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  Large scale expression, purification and 2D crystallization of recombinant plant plasma membrane H+-ATPase

Jahn, T., Dietrich, J., Andersen, B., Leidvik, B., Otter, C., Briving, C., et al. (2001). Large scale expression, purification and 2D crystallization of recombinant plant plasma membrane H+-ATPase. Journal of Molecular Biology (London), 309(2), 465-476. doi:10.1006/jmbi.2001.4688.

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Genre: Journal Article

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 Creators:
Jahn, Thomas1, Author
Dietrich, Jens2, Author           
Andersen, Birgitte1, Author
Leidvik, Brith3, Author
Otter, Charlotta3, Author
Briving, Carin3, Author
Kühlbrandt, Werner2, Author           
Palmgren, Michael Gjedde1, Author
Affiliations:
1Department of Plant Biology The Royal Veterinary and Agricultural University Thorvaldsensvej 40 DK-1871 Frederiksberg C, Copenhagen Denmark, ou_persistent22              
2Department of Structural Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068291              
3Astra Zeneca R&D Mölndal S-431 83 Mölndal, Sweden, ou_persistent22              

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Free keywords: Arabidopsis thaliana; 14-3-3 protein; fusicoccin; proton pump; Saccharomyces cerevisiae
 Abstract: P-type ATPases convert chemical energy into electrochemical gradients that are used to energize secondary active transport. Analysis of the structure and function of P-type ATPases has been limited by the lack of active recombinant ATPases in quantities suitable for crystallographic studies aiming at solving their three-dimensional structure. We have expressed Arabidopsis thaliana plasma membrane H+-ATPase isoform AHA2, equipped with a His6-tag, in the yeast Saccharomyces cerevisiae. The H+-ATPase could be purified both in the presence and in the absence of regulatory 14-3-3 protein depending on the presence of the diterpene fusicoccin which specifically induces formation of the H+-ATPase/14-3-3 protein complex. Amino acid analysis of the purified complex suggested a stoichiometry of two 14-3-3 proteins per H+-ATPase polypeptide. The purified H+-ATPase readily formed two-dimensional crystals following reconstitution into lipid vesicles. Electron cryo-microscopy of the crystals yielded a projection map at ∼8 Å resolution, the p22121 symmetry of which suggests a dimeric protein complex. Three distinct regions of density of approximately equal size are apparent and may reflect different domains in individual molecules of AHA2.

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Language(s): eng - English
 Dates: 2001-04-022000-11-212001-04-022002-05-252001-06-01
 Publication Status: Published in print
 Pages: 12
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1006/jmbi.2001.4688
 Degree: -

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Title: Journal of Molecular Biology (London)
  Other : J Mol Biol
Source Genre: Journal
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Publ. Info: London : Academic Press
Pages: - Volume / Issue: 309 (2) Sequence Number: - Start / End Page: 465 - 476 Identifier: ISSN: 0022-2836
CoNE: https://pure.mpg.de/cone/journals/resource/954922646042