Cryo-electron tomography (cryo-ET) provides a promising approach to study intact structures of macromolecules in situ, but the efficient preparation of high-quality cryosections represents a bottleneck. Although cryo-focused ion beam (cryo-FIB) milling has emerged for large and flat cryo-lamella preparation, its application to tissue specimens remains challenging. Here, we report an integrated workflow, VHUT-cryo-FIB, for efficiently preparing frozen hydrated tissue lamella that can be readily used in subsequent cryo-ET studies. The workflow includes vibratome slicing, high-pressure freezing, ultramicrotome cryo-trimming and cryo-FIB milling. Two strategies were developed for loading cryo-lamella via a side-entry cryo-holder or an FEI AutoGrid. The workflow was validated by using various tissue specimens, including rat skeletal muscle, rat liver and spinach leaf specimens, and in situ structures of ribosomes were obtained at nanometer resolution from the spinach and liver samples.
International Workshop of 3D Molecular Imaging by Cryo-Electron Microscopy, Third K. H. Kuo Summer School of Electron Microscopy and Crystallography in 2010.
International Workshop of Advanced Image Processing of Cryo-Electron Microscopy, 2013
Get acquainted with Cryo-Electron Microscopy: First Chinese Workshop for Structural Biologists, 2015
International Workshop of Advanced Image Processing of Cryo-Electron Microscopy, 2015
Instutions
Instutions
Institute of Biophysics, Chinese Academy of Sciences
The Scripps Research Institute
Max Planck Institute of Biochemistry
Database
Database
National Center for Biotechnology Information(NCBI)
Protein Data Bank
The Electron Microscopy Data Bank
ExPASy Proteomics Server
Pfam
3D EM
3DEM
Tools and Softwars
Tools and Softwars
CCP4
CCP-EM
MOLE 2.0 (characterization of channels and pores in protein complex)
