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- EMDB-8992: Cryo-electron tomogram of mouse rod connecting cilium and mother ... -

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Basic information

Entry
Database: EMDB / ID: EMD-8992
TitleCryo-electron tomogram of mouse rod connecting cilium and mother centriole with C9 sub-tomogram averaging
Map dataMap derived from C9 sub-tomogram averaging of B04.mrc. Used to generate Fig. 1a(1,3) and 1b-1h.
Sample
  • Organelle or cellular component: Base of connecting cilium and mother centriole of mouse rod cell.
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsZhang Z / Liu J / Schmid MF / Wensel TG
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Eye InstituteR01-EY026545 United States
National Institutes of Health/National Institute of General Medical SciencesP41-GM103832 United States
National Institutes of Health/National Eye InstituteR01-EY007981 United States
Robert A. Welch FoundationQ-0035 United States
Robert A. Welch FoundationAU-1714 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Defining the layers of a sensory cilium with STORM and cryoelectron nanoscopy.
Authors: Michael A Robichaux / Valencia L Potter / Zhixian Zhang / Feng He / Jun Liu / Michael F Schmid / Theodore G Wensel /
Abstract: Primary cilia carry out numerous signaling and sensory functions, and defects in them, "ciliopathies," cause a range of symptoms, including blindness. Understanding of their nanometer-scale ciliary ...Primary cilia carry out numerous signaling and sensory functions, and defects in them, "ciliopathies," cause a range of symptoms, including blindness. Understanding of their nanometer-scale ciliary substructures and their disruptions in ciliopathies has been hindered by limitations of conventional microscopic techniques. We have combined cryoelectron tomography, enhanced by subtomogram averaging, with superresolution stochastic optical reconstruction microscopy (STORM) to define subdomains within the light-sensing rod sensory cilium of mouse retinas and reveal previously unknown substructures formed by resident proteins. Domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of subcompartments, including the lumen and walls of the axoneme, the membrane glycocalyx, and the intervening cytoplasm. Within this framework, we report spatial distributions of key proteins in wild-type (WT) mice and the effects on them of genetic deficiencies in 3 models of Bardet-Biedl syndrome.
History
DepositionJul 25, 2018-
Header (metadata) releaseOct 2, 2019-
Map releaseOct 2, 2019-
UpdateDec 4, 2019-
Current statusDec 4, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.16
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.16
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8992.png

Downloads & links

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Map

FileDownload / File: emd_8992.map.gz / Format: CCP4 / Size: 1.9 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap derived from C9 sub-tomogram averaging of B04.mrc. Used to generate Fig. 1a(1,3) and 1b-1h.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
8.9 Å/pix.
x 800 pix.
= 7120. Å		8.9 Å/pix.
x 800 pix.
= 7120. Å		8.9 Å/pix.
x 800 pix.
= 7120. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 8.9 Å
Density

Histogram

Histogram (log scale)
Contour LevelMovie #1: 4.16
Minimum - Maximum-0.00000565859 - 4.4027042
Average (Standard dev.)2.3207843 (±1.6486316)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin27-160
Dimensions800800800
Spacing800800800
CellA=B=C: 7119.9995 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z8.98.98.9
M x/y/z800800800
origin x/y/z0.0000.0000.000
length x/y/z7120.0007120.0007120.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS-16270
NC/NR/NS800800800
D min/max/mean-0.0004.4032.321

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Supplemental data

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Additional map: Map constructed from IMOD. Used to generate Fig.1i (1).

Fileemd_8992_additional_1.map
AnnotationMap constructed from IMOD. Used to generate Fig.1i (1).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Map constructed from IMOD. Used to generate Fig. 1a(1,3).

Fileemd_8992_additional_2.map
AnnotationMap constructed from IMOD. Used to generate Fig. 1a(1,3).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Map constructed from IMOD. Used to generate Fig. 1i(3,4).

Fileemd_8992_additional_3.map
AnnotationMap constructed from IMOD. Used to generate Fig. 1i(3,4).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Base of connecting cilium and mother centriole of mouse rod cell.

EntireName: Base of connecting cilium and mother centriole of mouse rod cell.
Components
  • Organelle or cellular component: Base of connecting cilium and mother centriole of mouse rod cell.

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Supramolecule #1: Base of connecting cilium and mother centriole of mouse rod cell.

SupramoleculeName: Base of connecting cilium and mother centriole of mouse rod cell.
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Mouse rod cell fragments were isolated by density gradient centrifugation and applied to EM grid for plunge freezing.
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57Bl6/J / Organ: eye / Tissue: retina / Organelle: connecting cilium and basal body / Location in cell: base of outer segment

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4 / Details: Ringer's buffer
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK III
DetailsIsolate rod cell fragments applied to grid.
Cryo protectantNone- aqueous buffer
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Electron Microscopy Sciences / Diameter: 15 nm

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Frames/image: 1-8 / Average electron dose: 1.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus min: 0.009 µm / Nominal magnification: 9400
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware: (Name: IMOD, EMAN2, UCSF Chimera) / Number images used: 35

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