the catalytic interface of both MoFeP and FeP! Preventing nitrogen fixation while also protecting the oxygen sensitive metal clusters harbored by both MoFeP and FeP.
3) We show that Shetna is capable of binding both the catalytic (MoFeP) and reductant (FeP) subunits, creating oxygen protecting complex. Fascinatingly, this complex is capable of forming higher order complexes and ultimately filaments!
2) The only known nitrogen fixing enzyme employed by microbes is nitrogenase, a highly oxygen sensitive protein complex. In this study we were able to determine multiple structures of Nitrogenase in complex with the small protein Shethna (FeSII), solving a decades long mystery.
1) Since I am the only Herzik lab member on @bsky.app, I get the privilege to announce our most recent publication from the Herzik/Tezcan labs. I got the wonderful opportunity to contribute on the cryoEM study of the oxygen protection mechansim employed by cells to protect Nitrogenase!
