Cryo-EM/ET Methods

 

Cryogenic Electron Microscopy (cryoEM) is an electron microscopy (EM) technique applied on samples cooled to cryogenic temperatures and embedded in an environment of vitreous water. In most cases, we want molecules to be evenly distributed without overlapping in the thin ice. However, in the example shown here, the in vitro reconstitution of outer-arm dynein(OAD) and microtubule doublets (MTD), OAD and MTD unavoidably overlap. Because of symmetry mismatch and the dominant signal from tubulin-lattice, the OAD particles can not be properly aligned.  We have developed a reference-free approach to first model the tubulin-lattice signals and then subtract them in raw micrographs, which allows the accurate alignment of OAD particles. The principle can be easily extended to other similar biological systems. 

 

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