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Purification and cryoelectron microscopy structure determination of human V-ATPase.

ABSTRACT: Vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases) are multi-component, ATP-driven proton pumps, which play important roles in many physiological processes by acidifying intracellular vesicles, organelles, and the extracellular milieu. Long-standing challenges in purifying mammalian V-ATPases have limited the biochemical and structural study of mammalian V-ATPase. Here, we provide a protocol for purifying milligrams of human V-ATPase and detail procedures for the reconstruction of its structure by cryo-EM. Our method can be applied to any biochemical and biophysical study of human V-ATPase. For complete details on the use and execution of this protocol, please refer to Wang et al. (2020).

SUBMITTER: Wang L

PROVIDER: S-EPMC7902551 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Purification and cryoelectron microscopy structure determination of human V-ATPase.

Wang Longfei L Chen Zhenhang Z Wu Hao H Fu Tian-Min TM

STAR protocols 20210216 1

Vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases) are multi-component, ATP-driven proton pumps, which play important roles in many physiological processes by acidifying intracellular vesicles, organelles, and the extracellular milieu. Long-standing challenges in purifying mammalian V-ATPases have limited the biochemical and structural study of mammalian V-ATPase. Here, we provide a protocol for purifying milligrams of human V-ATPase and detail procedures for the reconstruction of ...[more]

PMID: 33665630

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