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Yorodumi- EMDB-6335: Three-Dimensional Reconstruction of Lipid Nanodisc Reconstituted ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6335 | |||||||||
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Title | Three-Dimensional Reconstruction of Lipid Nanodisc Reconstituted Yeast V-ATPase Membrane Sector | |||||||||
Map data | Reconstruction of Lipid Nanodisc Reconstituted Yeast V-ATPase Membrane Sector | |||||||||
Sample |
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Biological species | Saccharomyces cerevisiae (brewer's yeast) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 16.3 Å | |||||||||
Authors | Stam NJ / Wilkens S | |||||||||
Citation | Journal: J Biol Chem / Year: 2017 Title: Structure of the Lipid Nanodisc-reconstituted Vacuolar ATPase Proton Channel: DEFINITION OF THE INTERACTION OF ROTOR AND STATOR AND IMPLICATIONS FOR ENZYME REGULATION BY REVERSIBLE DISSOCIATION. Authors: Nicholas J Stam / Stephan Wilkens / Abstract: Eukaryotic vacuolar H-ATPase (V-ATPase) is a multisubunit enzyme complex that acidifies subcellular organelles and the extracellular space. V-ATPase consists of soluble V-ATPase and membrane-integral ...Eukaryotic vacuolar H-ATPase (V-ATPase) is a multisubunit enzyme complex that acidifies subcellular organelles and the extracellular space. V-ATPase consists of soluble V-ATPase and membrane-integral V proton channel sectors. To investigate the mechanism of V-ATPase regulation by reversible disassembly, we recently determined a cryo-EM reconstruction of yeast V The structure indicated that, when V is released from V, the N-terminal cytoplasmic domain of subunit a (a) changes conformation to bind rotor subunit d However, insufficient resolution precluded a precise definition of the a-d interface. Here we reconstituted V into lipid nanodiscs for single-particle EM. 3D reconstructions calculated at ∼15-Å resolution revealed two sites of contact between a and d that are mediated by highly conserved charged residues. Alanine mutagenesis of some of these residues disrupted the a-d interaction, as shown by isothermal titration calorimetry and gel filtration of recombinant subunits. A recent cryo-EM study of holo V-ATPase revealed three major conformations corresponding to three rotational states of the central rotor of the enzyme. Comparison of the three V-ATPase conformations with the structure of nanodisc-bound V revealed that V is halted in rotational state 3. Combined with our prior work that showed autoinhibited V-ATPase to be arrested in state 2, we propose a model in which the conformational mismatch between free V and V functions to prevent unintended reassembly of holo V-ATPase when activity is not needed. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6335.map.gz | 312.3 KB | EMDB map data format | |
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Header (meta data) | emd-6335-v30.xml emd-6335.xml | 9.6 KB 9.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_6335_fsc.xml | 4.1 KB | Display | FSC data file |
Images | 400_6335.gif 80_6335.gif | 57.7 KB 12.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6335 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6335 | HTTPS FTP |
-Validation report
Summary document | emd_6335_validation.pdf.gz | 77.9 KB | Display | EMDB validaton report |
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Full document | emd_6335_full_validation.pdf.gz | 77 KB | Display | |
Data in XML | emd_6335_validation.xml.gz | 493 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6335 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6335 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_6335.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of Lipid Nanodisc Reconstituted Yeast V-ATPase Membrane Sector | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.5 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : V-ATPase membrane sector (Vo) isolated from yeast membranes and r...
Entire | Name: V-ATPase membrane sector (Vo) isolated from yeast membranes and reconstituted with lipid nanodisc |
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Components |
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-Supramolecule #1000: V-ATPase membrane sector (Vo) isolated from yeast membranes and r...
Supramolecule | Name: V-ATPase membrane sector (Vo) isolated from yeast membranes and reconstituted with lipid nanodisc type: sample / ID: 1000 / Number unique components: 2 |
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Molecular weight | Theoretical: 400 KDa |
-Macromolecule #1: Vacuolar-type (V-) ATPase membrane sector (Vo)
Macromolecule | Name: Vacuolar-type (V-) ATPase membrane sector (Vo) / type: protein_or_peptide / ID: 1 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BY4743 / synonym: Yeast |
Molecular weight | Theoretical: 300 KDa |
-Macromolecule #2: MSP1E3D1
Macromolecule | Name: MSP1E3D1 / type: protein_or_peptide / ID: 2 / Name.synonym: membrane scaffold protein, nanodisc / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 30 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET28a |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.04 mg/mL |
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Buffer | pH: 7.4 / Details: 20 mM Tris-HCl, 100 mM NaCl, 0.5 mM EDTA |
Staining | Type: NEGATIVE Details: 2% w/v uranyl formate applied to grids with adsorbed protein |
Grid | Details: 200 mesh copper grid with thin carbon support, glow-discharged in air |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | JEOL 2100 |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification. |
Date | Nov 13, 2014 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Number real images: 390 / Average electron dose: 17 e/Å2 / Bits/pixel: 8 |
Electron beam | Acceleration voltage: 200 kV / Electron source: LAB6 |
Electron optics | Calibrated magnification: 85800 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.08 µm / Nominal defocus min: 1.125 µm / Nominal magnification: 60000 |
Sample stage | Specimen holder model: JEOL |