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TitleNear-atomic resolution reconstructions from in situ revitrified cryo samples.
Journal, issue, pagesActa Crystallogr D Struct Biol, Vol. 79, Issue Pt 6, Page 473-478, Year 2023
Publish dateJun 1, 2023
AuthorsGabriele Bongiovanni / Oliver F Harder / Jonathan M Voss / Marcel Drabbels / Ulrich J Lorenz /
PubMed AbstractA microsecond time-resolved version of cryo-electron microscopy (cryo-EM) has recently been introduced to enable observation of the fast conformational motions of proteins. The technique involves ...A microsecond time-resolved version of cryo-electron microscopy (cryo-EM) has recently been introduced to enable observation of the fast conformational motions of proteins. The technique involves locally melting a cryo sample with a laser beam to allow the proteins to undergo dynamics in the liquid phase. When the laser is switched off, the sample cools within just a few microseconds and revitrifies, trapping particles in their transient configurations, in which they can subsequently be imaged. Two alternative implementations of the technique have previously been described, using either an optical microscope or performing revitrification experiments in situ. Here, it is shown that it is possible to obtain near-atomic resolution reconstructions from in situ revitrified cryo samples. Moreover, the resulting map is indistinguishable from that obtained from a conventional sample within the spatial resolution. Interestingly, it is observed that revitrification leads to a more homogeneous angular distribution of the particles, suggesting that revitrification may potentially be used to overcome issues of preferred particle orientation.
External linksActa Crystallogr D Struct Biol / PubMed:37219589 / PubMed Central
MethodsEM (single particle)
Resolution1.59 - 1.61 Å
Structure data

EMDB-15715: Mouse heavy chain apoferritin in plunge-frozen vitreous ice
Method: EM (single particle) / Resolution: 1.61 Å

EMDB-15721: Mouse heavy chain apoferritin after laser melting and revitrification
Method: EM (single particle) / Resolution: 1.59 Å

Source
  • Mus musculus (house mouse)

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