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Updated: May 9, 2026
Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method
Published on: July 27, 2016
Protocol for purification, single-particle cryo-EM structure determination, and functional assays of the fungal
Yue-Ran Ni1, Zi-Long You1, Lin Bai1
1Department of Biophysics, School of Basic Medical Sciences, Peking University, Beijing, China; State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China.
Abstract:
The fungal plasma membrane H+-ATPase (Pma1) maintains intracellular pH and membrane potential, but its study has been limited by challenges in sample preparation and functional assays. Here, we present a protocol for the endogenous purification of Pma1 in Saccharomyces cerevisiae, assessment of ATPase activity, cryo-electron microscopy (cryo-EM) structure determination, generation of a conditional pma1 knockout strain, and the performance of growth assays on mutants. For complete details on the use and execution of this protocol, please refer to You et al.1.
Insights
This study introduces a new protocol to purify and study the fungal plasma membrane H+-ATPase (Pma1) in yeast. This method enables detailed structural and functional analysis of Pma1, crucial for cellular homeostasis.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- The fungal plasma membrane H+-ATPase (Pma1) is vital for maintaining intracellular pH and membrane potential.
- Previous research on Pma1 has been hindered by difficulties in sample preparation and functional assays.
Purpose of the Study:
- To develop and present a comprehensive protocol for the endogenous purification and characterization of Pma1 in Saccharomyces cerevisiae.
- To overcome existing limitations in studying Pma1's structure and function.
Main Methods:
- Endogenous purification of Pma1 from Saccharomyces cerevisiae.
- Assessment of Pma1 ATPase activity.
- Cryo-electron microscopy (cryo-EM) for structure determination.
- Generation of a conditional pma1 knockout strain.
- Growth assays on pma1 mutant strains.
Main Results:
- A robust protocol for Pma1 purification was established.
- The study enabled the determination of the cryo-EM structure of Pma1.
- Functional and growth assays provided insights into Pma1's role.
Conclusions:
- The presented protocol facilitates detailed investigation of Pma1.
- This work provides a foundation for further studies on Pma1's mechanism and regulation.
- Understanding Pma1 is critical for fungal cell physiology.
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