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- EMDB-9023: In situ structure of 96-nm repeat unit of Tetrahymena ciliary axo... -

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Basic information

Entry
Database: EMDB / ID: EMD-9023
TitleIn situ structure of 96-nm repeat unit of Tetrahymena ciliary axoneme by TYGRESS method
Map dataIn situ structure of 96 nm repeat unit from intact Tetrahymena ciliary axoneme resolved by a newly developed TYGRESS method
Sample
  • Organelle or cellular component: 96 nm repeat unit from Tetrahymena ciliary axoneme
Biological speciesTetrahymena thermophila CU428 (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.0 Å
AuthorsSong K / Shang Z / Fu X / Lou X / Grigorieff N / Nicastro D
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesRO1 GM111506 United States
Citation
Journal: Nat Methods / Year: 2020
Title: In situ structure determination at nanometer resolution using TYGRESS.
Authors: Kangkang Song / Zhiguo Shang / Xiaofeng Fu / Xiaochu Lou / Nikolaus Grigorieff / Daniela Nicastro /
Abstract: The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited owing to signal loss in, and misalignment of, the ...The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited owing to signal loss in, and misalignment of, the subtomograms. By contrast, single-particle cryo-electron microscopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We report a method called 'tomography-guided 3D reconstruction of subcellular structures' (TYGRESS) that is a hybrid of cryo-ET and SP-cryo-EM, and is able to achieve close-to-nanometer resolution of complexes inside crowded cellular environments. TYGRESS combines the advantages of SP-cryo-EM (images with good signal-to-noise ratio and contrast, as well as minimal radiation damage) and subtomogram averaging (three-dimensional alignment of macromolecules in a complex sample). Using TYGRESS, we determined the structure of the intact ciliary axoneme with up to resolution of 12 Å. These results reveal many structural details that were not visible by cryo-ET alone. TYGRESS is generally applicable to cellular complexes that are amenable to subtomogram averaging.
#1: Journal: biorXiv
Title: Structure of the ciliary axoneme at nanometer resolution reconstructed by TYGRESS
Authors: Song K / Shang Z / Fu X / Lou X / Grigorieff N / Nicastro D
History
DepositionAug 4, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseAug 21, 2019-
UpdateFeb 19, 2020-
Current statusFeb 19, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0078
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0078
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9023.png

Downloads & links

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Map

FileDownload / File: emd_9023.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ structure of 96 nm repeat unit from intact Tetrahymena ciliary axoneme resolved by a newly developed TYGRESS method
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.11 Å/pix.
x 600 pix.
= 1267.2 Å		2.11 Å/pix.
x 600 pix.
= 1267.2 Å		2.11 Å/pix.
x 600 pix.
= 1267.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.112 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0078 / Movie #1: 0.0078
Minimum - Maximum-0.016238 - 0.027837448
Average (Standard dev.)0.00016155842 (±0.0046692295)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-120-120-120
Dimensions600600600
Spacing600600600
CellA=B=C: 1267.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.1122.1122.112
M x/y/z600600600
origin x/y/z0.0000.0000.000
length x/y/z1267.2001267.2001267.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-532-20
NX/NY/NZ1019393
MAP C/R/S123
start NC/NR/NS-120-120-120
NC/NR/NS600600600
D min/max/mean-0.0160.0280.000

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Supplemental data

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Sample components

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Entire : 96 nm repeat unit from Tetrahymena ciliary axoneme

EntireName: 96 nm repeat unit from Tetrahymena ciliary axoneme
Components
  • Organelle or cellular component: 96 nm repeat unit from Tetrahymena ciliary axoneme

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Supramolecule #1: 96 nm repeat unit from Tetrahymena ciliary axoneme

SupramoleculeName: 96 nm repeat unit from Tetrahymena ciliary axoneme / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Tetrahymena thermophila CU428 (eukaryote) / Organelle: Cilia

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.0 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 9400 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: OTHER / Details: From subtomogram alignment
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: FSC 0.143 CUT-OFF / Details: TYGRESS was used for the reconstruction method / Number images used: 18857

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