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- EMDB-20047: Cryo-EM structure of the native rhodopsin dimer from rod photorec... -

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Basic information

Entry
Database: EMDB / ID: EMD-20047
TitleCryo-EM structure of the native rhodopsin dimer from rod photoreceptor cells
Map data3D reconstruction (with nanodisc density)
Sample
  • Complex: Rhodopsin
    • Protein or peptide: Rhodopsin
Function / homology
Function and homology information


Opsins / VxPx cargo-targeting to cilium / opsin binding / rod bipolar cell differentiation / sperm head plasma membrane / absorption of visible light / The canonical retinoid cycle in rods (twilight vision) / G protein-coupled opsin signaling pathway / photoreceptor inner segment membrane / podosome assembly ...Opsins / VxPx cargo-targeting to cilium / opsin binding / rod bipolar cell differentiation / sperm head plasma membrane / absorption of visible light / The canonical retinoid cycle in rods (twilight vision) / G protein-coupled opsin signaling pathway / photoreceptor inner segment membrane / podosome assembly / 11-cis retinal binding / G protein-coupled photoreceptor activity / rod photoreceptor outer segment / cellular response to light stimulus / G protein-coupled receptor complex / Inactivation, recovery and regulation of the phototransduction cascade / thermotaxis / Activation of the phototransduction cascade / phototransduction, visible light / outer membrane / detection of temperature stimulus involved in thermoception / response to light intensity / photoreceptor cell maintenance / arrestin family protein binding / photoreceptor outer segment membrane / G alpha (i) signalling events / phototransduction / response to light stimulus / photoreceptor outer segment / G-protein alpha-subunit binding / sperm midpiece / visual perception / guanyl-nucleotide exchange factor activity / microtubule cytoskeleton organization / photoreceptor disc membrane / cell-cell junction / gene expression / G protein-coupled receptor signaling pathway / Golgi membrane / zinc ion binding / identical protein binding / membrane / plasma membrane
Similarity search - Function
Rhodopsin, N-terminal / Amino terminal of the G-protein receptor rhodopsin / Rhodopsin / Opsin / Visual pigments (opsins) retinal binding site / Visual pigments (opsins) retinal binding site. / : / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like ...Rhodopsin, N-terminal / Amino terminal of the G-protein receptor rhodopsin / Rhodopsin / Opsin / Visual pigments (opsins) retinal binding site / Visual pigments (opsins) retinal binding site. / : / Serpentine type 7TM GPCR chemoreceptor Srsx / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family)
Similarity search - Domain/homology
Biological speciesBos taurus (cattle) / Bovine (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsZhao DY / Gulati S / Poege M / Baumeister W / Plitzko JM / Ernst OP / Palczewski K
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI) United States
CitationJournal: J Biol Chem / Year: 2019
Title: Cryo-EM structure of the native rhodopsin dimer in nanodiscs.
Authors: Dorothy Yanling Zhao / Matthias Pöge / Takefumi Morizumi / Sahil Gulati / Ned Van Eps / Jianye Zhang / Przemyslaw Miszta / Slawomir Filipek / Julia Mahamid / Jürgen M Plitzko / Wolfgang ...Authors: Dorothy Yanling Zhao / Matthias Pöge / Takefumi Morizumi / Sahil Gulati / Ned Van Eps / Jianye Zhang / Przemyslaw Miszta / Slawomir Filipek / Julia Mahamid / Jürgen M Plitzko / Wolfgang Baumeister / Oliver P Ernst / Krzysztof Palczewski /
Abstract: Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein- ...Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein-coupled receptor (GPCR), can organize as rows of dimers. GPCR dimerization and oligomerization offer possibilities for allosteric regulation of GPCR activity, but the detailed structures and mechanism remain elusive. In this investigation, we made use of the high rhodopsin density in the native disc membranes and of a bifunctional cross-linker that preserves the native rhodopsin arrangement by covalently tethering rhodopsins via Lys residue side chains. We purified cross-linked rhodopsin dimers and reconstituted them into nanodiscs for cryo-EM analysis. We present cryo-EM structures of the cross-linked rhodopsin dimer as well as a rhodopsin dimer reconstituted into nanodiscs from purified monomers. We demonstrate the presence of a preferential 2-fold symmetrical dimerization interface mediated by transmembrane helix 1 and the cytoplasmic helix 8 of rhodopsin. We confirmed this dimer interface by double electron-electron resonance measurements of spin-labeled rhodopsin. We propose that this interface and the arrangement of two protomers is a prerequisite for the formation of the observed rows of dimers. We anticipate that the approach outlined here could be extended to other GPCRs or membrane receptors to better understand specific receptor dimerization mechanisms.
History
DepositionMar 30, 2019-
Header (metadata) releaseApr 24, 2019-
Map releaseAug 21, 2019-
UpdateAug 12, 2020-
Current statusAug 12, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00993
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.00993
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ofj
  • Surface level: 0.00993
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6ofj
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20047.png

