A Passage Through the Ribosome by Cryo-EM - Biophysical Approaches to Translational Control of Gene Expression - page 28

Molecular Microscopy

Cryo-TEM

￿

￿ ￿

Grid

Plunger

Sample

Liquid
Ethan e

Cryo grid preparation Data collection Structural analysis,

by: Chimera, PyMOL, Coot

300 kV
Low e - dose

Thin ice 3D Structure

cryo-grid

[ Randomly oriented
individual molecules
frozen in the amorphous
ice in their native forms ]

Image processing
by: SPIDER, EMAN

2D images at ~50k mag

Fig. 2.2 Schematic showing the cryo sample preparation, cryo-Electron Microscopy, and
image-processing methods

and then finally back projected using suitable image-processing software to generate
a 3D cryo-EM density map (3D structure). To scoop out the functional information
from the resulting cryo-EM structure, generally a compound approach is taken, where
the entire or the portions of the 3D EM density map is interpreted at the atomic level
through fitting or docking into its identifiable components with corresponding avail-
able atomic structure obtained from X-ray crystallography, NMR, or homology mod-
els. Thus, through this powerful method it is becoming increasingly possible to
investigate the native forms and the dynamics of any molecular machines caught at
any event of its biological actions. For a schematic of cryo-EM methodology see
Fig. 2.2 .
During protein synthesis, ribosome undergoes multiple dynamic conformational
changes very rapidly which materialize not only from its inherent molecular char-
acter but also due to its interactions with numerous ligands. In order to grasp the
detailed knowledge of the translation mechanisms one needs to trap these dynamic
events in rapid successions and study the corresponding structures in great detail.
Due to the highly dynamic nature of the ribosome-ligand interactions, it is very
challenging task for the X-ray crystallographers to obtain crystals from the series of
structural forms during the translation events. On the other hand, since ribosome is
a very large (~2.5 MDa) macromolecular machine it is impossible to study by NMR,
which is otherwise a very useful technique for studying dynamic smaller molecules.