A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals

Abstract

Crystallization of membrane proteins, such as G protein-coupled receptors (GPCRs), is challenging and frequently requires the use of lipidic cubic phase (LCP) crystallization methods. These typically yield crystals that are too small for synchrotron X-ray crystallography, but ideally suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, the viscous nature of LCP makes sample preparation challenging. The LCP layer is often too thick for transmission electron microscopy (TEM), and crystals buried in LCP cannot be identified topologically using a focused ion-beam and scanning electron microscope (FIB/SEM). Therefore, the LCP needs to either be converted to the sponge phase or entirely removed from the path of the ion-beam to allow identification and milling of these crystals. Unfortunately, conversion of the LCP to sponge phase can also deteriorate the sample. Methods that avoid LCP conversion are needed. Here, we employ a novel approach using an integrated fluorescence light microscope (iFLM) inside of a FIB/SEM to identify fluorescently labelled crystals embedded deep in a thick LCP layer. The crystals are then targeted using fluorescence microscopy and unconverted LCP is removed directly using a plasma focused ion beam (pFIB). To assess the optimal ion source to prepare biological lamellae, we first characterized the four available gas sources on standard crystals of the serine protease, proteinase K. However, lamellae prepared using either argon and xenon produced the highest quality data and structures. Fluorescently labelled crystals of the human adenosine receptor embedded in thick LCP were placed directly onto EM grids without conversion to the sponge phase. Buried microcrystals were identified using iFLM, and deep lamellae were created using the xenon beam. Continuous rotation MicroED data were collected from the exposed crystalline lamella and the structure was determined using a single crystal. This study outlines a robust approach to identifying and milling LCP grown membrane protein crystals for MicroED using single microcrystals, and demonstrates plasma ion-beam milling as a powerful tool for preparing biological lamellae.

MicroED structure of the human vasopressin 1B receptor

“…These instruments were first demonstrated to be viable for milling embedded biological materials at room temperature using an oxygen beam (Gorelick et al, 2018). This was followed by the first cryogenic temperature milling of protein crystals, where pFIBs were also shown to remove material faster, and ultimately resulted in better quality data (Martynowycz et al, 2022b). pFIBs have since also been shown to be useful for the preparation of cellular lamellae for cryoET investigations (Berger et al, 2022).…”

Cryo-plasma FIB/SEM volume imaging of biological specimens

“…This effect was present despite the "smooth" surface of the lamella and was attributed to damage introduced by Ar colliding with and penetrating the sample. The dependency of obtainable resolution on the ion source used has been shown in electron diffraction (Martynowycz et al 2022). For Serial pFIB/SEM, the wattage used for argon 2-fold higher than that used for the final step of lamella polishing (200 pA at 20 kV vs 50 pA at 30 kV).…”