Nanodisc, amphipol or detergent belts in cryoEM reconstructions of membrane proteins are similar and correspond to a common ordered solvent layer

Abstract

SummaryTo maintain membrane proteins soluble in aqueous solution, amphipathic compounds are used to shield the hydrophobic patch of their membrane insertion, which forms a belt around the protein. This hydrophobic belt is seldom looked at due to the difficulty to visualize it. Cryo-EM is now offering this possibility, where belts are visible in 3D reconstructions. We investigated membrane proteins solved in nanodiscs, amphipols or detergents to analyze whether the nature of the amphipathic compound influences the belt size in 3D reconstructions. We identified belt boundaries in map-density distributions and measured distances for every reconstruction. We showed that all the belts create on average similar reconstructions, whether they originate from the same protein, or from protein from different shapes and structures. There is no difference among detergents or types of nanodisc used. These observations illustrate that the belt observed in 3D reconstructions corresponds to the minimum ordered layer around membrane proteins.

The Det.Belt Server: A Tool to Visualize and Estimate Amphipathic Solvent Belts around Membrane Proteins

“…This belt is shaped as a taurus. The one observed by cryoEM roughly matches the same taurus [ 20 ]. Both methods use an averaging procedure that results in seeing the commonality between proteins within the crystal or particles on the grid, respectively, thereby removing differences between individual proteins/particles.…”

“…However, the detergent belt is very mobile as was shown by molecular dynamics simulations of detergents [ 16 ]. The averaging process of SPA thus truncates a part of the detergent signal, resulting in belts smaller than they really are [ 20 ].…”

Simulation and Machine Learning Methods for Ion-Channel Structure Determination, Mechanistic Studies and Drug Design

“…While being essential for mechanistic studies and rational drug design, molecular structures for ion channels are more difficult to purify and crystallize than soluble proteins, mainly due to the necessity and difficulty of preserving the membrane-like environment. Detergents, amphipols, and nanodiscs have been commonly used to extract membrane proteins such as ion channels, and also serve as substitutes for the local membrane to stabilize the transmembrane domains (Zampieri et al, 2021). In recent years, the single-particle cryo-EM technique has rapidly evolved as a powerful method for structure determination for various ion channels (Martin et al, 2017;Basak et al, 2019;Dang et al, 2019;Masiulis et al, 2019;Zhao et al, 2019;Wang Q. et al, 2020;Sun and MacKinnon, 2020;Lin et al, 2021;Song et al, 2021;Yu et al, 2021).…”

“…Ion channels naturally reside in biological membranes which means preserving the amphiphilic local environment for the channel transmembrane domains is essential during cryo-EM sample preparation. Techniques such as nanodiscs can serve as good replacement for the membrane (Zampieri et al, 2021). However, the disordered nature of these membrane memetics also add difficulties to the cryo-EM structural analysis.…”