Demonstration of electron diffraction from membrane protein crystals grown in a lipidic mesophase after lamella preparation by focused ion beam milling at cryogenic temperatures

Abstract

Electron crystallography of sub-micrometre-sized 3D protein crystals has emerged recently as a valuable field of structural biology. In meso crystallization methods, utilizing lipidic mesophases, particularly lipidic cubic phases (LCPs), can produce high-quality 3D crystals of membrane proteins (MPs). A major step towards realizing 3D electron crystallography of MP crystals, grown in meso, is to demonstrate electron diffraction from such crystals. The first task is to remove the viscous and sticky lipidic matrix that surrounds the crystals without damaging the crystals. Additionally, the crystals have to be thin enough to let electrons traverse them without significant multiple scattering. In the present work, the concept that focused ion beam milling at cryogenic temperatures (cryo-FIB milling) can be used to remove excess host lipidic mesophase matrix is experimentally verified, and then the crystals are thinned to a thickness suitable for electron diffraction. In this study, bacteriorhodopsin (BR) crystals grown in a lipidic cubic mesophase of monoolein were used as a model system. LCP from a part of a hexagon-shaped plate-like BR crystal (∼10 µm in thickness and ∼70 µm in the longest dimension), which was flash-frozen in liquid nitrogen, was milled away with a gallium FIB under cryogenic conditions, and a part of the crystal itself was thinned into a ∼210 nm-thick lamella with the ion beam. The frozen sample was then transferred into an electron cryo-microscope, and a nanovolume of ∼1400 × 1400 × 210 nm of the BR lamella was exposed to 200 kV electrons at a fluence of ∼0.06 e Å−2. The resulting electron diffraction peaks were detected beyond 2.7 Å resolution (with an average peak height to background ratio of >2) by a CMOS-based Ceta 16M camera. The results demonstrate that cryo-FIB milling produces high-quality lamellae from crystals grown in lipidic mesophases and pave the way for 3D electron crystallography on crystals grown or embedded in highly viscous media.

Macromolecular crystallography using microcrystal electron diffraction

“…By inducing a transition from LCP to less viscous mesophases or sponge phases, it has recently been illustrated that LCP-grown microcrystals of G protein-coupled receptor (GPCR) could be analysed by MicroED (Zhu et al, 2020). An alternative way to deal with the high viscosity is by using cryo-focused ion-beam (cryo-FIB) milling to prepare thin crystalline lamella (see also Section 3.4), which has been demonstrated as a potential routine for lipid-embedded bacteriorhodopsin microcrystals (Polovinkin et al, 2020). However, these reports did not resolve a structural model of any lipid-embedded membrane protein.…”

“…For the final stages of the milling process, a fine polishing step using a low-current ion beam can be performed to make a smoother crystal surface for MicroED data collection (Martynowycz et al, 2019b). Cryo-FIB makes it possible to collect MicroED data from crystals with a wider range of sizes and morphologies, even those crystals embedded in a thick layer of vitreous ice or highly viscous sample conditions such as LCP (Zhu et al, 2020;Polovinkin et al, 2020;Martynowycz, Shiriaeva et al, 2020;Martynowycz, Khan et al, 2020). Furthermore, creating large thin crystal lamella with controlled specimen thickness can improve the data quality.…”

Refinements on electron diffraction data of β-glycine in MoPro: a quest for an improved structure model

“…Unlike X-ray diffraction, where the X-ray beam is scattered by the electron density, in 3D ED, the electron beam is scattered by the electrostatic (Coulomb) potential of the sample. Apart from advances in technology for electron diffraction instruments (Kolb et al, 2011;Wang et al, 2019;Hattne et al, 2019;Gemmi et al, 2019;Polovinkin et al, 2020), the major reason for drawing the attention of chemists and biologists to 3D ED is the nano-size of crystals. With 3D ED it is possible to obtain high-resolution data from nano-crystals with an accuracy approaching that of X-ray diffraction data and to determine the absolute configuration (Sawaya et al, 2016;Jones et al, 2018;Gruene et al, 2018;Krysiak et al, 2018;Clabbers et al, 2019;Brá zda et al, 2019;Xu & Zou, 2019), something that is impossible with X-ray diffraction.…”

A simple pressure-assisted method for MicroED specimen preparation

“…However, their addition makes the buffer viscous. Lipid cubic phase (LCP) is commonly used for crystallization of membrane protein crystals, and they are extremely viscous as toothpaste (Martynowycz et al, 2019;Caffrey, 2015;Polovinkin et al, 2020). It has been very challenging to prepare MicroED samples for crystals grown in viscous buffers (Nannenga & Gonen, 2019;Martynowycz et al, 2019;Shi et al, 2016a).…”

“…The pipetting-blotting-plunging rountine (Dubochet & McDowall, 1981), originally designed for single-particle cryoEM specimen preparation, has two major drawbacks for MicroED specimen preparation; 1) many microcrystals are removed by blotting and 2) it is insufficient in removing viscous liquids (Shi et al, 2016b). Manual back-side blotting (Shi et al, 2016b), direct crystallization on EM grids (Li et al, 2018), and cryo-FIB (Martynowycz et al, 2019;Beale et al, 2020;Polovinkin et al, 2020;Martynowycz et al, 2020) have been proposed as possible solutions to deal with the above problems. However, a simple and universal method for MicroED specimen preparation is still missing.…”