For a century X-ray crystallography and NMR were the only tools for three dimensional structural determination of macromolecular complexes. Due to new advances in Cryo-electron microscopy (Cryo-EM) there is a new tool in this process for scientists to use which is gaining increased interest and popularity. The most significant work done with the Cyro-EM technique was the achievement of a 3.4 Å resolution structure of a mammalian TRP channel (TRPV1)(1) and a 4.5 Å gamma-Secretase membrane embedded aspartyl protease.(2) This innovation broke the side-chain resolution barrier for membrane proteins without crystallization.
As Cryo-EM becomes more widely used some of the additional challenges also become apparent. One of the most evident concerns when working with membrane proteins is that they require a solubilizing and stabilizing agent, which is usually a detergent. Adding detergents into the equation allows the membrane protein to oscillate and could introduce structural variations which are not immediately seen when localized in a membrane. Another challenge is that when detergents are used, they affect water surface tension and the thinness of the ice when flash frozen during the Cryo-EM preparation process.
Anatrace offers a solution to both concerns by offering an amphipathic polymer, Amphipol A8-35, to be used in place of a detergent. Amphipol A8-35 binds tightly to the transmembrane portion of the protein, which allows the protein to remain soluble and stable in buffers(2). Additionally, an Amphipol-stabilized membrane protein lacks excess free, non-associated, Amphipol molecules in the buffer which can become a major challenge when working with detergents(3). Amphipol A8-35 conformationally restrains membrane proteins as well as reduces pressure on water surface tension leading to thinner ice buildup during the nitrogen flash freezing process required for Cryo-EM.
Anatrace Amphipol A8-35 should be your first choice when using Cryo-EM for solubilizing and stabilizing membrane proteins in three dimensional structural determination.
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