MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel

Abstract

A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.

Benchmarking ideal sample thickness in cryo-EM using MicroED

“…Twenty-one crystals over six grids were chosen for milling. Crystals were milled as described (23, 29, 30, 40, 41). Briefly, the milling was conducted in steps of: rough milling, fine milling, and polishing.…”

Protocol for the use of focused ion-beam milling to prepare crystalline lamellae for microcrystal electron diffraction (MicroED)

“…General instructions on the operation of the TEM and dual-beam instruments to prepare lamellae of non-crystalline materials are described elsewhere ( Marko et al., 2007 ; Medeiros et al., 2018 ; Wagner et al., 2020 ). The general workflows and approaches of milling samples prior to collecting cryo-tomography data are overall similar ( Buckley et al., 2020 ; Gorelick et al., 2019 ; Zachs et al., 2020 ), but there are differences when preparing crystalline lamellae ( Martynowycz et al., 2020a ). Focused ion-beam milling is a general approach to sample preparation for MicroED samples ( Beale et al, 2020 ; Duyvesteyn et al., 2018 ; Li et al., 2018 ; Martynowycz et al., 2019a , 2019b ; Polovinkin et al., 2020 ; Zhou et al., 2019 ).…”

“…Blotting grids with protein crystals can be sensitive to the conditions of the blotting chamber, such as the relative humidity, temperature, blotting time, blotting force, and the method of blotting (back blotting, front blotting, etc. )( Martynowycz et al., 2020a , 2020b ). The protocol we present is the typical approach used in the Gonen lab, and works in most cases.…”

“…Crystals may also be flat sheets that are very difficult to detect. In these cases, SEM imaging may help locate crystals ( Martynowycz et al., 2020a ).…”

“…For complete details on the use and execution of this protocol, please refer to Martynowycz et al. (2019a , 2020a) .…”