Preparation of macromolecular complexes for cryo-electron microscopy.
This protocol describes the preparation of frozen-hydrated single-particle specimens of macromolecular complexes. First, it describes how to create a grid surface coated with holey carbon by first inducing holes in a Formvar film to act as a template for the holey carbon that is stable under cryo-electron microscopy (cryo-EM) conditions and is sample-friendly. The protocol then describes the steps required to deposit the homogeneous sample on the grid and to plunge-freeze the grid into liquid ethane at the temperature of liquid nitrogen, so that it is suitable for cryo-EM visualization. It takes 4-5 h to make several hundred holey carbon grids and about 1 h to make the frozen-hydrated grids. The time required for sample purification varies from hours to days, depending on the sample and the specific procedure required. A companion protocol details how to collect cryo-EM data using an FEI Tecnai transmission electron microscope that can subsequently be processed to obtain a three-dimensional reconstruction of the macromolecular complex.
Grassucci
Robert A
RA
Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032, USA.
Taylor
Derek J
DJ
Frank
Joachim
J
D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods*; D003451:Cryoprotective Agents; D005615:Freezing; D007053:Ice; D046911:Macromolecular Substances
D003451:Cryoprotective Agents; D007053:Ice; D046911:Macromolecular Substances