A Passage Through the Ribosome by Cryo-EM - Biophysical Approaches to Translational Control of Gene Expression - page 27

Fig. 2.1 A 5.5 Å resolution cryo-EM map of Triticum asestivum 80S ribosome. Source : From the
“Fig 1, A” of the “Supporting Information” section of Armache et al. ( 2010 )

2.1.1 Why Cryo-EM is a Sought After Technique

in the Ribosome Field?

Cryo-electron microscopy is a powerful technique which is predominantly used in
studying the 3D structure of large macromolecules in different functional states.
Using liquid ethane, a dilute solution of a biological macromolecular complex is
flash frozen in a thin layer of vitreous ice on an EM grid, and then single molecular
entities are imaged under cryo-Transmission Electron Microscope (cryo-TEM) at
low electron dose condition. Due to the flash freezing in noncrystalline ice, the ran-
domly oriented individual macromolecules (particles) become trapped in their native
forms. Two-dimensional (2D) projection images of those particles are collected under
high (~50 K or more) magnification in the cryo-TEM. Since no heavy metal stains
are used in the sample preparation, the signal to noise ratio of the images of the par-
ticles to the background ice becomes very low, resulting in low contrast images. To
address this issue and to obtain a full spectrum of information, hundreds of thousands
of individual images are collected at a series of under-defocus ranges in the cryo-
TEM. From the digital images of those collected and subsequently selected good
particles, contrast transfer functions (CTF) are calculated. Particles falling in the
same orientations are classified, averaged, merged to other class averaged particles