8FYO

MicroED structure of Proteinase K from lamellae milled from multiple plasma sources

8FYO の概要

• エントリーDOI: 10.2210/pdb8fyo/pdb
• EMDBエントリー: 29587
• 分子名称: Proteinase K, NITRATE ION, CALCIUM ION, ... (4 entities in total)
• 機能のキーワード: hydrolase
• 由来する生物種: Parengyodontium album
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 29100.95

構造登録者

Martynowycz, M.W.,Shiriaeva, A.,Clabbers, M.T.B.,Nicolas, W.J.,Weaver, S.J.,Hattne, J.,Gonen, T. (登録日: 2023-01-26, 公開日: 2023-05-24, 最終更新日: 2024-10-23)

主引用文献

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

実験手法

ELECTRON CRYSTALLOGRAPHY (1.39 Å)