Purification and cryoelectron microscopy structure determination of human V-ATPase

STAR Protoc. 2021 Feb 16;2(1):100350. doi: 10.1016/j.xpro.2021.100350. eCollection 2021 Mar 19.

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).

Keywords: Biophysics; Cryo-EM; Molecular biology; Protein biochemistry; Protein expression and purification; Structural biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryoelectron Microscopy*
  • HEK293 Cells
  • Humans
  • Protein Structure, Quaternary
  • Vacuolar Proton-Translocating ATPases* / biosynthesis
  • Vacuolar Proton-Translocating ATPases* / chemistry
  • Vacuolar Proton-Translocating ATPases* / isolation & purification
  • Vacuolar Proton-Translocating ATPases* / ultrastructure

Substances

  • Vacuolar Proton-Translocating ATPases