A Robust Approach for MicroED Sample Preparation of Lipidic Cubic Phase Embedded Membrane Protein Crystals

Overview

Journal Nat Commun

Specialty Biology

Date 2023 Feb 25

PMID 36841804

Authors

Michael W Martynowycz

Anna Shiriaeva

Max T B Clabbers

William J Nicolas

Sara J Weaver

Johan Hattne

Tamir Gonen

Affiliations

Soon will be listed here.

Abstract

Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae.

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