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- EMDB-2318: Single particle cryo-microscopy reconstruction of the isolated Ma... -

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Basic information

Entry
Database: EMDB / ID: EMD-2318
TitleSingle particle cryo-microscopy reconstruction of the isolated Manduca sexta V1 domain
Map dataV1 domain without the C subunit bound
Sample
  • Sample: M. sexta isolated V1 domain without subunit C
  • Protein or peptide: Vacuolar ATPase isolated V1 domain
KeywordsV-ATPase / atp synthase / membrane complex
Biological speciesManduca sexta (tobacco hornworm)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 20.0 Å
AuthorsMuench SP / Scheres SHW / Huss M / Phillips C / Vitavska O / Wieczorek H / Trinick J / Harrison MA
CitationJournal: J Mol Biol / Year: 2014
Title: Subunit positioning and stator filament stiffness in regulation and power transmission in the V1 motor of the Manduca sexta V-ATPase.
Authors: Stephen P Muench / Sjors H W Scheres / Markus Huss / Clair Phillips / Olga Vitavska / Helmut Wieczorek / John Trinick / Michael A Harrison /
Abstract: The vacuolar H(+)-ATPase (V-ATPase) is an ATP-driven proton pump essential to the function of eukaryotic cells. Its cytoplasmic V1 domain is an ATPase, normally coupled to membrane-bound proton pump ...The vacuolar H(+)-ATPase (V-ATPase) is an ATP-driven proton pump essential to the function of eukaryotic cells. Its cytoplasmic V1 domain is an ATPase, normally coupled to membrane-bound proton pump Vo via a rotary mechanism. How these asymmetric motors are coupled remains poorly understood. Low energy status can trigger release of V1 from the membrane and curtail ATP hydrolysis. To investigate the molecular basis for these processes, we have carried out cryo-electron microscopy three-dimensional reconstruction of deactivated V1 from Manduca sexta. In the resulting model, three peripheral stalks that are parts of the mechanical stator of the V-ATPase are clearly resolved as unsupported filaments in the same conformations as in the holoenzyme. They are likely therefore to have inherent stiffness consistent with a role as flexible rods in buffering elastic power transmission between the domains of the V-ATPase. Inactivated V1 adopted a homogeneous resting state with one open active site adjacent to the stator filament normally linked to the H subunit. Although present at 1:1 stoichiometry with V1, both recombinant subunit C reconstituted with V1 and its endogenous subunit H were poorly resolved in three-dimensional reconstructions, suggesting structural heterogeneity in the region at the base of V1 that could indicate positional variability. If the position of H can vary, existing mechanistic models of deactivation in which it binds to and locks the axle of the V-ATPase rotary motor would need to be re-evaluated.
History
DepositionFeb 20, 2013-
Header (metadata) releaseMar 20, 2013-
Map releaseOct 9, 2013-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image2318.png

Downloads & links

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Map

FileDownload / File: emd_2318.map.gz / Format: CCP4 / Size: 825.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationV1 domain without the C subunit bound
Voxel sizeX=Y=Z: 4.36 Å
Density
Contour LevelBy AUTHOR: 1.2 / Movie #1: 1.2
Minimum - Maximum-1.46844101 - 6.18527985
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-30-30-30
Dimensions606060
Spacing606060
CellA=B=C: 261.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.364.364.36
M x/y/z606060
origin x/y/z0.0000.0000.000
length x/y/z261.600261.600261.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-12-40
NX/NY/NZ732581
MAP C/R/S123
start NC/NR/NS-30-30-30
NC/NR/NS606060
D min/max/mean-1.4686.185-0.000

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Supplemental data

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Sample components

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Entire : M. sexta isolated V1 domain without subunit C

EntireName: M. sexta isolated V1 domain without subunit C
Components
  • Sample: M. sexta isolated V1 domain without subunit C
  • Protein or peptide: Vacuolar ATPase isolated V1 domain

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Supramolecule #1000: M. sexta isolated V1 domain without subunit C

SupramoleculeName: M. sexta isolated V1 domain without subunit C / type: sample / ID: 1000
Details: The sample was monodisperse and not freeze thawed to maintain V1 integrity
Oligomeric state: One V1 bound to a monomer of subunit C / Number unique components: 1
Molecular weightExperimental: 650 KDa / Theoretical: 650 KDa / Method: Mass Spec

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Macromolecule #1: Vacuolar ATPase isolated V1 domain

MacromoleculeName: Vacuolar ATPase isolated V1 domain / type: protein_or_peptide / ID: 1 / Name.synonym: V1 / Details: No C subunit / Number of copies: 1 / Oligomeric state: 3A,B,E,G subunits and 1 D,F,H subunit / Recombinant expression: No
Source (natural)Organism: Manduca sexta (tobacco hornworm) / synonym: Tobacco Hornworm / Tissue: midgut epithelium
Molecular weightExperimental: 650 KDa / Theoretical: 650 KDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 8.1 / Details: 150mM NaCl, 20mM Tris-HCL, 0.01% C12E10
StainingType: NEGATIVE
Details: negative stain data was collected using 1% w/v uranyl acetate for 1 minute.
GridDetails: UV glow discharged grids, 400 mesh
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV
Method: Grids were blotted for 6 seconds with a 6 second drain time

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 69000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.0035 µm / Nominal defocus min: 0.001 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Nitrogen cooled 3350 holder / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 90 K / Max: 110 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: Astigmatism corrected for each image in focus mode at 100,000 times magnification
DetailsFEI Low dose mode
DateApr 14, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Average electron dose: 16 e/Å2 / Details: All collected on Gatan 4K x 4K CCD
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Relion
Final angle assignmentDetails: Relion angular sampling 10 degrees
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: OTHER / Software - Name: Relion
Details: Maximum likelihood in Relion using the MLF3D protocol
Number images used: 16500
DetailsThe particles were handpicked in BOXER

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Atomic model buiding 1

Initial modelPDB ID:

3a5d


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: G / Chain - #7 - Chain ID: H
SoftwareName: Chimera
DetailsThe domains were fitted using Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: best fit

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