Downloads & links

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Map

FileDownload / File: emd_20047.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction (with nanodisc density)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 192 pix.
= 155.52 Å		0.81 Å/pix.
x 192 pix.
= 155.52 Å		0.81 Å/pix.
x 192 pix.
= 155.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.81 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00993 / Movie #1: 0.00993
Minimum - Maximum-0.011859175 - 0.03948573
Average (Standard dev.)0.0004611074 (±0.0024855642)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 155.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.810.810.81
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z155.520155.520155.520
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ254265109
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0120.0390.000

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Supplemental data

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Mask #1

Fileemd_20047_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 3D reconstruction (with nanodisc density)

Fileemd_20047_additional.map
Annotation3D reconstruction (with nanodisc density)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half 2

Fileemd_20047_half_map_1.map
AnnotationHalf 2
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half 1

Fileemd_20047_half_map_2.map
AnnotationHalf 1
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 : Rhodopsin

EntireName: Rhodopsin
Components
  • Complex: Rhodopsin
    • Protein or peptide: Rhodopsin

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Supramolecule #1: Rhodopsin

SupramoleculeName: Rhodopsin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bos taurus (cattle) / Organ: Eye / Tissue: Retina

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Macromolecule #1: Rhodopsin

MacromoleculeName: Rhodopsin / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bovine (cattle) / Organ: Eye / Tissue: Retina
Molecular weightTheoretical: 39.031457 KDa
SequenceString: MNGTEGPNFY VPFSNKTGVV RSPFEAPQYY LAEPWQFSML AAYMFLLIML GFPINFLTLY VTVQHKKLRT PLNYILLNLA VADLFMVFG GFTTTLYTSL HGYFVFGPTG CNLEGFFATL GGEIALWSLV VLAIERYVVV CKPMSNFRFG ENHAIMGVAF T WVMALACA ...String:
MNGTEGPNFY VPFSNKTGVV RSPFEAPQYY LAEPWQFSML AAYMFLLIML GFPINFLTLY VTVQHKKLRT PLNYILLNLA VADLFMVFG GFTTTLYTSL HGYFVFGPTG CNLEGFFATL GGEIALWSLV VLAIERYVVV CKPMSNFRFG ENHAIMGVAF T WVMALACA APPLVGWSRY IPEGMQCSCG IDYYTPHEET NNESFVIYMF VVHFIIPLIV IFFCYGQLVF TVKEAAAQQQ ES ATTQKAE KEVTRMVIIM VIAFLICWLP YAGVAFYIFT HQGSDFGPIF MTIPAFFAKT SAVYNPVIYI MMNKQFRNCM VTT LCCGKN PLGDDEASTT VSKTETSQVA PA

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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

Concentration1 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 762000
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER

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Atomic model buiding 1

RefinementOverall B value: 100
Output model

PDB-6ofj:
Cryo-EM structure of the native rhodopsin dimer from rod photoreceptor cells

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