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- PDB-6ofj: Cryo-EM structure of the native rhodopsin dimer from rod photorec... -

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Entry
Database: PDB / ID: 6ofj
TitleCryo-EM structure of the native rhodopsin dimer from rod photoreceptor cells
ComponentsRhodopsin
KeywordsSIGNALING PROTEIN / G protein-coupled receptor / native dimer / rod outer segment
Function / homology
Function and homology information


Opsins / VxPx cargo-targeting to cilium / rod photoreceptor outer segment / rod bipolar cell differentiation / sperm head plasma membrane / podosome assembly / absorption of visible light / opsin binding / The canonical retinoid cycle in rods (twilight vision) / : ...Opsins / VxPx cargo-targeting to cilium / rod photoreceptor outer segment / rod bipolar cell differentiation / sperm head plasma membrane / podosome assembly / absorption of visible light / opsin binding / The canonical retinoid cycle in rods (twilight vision) / : / G protein-coupled photoreceptor activity / photoreceptor inner segment membrane / rhodopsin mediated signaling pathway / 11-cis retinal binding / cellular response to light stimulus / G protein-coupled receptor complex / Inactivation, recovery and regulation of the phototransduction cascade / phototransduction, visible light / thermotaxis / Activation of the phototransduction cascade / detection of temperature stimulus involved in thermoception / outer membrane / arrestin family protein binding / photoreceptor cell maintenance / photoreceptor outer segment membrane / G alpha (i) signalling events / response to light stimulus / phototransduction / 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 / membrane / identical protein binding / 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 / GPCR, rhodopsin-like, 7TM ...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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsZhao, D.Y. / Gulati, S. / Ernst, O.P. / Palczewski, K.
Funding support United States, 1items
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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 21, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 9, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Assembly

Deposited unit
A: Rhodopsin
B: Rhodopsin


Theoretical massNumber of molelcules
Total (without water)78,0632
Polymers78,0632
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Rhodopsin /


Mass: 39031.457 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: Eye / Tissue: Retina / References: UniProt: P02699

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Rhodopsin / Type: COMPLEX / Entity ID: all / Source: NATURAL
Source (natural)Organism: Bos taurus (cattle) / Organ: Eye / Tissue: Retina
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsPhase plate: Volta phase plate

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Processing

SoftwareName: PHENIX / Version: 1.15rc1_3420: / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
12RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 762000 / Symmetry type: POINT
Atomic model buildingB value: 100
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00510474
ELECTRON MICROSCOPYf_angle_d0.94118890
ELECTRON MICROSCOPYf_dihedral_angle_d6.3644282
ELECTRON MICROSCOPYf_chiral_restr0.046808
ELECTRON MICROSCOPYf_plane_restr0.0041516

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