Preparation of macromolecular complexes for cryo-electron microscopy

Abstract

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 threedimensional reconstruction of the macromolecular complex.

Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-EM

“…A ∼5-nm-thick layer of continuous carbon was deposited onto freshly cleaved mica using a Bal-Tec MED 020 evaporator (Leica Microsystems). The carbon was floated onto R2/4 Quantifoil grids (Micro Tools GmbH), and the grids were glow-discharged for 30 s, using a plasma cleaner (Harrick PKC-3XG; Harrick Scientific Corp.) to render them hydrophilic (36). Two micromoles of each 30S and 50S subunit (for a final concentration of 1 μM after mixing) in Hepes-K buffer with 12 mM MgCl 2 were preincubated at 37°C for 45 min before being used.…”

Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis

“…The sample consisted of preaccommodated E. coli 70S ribosome bound to the Phe-tRNA Phe ⅐EF-Tu⅐GDP ternary complex stalled by the antibiotic kirromycin (kir) (11) by using a standard preparation (31). The map was obtained from Ϸ130,000 particles, recorded with Ϸ58,000ϫ magnification on a FEI Tecnai F30 Polara electron microscope operated at 300 kV at liquid-nitrogen temperature (80 K) and processed by using SPIDER.…”

Structure and Cellular Dynamics of Deinococcus radiodurans Single-stranded DNA (ssDNA)-binding Protein (SSB)-DNA Complexes

“…Carbon films, mounted on 400-mesh EM grids, were first activated by a brief glow-discharge treatment (39). Activated grids were used immediately for spreading.…”