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Controlled bacterial lysis for electron tomography of native cell membranes.


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 ?X174 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.

SUBMITTER: Fu X 

PROVIDER: S-EPMC4255137 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Controlled bacterial lysis for electron tomography of native cell membranes.

Fu Xiaofeng X   Himes Benjamin A BA   Ke Danxia D   Rice William J WJ   Ning Jiying J   Zhang Peijun P  

Structure (London, England : 1993) 20141120 12


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 rapidl  ...[more]

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