Format

Send to

Choose Destination
Structure. 2014 Dec 2;22(12):1875-1882. doi: 10.1016/j.str.2014.09.017. Epub 2014 Nov 20.

Controlled bacterial lysis for electron tomography of native cell membranes.

Author information

1
Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.
2
New York Structural Biology Center, 89 Convent Avenue, New York, NY 10027, USA.
3
Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA; Department of Mechanical Engineering and Materials Science, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA 15260, USA. Electronic address: pez7@pitt.edu.

Abstract

Cryo-electron tomography (cryoET) has become a powerful tool for direct visualization of 3D structures of native biological specimens at molecular resolution, but its application is limited to thin specimens (<300 nm). Recently, vitreous sectioning and cryoFIB milling technologies were developed to physically reduce the specimen thickness; however, cryoET analysis of membrane protein complexes within native cell membranes remains a great challenge. Here, we use phage ΦX174 lysis gene E to rapidly produce native, intact, bacterial cell membranes for high resolution cryoET. We characterized E gene-induced cell lysis using FIB/SEM and cryoEM and showed that the bacteria cytoplasm was largely depleted through spot lesion, producing ghosts with the cell membranes intact. We further demonstrated the utility of E-gene-induced lysis for cryoET using the bacterial chemotaxis receptor signaling complex array. The described method should have a broad application for structural and functional studies of native, intact cell membranes and membrane protein complexes.

PMID:
25456413
PMCID:
PMC4255137
DOI:
10.1016/j.str.2014.09.017
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center