It’s the month for fall favorites.
Like the growing success of GDN.

If you’ve been keeping up with our newsletters, you know that we get really excited when we get the opportunity to highlight our customers' remarkable research. This September has been a particularly exciting month. As of this writing, there have been four Cryo-EM structures of membrane proteins published this month using glyco-diosgenin (GDN)(1)!  
In our earlier newsletters highlighting GDN success stories (see: Nov, 2018 and Mar, 2018), we discussed how GDN is gaining in popularity as a synthetic drop-in substitute for Digitonin(2). There are many advantages to using GDN over Digitonin, including high solubility, no batch-to-batch variability, no toxic byproducts, and price. This gaining popularity is evidenced by the publication of over 10 Cryo-EM structures determined using GDN thus far in 2019.

The four Cryo-EM structures published this September include:

 
  • The mouse PIEZO2 mechanosensitive ion channel (PDB: 6KG7) published in the September 12th issue of Nature by the labs of Xueming Li and Bailong Xiao at Tsinghua University in Beijing(3). Comprising 114 transmembrane helices, the PIEZO2 trimer plays a role in converting mechanical forces from the environment (e.g. touch and pain) into biological signals. To prepare the PIEZO2 protein for Cryo-EM structure determination, membranes were solubilized using 0.012% GDN, 0.1% C12E9, 1.0% CHAPS, and 0.5% Phosphatidylcholine, and the protein was purified using 0.02% GDN. To improve the vitrification process, 0.65 mM fluorinated Fos-Choline-8 was added immediately before freezing.
  • The human TRPM2 channel (PDB: 6PUO) published on September 12th in the journal eLife by the labs of Wei Lu and Juan Du at the Van Andel Institute(4). The TRPM2 protein is a transient receptor potential cation channel involved in a number of key physiological processes in the body. Here, TRPM2 was solubilized using 10 mM GDN, and further purified using 0.2 mM GDN for Cryo-EM structure determination.
  • The human ATP8A1-CSC50a complex (PDB:6K7G), a P4-ATP flippase, published in the September 13th issue of Science by the labs of Tomohiro Nishizawa and Osamu Nureki at the University of Tokyo(5). This series of structures show how lipids are translocated from the outer to the inner leaflet of a cell membrane. In this study, the ATP8A1 protein was solubilized using 1.5% / 0.15% DDM/CHS and purified using 0.06% GDN for Cryo-EM structure determination.
  • The C. reinhardtii thermosensitive TRP channel TRP1 published on September 13th in Nature Communications by the lab of Alex Sobolevsky at Columbia University(6). The structure of TRP1 has a unique two-fold symmetry and differs from other TRP channel structures. To determine this structure, the TRP1 protein was solubilized using 2% digitonin, and exchanged into 0.01% GDN for Cryo-EM.

Want even more GDN success stories? Check out other recently published structures including: the mouse epithelial anion transporter, Slc26a9 (PDB: 6RTC) published by the lab of Raimund Dutzler at the University of Zurich in July(7); the human LAT1-4F2hc heteromeric amino acid transporter complex (PDB: 6IRS) published by the lab of Qiang Zhou at Westlake University in China in April(8); and the human TPC2 channel (PDB: 6NQ1) published by the labs of Xiao-chen Bai and Youxing Jiang at UTSW in March(9).

Do you have a success story using one of our products you would like us to highlight in a future newsletter? Let us know!

Tools for Cryo-EM:

Anatrace and Molecular Dimensions are continually developing tools and reagents to support the membrane and soluble protein Cryo-EM workflow. These include fluorinated surfactants to improve vitrification, amphipols, protein stability screens, grid boxes and grid box storage pucks, and more. Check out our Cryo-EM workflow page to learn more about all of the latest tools we have to support your Cryo-EM experiments!

 
 References:
  1. Chae, P. S., et al. (2012) Chemistry 18(31), 9485-9490.
  2. Autzen, H.E. et al. (2019) Curr Opin Struct Biol. doi: 10.1016/j.sbi.2019.05.022.
  3. Wang, L. et al. (2019) Nature. doi: 10.1038/s41586-019-1505-8.
  4. Huang, Y. et al. (2019) eLife. 8:e50175. doi: 10.7554/eLife.50175.
  5. Hiraizumi, M. et al. (2019) Science. doi: 10.1126/science.aay3353.
  6. McGoldrick, L.L, et al. (2019) Nat Commun. doi: 10.1038/s41467-019-12121-9.
  7. Walter, J.D., et al. (2019) eLife. 8:e46986. doi: 10.7554/eLife.46986.
  8. Yan, R., et al. (2019) Nature. 568(7750):127-130.
  9. She, J., et al. (2019) eLife. 8:e45222 doi: 10.7554/eLife.45222.