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Principles & ProtocolsPrinciples - Plunge FreezingJump to ProtocolPreservation of structure for electron microsopyThe objective of any structural biology technique is to produce, for the system of interest, a structural model that most closely represents its native conformation. Non-physiological buffer conditions, such as exist in negative-stain preparations for EM as well as in many crystals for X-ray crystallography, may produce distortions in the structures under study. It is thus highly desirable to study the specimens under conditions that are physiological or native-like. 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.ProtocolsManualPlungeFreezingAutomated |