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- EMDB-3055: The vacuolar H+-ATPase masked around Vo -

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Basic information

Entry
Database: EMDB / ID: EMD-3055
TitleThe vacuolar H+-ATPase masked around Vo
Map dataReconstruction of the Vo domain from the V-ATPase using masking
Sample
  • Sample: Manduca sexta vacuolar ATPase complex
  • Organelle or cellular component: Vacuolar ATPase
KeywordsRotary ATPase / vacuolar ATPase
Biological speciesManduca sexta (tobacco hornworm)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.8 Å
AuthorsRawson S / Tiburcy F / Trinick J / Wieczorek H / Harrison MA / Muench SP
CitationJournal: Methods / Year: 2016
Title: Methods to account for movement and flexibility in cryo-EM data processing.
Authors: S Rawson / M G Iadanza / N A Ranson / S P Muench /
Abstract: Recent advances in direct electron detectors and improved CMOS cameras have been accompanied by the development of a range of software to take advantage of the data they produce. In particular they ...Recent advances in direct electron detectors and improved CMOS cameras have been accompanied by the development of a range of software to take advantage of the data they produce. In particular they allow for the correction of two types of motion in cryo electron microscopy samples: motion correction for movements of the sample particles in the ice, and differential masking to account for heterogeneity caused by flexibility within protein complexes. Here we provide several scripts that allow users to move between RELION and standalone motion correction and centring programs. We then compare the computational cost and improvements in data quality with each program. We also describe our masking procedures to account for conformational flexibility. For the different elements of this study we have used three samples; a high symmetry virus, flexible protein complex (∼1MDa) and a relatively small protein complex (∼550kDa), to benchmark four widely available motion correction packages. Using these as test cases we demonstrate how motion correction and differential masking, as well as an additional particle re-centring protocol can improve final reconstructions when used within the RELION image-processing package.
History
DepositionJun 19, 2015-
Header (metadata) releaseJul 8, 2015-
Map releaseJul 6, 2016-
UpdateAug 10, 2016-
Current statusAug 10, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Vo_13499_305...

Downloads & links

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Map

FileDownload / File: emd_3055.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the Vo domain from the V-ATPase using masking
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 320 pix.
= 432. Å		1.35 Å/pix.
x 320 pix.
= 432. Å		1.35 Å/pix.
x 320 pix.
= 432. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.07646108 - 0.18398903
Average (Standard dev.)0.0007246 (±0.00512674)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 432.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z432.000432.000432.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0760.1840.001

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Supplemental data

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Sample components

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Entire : Manduca sexta vacuolar ATPase complex

EntireName: Manduca sexta vacuolar ATPase complex
Components
  • Sample: Manduca sexta vacuolar ATPase complex
  • Organelle or cellular component: Vacuolar ATPase

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Supramolecule #1000: Manduca sexta vacuolar ATPase complex

SupramoleculeName: Manduca sexta vacuolar ATPase complex / type: sample / ID: 1000 / Details: monodisperse / Oligomeric state: monomeric / Number unique components: 1
Molecular weightExperimental: 900 KDa / Method: mass spec

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Supramolecule #1: Vacuolar ATPase

SupramoleculeName: Vacuolar ATPase / type: organelle_or_cellular_component / ID: 1 / Name.synonym: V-ATPase / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Manduca sexta (tobacco hornworm) / synonym: tobacco hornworm
Molecular weightExperimental: 900 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.025 mg/mL
BufferpH: 8.1
Details: 150 mM NaCl, 20 mM Tris-HCl, 9.6 mM 2-mercaptoethanol, 0.01% C12E10
GridDetails: 400 mesh Quantifoil R2.0/2.0 grids with thin carbon (10 nm) coating, glow discharged in air.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV / Method: Grids were blotted for 7.5 seconds

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailsCollected with FEI EPU software
DateFeb 27, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 1366 / Average electron dose: 60 e/Å2
Details: Each micrograph is sum of 34 frames recorded by direct detector.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 103704 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.5 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsStandard procedures in RELION1.3, using mask around Vo
CTF correctionDetails: Relion
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.8 Å / Resolution method: OTHER / Software - Name: Relion
Details: Maximum likelihood in Relion using 3D auto-refine and a mask around Vo. The particles were handpicked in BOXER
Number images used: 13083

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