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- EMDB-13322: Peripheral row of the protein scaffold at rod outer segment disk ... -

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Basic information

Entry
Database: EMDB / ID: EMD-13322
TitlePeripheral row of the protein scaffold at rod outer segment disk rims in wild type mice (conventional defocused data).
Map data
Sample
  • Complex: Rod outer segment disk rim scaffold
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 19.3 Å
AuthorsPoege M / Mahamid J / Imanishi SS / Plitzko JM / Palczewski K / Baumeister W
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Elife / Year: 2021
Title: Determinants shaping the nanoscale architecture of the mouse rod outer segment.
Authors: Matthias Pöge / Julia Mahamid / Sanae S Imanishi / Jürgen M Plitzko / Krzysztof Palczewski / Wolfgang Baumeister /
Abstract: The unique membrane organization of the rod outer segment (ROS), the specialized sensory cilium of rod photoreceptor cells, provides the foundation for phototransduction, the initial step in vision. ...The unique membrane organization of the rod outer segment (ROS), the specialized sensory cilium of rod photoreceptor cells, provides the foundation for phototransduction, the initial step in vision. ROS architecture is characterized by a stack of identically shaped and tightly packed membrane disks loaded with the visual receptor rhodopsin. A wide range of genetic aberrations have been reported to compromise ROS ultrastructure, impairing photoreceptor viability and function. Yet, the structural basis giving rise to the remarkably precise arrangement of ROS membrane stacks and the molecular mechanisms underlying genetically inherited diseases remain elusive. Here, cryo-electron tomography (cryo-ET) performed on native ROS at molecular resolution provides insights into key structural determinants of ROS membrane architecture. Our data confirm the existence of two previously observed molecular connectors/spacers which likely contribute to the nanometer-scale precise stacking of the ROS disks. We further provide evidence that the extreme radius of curvature at the disk rims is enforced by a continuous supramolecular assembly composed of peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) oligomers. We suggest that together these molecular assemblies constitute the structural basis of the highly specialized ROS functional architecture. Our Cryo-ET data provide novel quantitative and structural information on the molecular architecture in ROS and substantiate previous results on proposed mechanisms underlying pathologies of certain PRPH2 mutations leading to blindness.
History
DepositionAug 3, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateJan 19, 2022-
Current statusJan 19, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.16
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.16
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13322.png

Downloads & links

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Map

FileDownload / File: emd_13322.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 2.62 Å
Density
Contour LevelBy AUTHOR: 0.16 / Movie #1: 0.16
Minimum - Maximum-0.16303219 - 0.3968682
Average (Standard dev.)0.01732243 (±0.09088331)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 335.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.622.622.62
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z335.360335.360335.360
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.1630.3970.017

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Supplemental data

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Mask #1

Fileemd_13322_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Additional map: Masked volume focusing on 4 repeats along the...

Fileemd_13322_additional_1.map
AnnotationMasked volume focusing on 4 repeats along the peripheral row with its connections to the central row.
Projections & Slices
AxesZYX

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Histogram (log scale)

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Half map: Unfiltered half map one as output of RELION Refine3D.

Fileemd_13322_half_map_1.map
AnnotationUnfiltered half map one as output of RELION Refine3D.
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map two as output of RELION Refine3D.

Fileemd_13322_half_map_2.map
AnnotationUnfiltered half map two as output of RELION Refine3D.
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Rod outer segment disk rim scaffold

EntireName: Rod outer segment disk rim scaffold
Components
  • Complex: Rod outer segment disk rim scaffold

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Supramolecule #1: Rod outer segment disk rim scaffold

SupramoleculeName: Rod outer segment disk rim scaffold / type: complex / ID: 1 / Parent: 0
Details: Protein scaffold located at the outer periphery of rod outer segment disk rims in wild type mice - the peripheral row.
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6J / Organ: Eye / Tissue: Retina / Location in cell: Rod outer segment

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4 / Details: Ringers buffer
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Chamber temperature: 310 K / Instrument: FEI VITROBOT MARK IV
DetailsRod outer segments were isolated by retinal detachment with a single physical disruption, applied to EM-grids and plunge frozen.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.8 e/Å2
Details: The total accumulated dose per tilt series was ~ 100e-/A^2.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 48 / Number images used: 106000
Final 3D classificationSoftware - Name: RELION (ver. 3.0.5)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.5)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 19.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 3.0.5) / Number subtomograms used: 48000
FSC plot (resolution estimation)

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