Premium
Microtubule structure studied by quick freezing: Cryo‐electron microscopy and freeze fracture
Author(s) -
Mandelkow EvaMaria,
Rapp Roberto,
Mandelkow Eckhard
Publication year - 1986
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1986.tb02729.x
Subject(s) - electron microscope , liquid nitrogen , cryo electron microscopy , microtubule , resolution (logic) , microscopy , transmission electron microscopy , materials science , crystallography , chemistry , optics , nanotechnology , physics , biochemistry , organic chemistry , artificial intelligence , computer science , biology , microbiology and biotechnology
SUMMARY Microtubules have been quickly frozen and examined by electron microscopy using several techniques: (1) freezing of a thin layer of solution by plunging into cryogen, followed by cryo‐electron microscopy of the unstained vitrified samples; (2) freezing by the propane‐jet method, followed by freeze fracturing and metal replication. The unstained frozen‐hydrated microtubules show a structure in agreement with X‐ray diffraction data; they differ from negatively stained particles mainly by the better preservation of cylindrical shape. Secondly, they reveal a supertwist of the profilaments that is not detected reliably by other methods. This allows a determination of the number of protofilaments and the polarity. The structural resolution of unstained microtubules is similar to that of stained ones (about 2–3 nm); it is limited by low contrast and lack of crystalline order. Propane‐jet or cryo‐block freezing followed by freeze fracturing reveals the structures of the inner and outer surfaces of the microtubule wall at a resolution of 4 nm or better. The outside is dominated by the longitudinal protofilaments whereas on the inside one observes tilted cross‐striations. Although the freezing temperatures of the two methods are different (liquid nitrogen or helium) they yield similar results for the case of thin layers of protein solution.