How do you prepare samples for cryo-EM?
Cryo-EM samples are typically prepared using several microliters of protein solution at a concentration ranging between 50 nM and 5 μM, depending on the sample, EM grids and conditions used. The studied biological specimen should remain active in the in vivo optimized conditions (buffer composition etc.)
How much sample do you need for cryo-EM?
Conventional sample preparation for cryo-EM requires several microliters of a purified protein solution at a concentration of ∼1 mg/mL per grid, from which extensive filter-paper blotting later removes the vast majority of protein particles (9⇓⇓–12).
Does cryo-EM destroy the sample?
In Cryo-EM, samples are plunged frozen to cryogenic temperatures and embedded in vitreous water. Then a softer electron beam is used to interact with the sample which does not destroy the sample or evaporate the water around it (unlike TEMs). This does not require crystallization either, unlike x-ray crystallography.
How do you make cryo-EM grids?
Cryo-EM Grid Preparation: Plunge Freezing by Blotting A few microliters of purified sample are adsorbed onto a grid, blotted with filter paper to make a thin aqueous layer, and then plunge frozen into the cryogen. The vitrified ice should be as thin as possible to support the particle.
What is vitrification in cryo-EM?
Samples are prepared for cryo-EM by plunge freezing in liquid ethane. This results in rapid freezing of the water into vitreous ice containing the suspended samples.
Why is cryo-EM so important?
PROS. A major advantage of cryo-EM over x-ray crystallography is that the molecule of interest does not have to be crystallised. Some proteins or important macromolecules simply can not be crystallised; others have their structures irreversibly changed by crystallisation.
What is cryo-EM grid?
Cryogenic sample-grid preparation allows fixing biological samples by rapidly transferring and cooling them in liquid ethane (−188°C). Under these conditions, ice crystals are unable to form, thus preserving the specimen integrity.
Does Cryo require crystalline samples?
Unlike X-ray crystallography, cryo-EM does not require crystallized samples. This freezing—or rather vitrifying—is so rapid that sample molecules are immobilized with their structure preserved and without ice crystals that interfere with transmission electron microscopy (TEM).
How is cryo-EM done?
The technique involves flash-freezing solutions of proteins or other biomolecules and then bombarding them with electrons to produce microscope images of individual molecules. These are used to reconstruct the 3D shape, or structure, of the molecule.