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  • Title: Microtubule structure studied by quick freezing: cryo-electron microscopy and freeze fracture.
    Author: Mandelkow EM, Rapp R, Mandelkow E.
    Journal: J Microsc; 1986 Mar; 141(Pt 3):361-73. PubMed ID: 3517351.
    Abstract:
    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 cryoelectron 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.
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