PMID- 36841804
OWN - NLM
STAT- MEDLINE
DCOM- 20230228
LR  - 20230311
IS  - 2041-1723 (Electronic)
IS  - 2041-1723 (Linking)
VI  - 14
IP  - 1
DP  - 2023 Feb 25
TI  - A robust approach for MicroED sample preparation of lipidic cubic phase embedded 
      membrane protein crystals.
PG  - 1086
LID - 10.1038/s41467-023-36733-4 [doi]
LID - 1086
AB  - 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.
CI  - (c) 2023. The Author(s).
FAU - Martynowycz, Michael W
AU  - Martynowycz MW
AD  - Howard Hughes Medical Institute, University of California, Los Angeles, CA, 
      90095, USA.
AD  - Department of Biological Chemistry, University of California, Los Angeles, CA, 
      90095, USA.
FAU - Shiriaeva, Anna
AU  - Shiriaeva A
AUID- ORCID: 0000-0002-4082-7884
AD  - Howard Hughes Medical Institute, University of California, Los Angeles, CA, 
      90095, USA.
AD  - Department of Biological Chemistry, University of California, Los Angeles, CA, 
      90095, USA.
FAU - Clabbers, Max T B
AU  - Clabbers MTB
AUID- ORCID: 0000-0002-5466-6508
AD  - Howard Hughes Medical Institute, University of California, Los Angeles, CA, 
      90095, USA.
AD  - Department of Biological Chemistry, University of California, Los Angeles, CA, 
      90095, USA.
FAU - Nicolas, William J
AU  - Nicolas WJ
AD  - Howard Hughes Medical Institute, University of California, Los Angeles, CA, 
      90095, USA.
AD  - Department of Biological Chemistry, University of California, Los Angeles, CA, 
      90095, USA.
FAU - Weaver, Sara J
AU  - Weaver SJ
AD  - Howard Hughes Medical Institute, University of California, Los Angeles, CA, 
      90095, USA.
AD  - Department of Biological Chemistry, University of California, Los Angeles, CA, 
      90095, USA.
FAU - Hattne, Johan
AU  - Hattne J
AUID- ORCID: 0000-0002-8936-0912
AD  - Howard Hughes Medical Institute, University of California, Los Angeles, CA, 
      90095, USA.
AD  - Department of Biological Chemistry, University of California, Los Angeles, CA, 
      90095, USA.
FAU - Gonen, Tamir
AU  - Gonen T
AUID- ORCID: 0000-0002-9254-4069
AD  - Howard Hughes Medical Institute, University of California, Los Angeles, CA, 
      90095, USA. tgonen@g.ucla.edu.
AD  - Department of Biological Chemistry, University of California, Los Angeles, CA, 
      90095, USA. tgonen@g.ucla.edu.
AD  - Department of Physiology, University of California, Los Angeles, CA, 90095, USA. 
      tgonen@g.ucla.edu.
LA  - eng
GR  - P41 GM136508/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20230225
PL  - England
TA  - Nat Commun
JT  - Nature communications
JID - 101528555
RN  - 0 (Membrane Proteins)
RN  - EC 3.4.21.64 (Endopeptidase K)
RN  - 0 (Lipids)
SB  - IM
MH  - *Membrane Proteins
MH  - *Electrons
MH  - Cryoelectron Microscopy/methods
MH  - Endopeptidase K
MH  - Lipids/chemistry
PMC - PMC9968316
COIS- The authors declare no competing interests.
EDAT- 2023/02/26 06:00
MHDA- 2023/03/03 06:00
PMCR- 2023/02/25
CRDT- 2023/02/25 23:40
PHST- 2022/08/08 00:00 [received]
PHST- 2023/02/15 00:00 [accepted]
PHST- 2023/02/25 23:40 [entrez]
PHST- 2023/02/26 06:00 [pubmed]
PHST- 2023/03/03 06:00 [medline]
PHST- 2023/02/25 00:00 [pmc-release]
AID - 10.1038/s41467-023-36733-4 [pii]
AID - 36733 [pii]
AID - 10.1038/s41467-023-36733-4 [doi]
PST - epublish
SO  - Nat Commun. 2023 Feb 25;14(1):1086. doi: 10.1038/s41467-023-36733-4.