Principles
Vitreous ice and preservation of structure
The objective of any structural biology technique is to obtain for the complex of interest
a structural model that most closely represents the complex in its native conformation.
Non-physiological buffer conditions, as existing in negative-stain preparations for EM as well
as in many crystals for X-ray crystallography, may produce artifactual distortions in the
complexes under study. It is thus highly desirable to acquire structural data for a sample
under physiological or native-like conditions.
Plunge freezing a thin layer of sample adhering to an EM grid ensures that due to the rapid
decrease in temperature the disordered nature of the aqueous solvent is preserved. Disordered
frozen water is referred to as vitreous ice and allows similar hydrogen-bonding networks
(intra-complex and complex-solvent) as does liquid water. Thus, complexes embedded in
vitreous ice
preserve their native-like structure(s). In addition, the creation of vitreous ice during the
process of plunge freezing the sample minimizes structural damage to the complexes that would
otherwise occur due to the formation of ordered ice crystals.
For 3-D reconstruction using single particle analysis plunge freezing the sample of interest
without the use of stain, cryo-protectants, or other non-physiological agents, is the most
ideal method of choice for preservation and subsequent study. Since complexes are rapidly frozen
while in solution, i.e. the complexes are in a frozen-hydrated state, possible structural
disortions due to crystal packing are also avoided.
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Protocols
Protocols
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