8FYR

MicroED structure of Proteinase K from oxygen milled lamellae

Summary for 8FYR

• Entry DOI: 10.2210/pdb8fyr/pdb
• EMDB information: 29595
• Descriptor: Proteinase K, NITRATE ION, CALCIUM ION, ... (4 entities in total)
• Functional Keywords: hydrolase
• Biological source: Parengyodontium album
• Total number of polymer chains: 1
• Total formula weight: 29162.96

Authors

Martynowycz, M.W.,Shiriaeva, A.,Clabbers, M.T.B.,Nicolas, W.J.,Weaver, S.J.,Hattne, J.,Gonen, T. (deposition date: 2023-01-26, release date: 2023-03-08, Last modification date: 2024-10-23)

Primary citation

Martynowycz, M.W.,Shiriaeva, A.,Clabbers, M.T.B.,Nicolas, W.J.,Weaver, S.J.,Hattne, J.,Gonen, T.
A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals.
Nat Commun, 14:1086-1086, 2023

PubMed 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.

PubMed: 36841804
DOI: 10.1038/s41467-023-36733-4

Experimental method

ELECTRON CRYSTALLOGRAPHY (1.5 Å)