Cryo-electron microscope analysis of frozen-hydrated crystals of Na, K-ATPase.

Maunsbach, Arvid B.; Hebert, Hans; Kavéus, Urban

doi:10.1267/ahc.25.279

Cited by 8 publications

(4 citation statements)

References 21 publications

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“…The specific Na,K‐ATPase activity of the enzyme peaks recovered from the zonal gradient and used for crystallization was 20–30 μmol P i /min·mg protein. The purified membrane fragments were dialyzed against 1 mM NH 4 VO 3 , 5 mM MgCl 2 , 5 mM CaCl 2 , and 0.33 mM phospholipase A 2 in 10 mM imidazole‐HCl, pH 7.3, at 4°C for 1 day 15,22…”

Section: Methodsmentioning

confidence: 99%

“…The purified membrane fragments were dialyzed against 1 mM NH 4 VO 3 , 5 mM MgCl 2 , 5 mM CaCl 2 , and 0.33 mM phospholipase A 2 in 10 mM imidazole-HCl, pH 7.3, at 4 o C for 1 day. 15,22…”

Section: Enzyme Purificationmentioning

confidence: 99%

“…14). Cryo-electron microscopy has more recently been applied both to flat 2-D crystals obtained from renal Na,K-ATPase [15][16][17] and tubular crystals prepared from duck salt glands, 18 but 3-D crystals of Na,K-ATPase suitable for X-ray crystallography have not yet been obtained.…”

Section: Introductionmentioning

confidence: 99%

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Renal Na,K‐ATPase Structure from Cryo‐electron Microscopy of Two‐Dimensional Crystals

Hebert

Purhonen

Thomsen³

et al. 2003

Annals of the New York Academy of Sciences

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The molecular structure of Na,K-ATPase was determined by electron crystallography from two-dimensional crystals induced in purified membranes isolated from the outer medulla of pig kidney. The P2 type unit cell contains two protomers in the E(2) conformation, each of them with a size of 65 x 75 x 150 A(3). The alpha, beta, and gamma subunits in the membrane crystals were demonstrated in the crystals with Western blotting and related to distinct domains in the density map. The alpha subunit corresponds to most of the density in the transmembrane region as well as to the large hydrophilic headpiece on the cytoplasmic side of the membrane. The headpiece is divided into three separated domains. One of these gives rise to an elongated projection onto the membrane plane, while the putative nucleotide binding and phosphorylation domains form compact densities in the rest of the cytoplasmic part of the structure. Density on the extracellular face corresponds to the protein part of the beta subunit. Ten helices from the catalytic a subunit correspond to two groups of distinct densities in the transmembrane region. The structure of the lipid bilayer spanning part also suggests positions for the transmembrane helices from the beta and gamma subunits. The overall structure of the alpha subunit of Na,K-ATPase as determined here by cryo-electron microscopy is similar to the X-ray structure of Ca-ATPase. However, conformational changes between the E(1) and E(2) forms are suggested by different relative positions of cytoplasmic domains.

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Section: Methodsmentioning

confidence: 99%

“…The purified membrane fragments were dialyzed against 1 mM NH 4 VO 3 , 5 mM MgCl 2 , 5 mM CaCl 2 , and 0.33 mM phospholipase A 2 in 10 mM imidazole-HCl, pH 7.3, at 4 o C for 1 day. 15,22…”

Section: Enzyme Purificationmentioning

confidence: 99%

See 1 more Smart Citation

Renal Na,K‐ATPase Structure from Cryo‐electron Microscopy of Two‐Dimensional Crystals

Hebert

Purhonen

Thomsen³

et al. 2003

Annals of the New York Academy of Sciences

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“…As a model system we have used twodimensional membrane crystals of Na,K-ATPase, which we have previously characterized with respect to molecular organization by means of different electron microscope methods (16,17). The results illustrate the usefulness of double immunonegative staining for epitope mapping on isolated Na,K-ATPase membranes and suggest that it is generally applicable for isolated membranes.…”

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confidence: 94%