Label-free single-instance protein detection in vitrified cells
| Autor: | Rickgauer Jp, Heejun Choi, Winfried Denk, Jennifer Lippincott-Schwartz |
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| Rok vydání: | 2020 |
| Předmět: | |
| DOI: | 10.1101/2020.04.22.053868 |
| Popis: | A general method to map molecular interactions and conformational states in structurally intact cells would find wide application in biochemistry and cell biology. We used a library of images— calculated on the basis of known structural data—as search templates to detect targets as small as the “head” domain (350 kDa) of the ribosome’s small subunit in single-tilt electron cryo-micrographs by cellular high resolution template matching (cHRTM). Atomically precise position and orientation estimates reveal the conformation of individual ribosomes and enable the detection of specifically bound ligands down to 24 kDa. We show that highly head-swivelled states are likely to play a role in mRNA translocation in living cells. cHRTM outperforms cryo-electron tomography three-fold in sensitivity and completely avoids the vicissitudes of exogenous labelling. |
| Databáze: | OpenAIRE |
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