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- EMDB-20223: Structure of the Rhodopsin-Transducin-Nanobody Complex -

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Entry
Database: EMDB / ID: EMD-20223
TitleStructure of the Rhodopsin-Transducin-Nanobody Complex
Map dataRhodopsin-Transducin-Nanobody Complex
Sample
  • Complex: Rhodopsin-Transducin-Nanobody Complex
    • Complex: Transducin
      • Protein or peptide: Gt-alpha/Gi1-alpha chimera
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
      • Protein or peptide: Guanine nucleotide-binding protein G(T) subunit gamma-T1
    • Complex: Rhodopsin
      • Protein or peptide: Rhodopsin
    • Complex: Nanobody
      • Protein or peptide: Camelid antibody VHH fragment
  • Ligand: RETINAL
KeywordsGPCR / G protein / Complex / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


negative regulation of cyclic-nucleotide phosphodiesterase activity / Opsins / VxPx cargo-targeting to cilium / sperm head plasma membrane / rod bipolar cell differentiation / absorption of visible light / opsin binding / The canonical retinoid cycle in rods (twilight vision) / G protein-coupled opsin signaling pathway / Olfactory Signaling Pathway ...negative regulation of cyclic-nucleotide phosphodiesterase activity / Opsins / VxPx cargo-targeting to cilium / sperm head plasma membrane / rod bipolar cell differentiation / absorption of visible light / opsin binding / The canonical retinoid cycle in rods (twilight vision) / G protein-coupled opsin signaling pathway / Olfactory Signaling Pathway / podosome assembly / Sensory perception of sweet, bitter, and umami (glutamate) taste / 11-cis retinal binding / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / G protein-coupled photoreceptor activity / photoreceptor inner segment membrane / rod photoreceptor outer segment / cellular response to light stimulus / eye photoreceptor cell development / detection of light stimulus involved in visual perception / G protein-coupled receptor complex / Inactivation, recovery and regulation of the phototransduction cascade / thermotaxis / Activation of the phototransduction cascade / outer membrane / detection of temperature stimulus involved in thermoception / response to light intensity / photoreceptor cell maintenance / arrestin family protein binding / Activation of G protein gated Potassium channels / G-protein activation / G beta:gamma signalling through PI3Kgamma / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through PLC beta / ADP signalling through P2Y purinoceptor 1 / Thromboxane signalling through TP receptor / Presynaptic function of Kainate receptors / G beta:gamma signalling through CDC42 / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G alpha (12/13) signalling events / Glucagon-type ligand receptors / G beta:gamma signalling through BTK / ADP signalling through P2Y purinoceptor 12 / Adrenaline,noradrenaline inhibits insulin secretion / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Ca2+ pathway / Thrombin signalling through proteinase activated receptors (PARs) / G alpha (z) signalling events / Extra-nuclear estrogen signaling / photoreceptor outer segment membrane / G alpha (s) signalling events / G alpha (q) signalling events / G alpha (i) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / Vasopressin regulates renal water homeostasis via Aquaporins / acyl binding / response to light stimulus / phototransduction, visible light / G-protein alpha-subunit binding / phototransduction / photoreceptor outer segment / sperm midpiece / photoreceptor inner segment / visual perception / guanyl-nucleotide exchange factor activity / G protein-coupled receptor binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / microtubule cytoskeleton organization / G-protein beta/gamma-subunit complex binding / photoreceptor disc membrane / cell-cell junction / GDP binding / cellular response to catecholamine stimulus / intracellular protein localization / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / sensory perception of taste / signaling receptor complex adaptor activity / retina development in camera-type eye / GTPase binding / phospholipase C-activating G protein-coupled receptor signaling pathway / gene expression / cell population proliferation / G protein-coupled receptor signaling pathway / Golgi membrane / GTPase activity / synapse / protein kinase binding / GTP binding / protein-containing complex binding / zinc ion binding / metal ion binding / identical protein binding / membrane / plasma membrane / cytoplasm
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. / : / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / Guanine nucleotide binding protein (G-protein), alpha subunit ...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. / : / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / G protein beta WD-40 repeat protein / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / 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) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Guanine nucleotide-binding protein G(T) subunit gamma-T1 / Rhodopsin / Guanine nucleotide-binding protein G(t) subunit alpha-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
Similarity search - Component
Biological speciesBos taurus (domestic cattle) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGao Y / Hu H
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA201402 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM122575 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01 NS092695 United States
CitationJournal: Mol Cell / Year: 2019
Title: Structures of the Rhodopsin-Transducin Complex: Insights into G-Protein Activation.
Authors: Yang Gao / Hongli Hu / Sekar Ramachandran / Jon W Erickson / Richard A Cerione / Georgios Skiniotis /
Abstract: Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (G) by catalyzing GDP-GTP exchange on its α subunit (Gα). To elucidate the ...Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (G) by catalyzing GDP-GTP exchange on its α subunit (Gα). To elucidate the determinants of G coupling and activation, we obtained cryo-EM structures of a fully functional, light-activated Rho-G complex in the presence and absence of a G-protein-stabilizing nanobody. The structures illustrate how G overcomes its low basal activity by engaging activated Rho in a conformation distinct from other GPCR-G-protein complexes. Moreover, the nanobody-free structures reveal native conformations of G-protein components and capture three distinct conformers showing the Gα helical domain (αHD) contacting the Gβγ subunits. These findings uncover the molecular underpinnings of G-protein activation by visual rhodopsin and shed new light on the role played by Gβγ during receptor-catalyzed nucleotide exchange.
History
DepositionMay 14, 2019-
Header (metadata) releaseJun 19, 2019-
Map releaseJul 24, 2019-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6oya
  • Surface level: 4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20223.png

Downloads & links

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Map

FileDownload / File: emd_20223.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRhodopsin-Transducin-Nanobody Complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 240 pix.
= 254.4 Å		1.06 Å/pix.
x 240 pix.
= 254.4 Å		1.06 Å/pix.
x 240 pix.
= 254.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 4.0 / Movie #1: 4
Minimum - Maximum-6.4182916 - 15.021849
Average (Standard dev.)-0.022678247 (±0.46434137)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 254.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z254.400254.400254.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-420-29
NX/NY/NZ887886
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-6.41815.022-0.023

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Supplemental data

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Sample components

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Entire : Rhodopsin-Transducin-Nanobody Complex

EntireName: Rhodopsin-Transducin-Nanobody Complex
Components
  • Complex: Rhodopsin-Transducin-Nanobody Complex
    • Complex: Transducin
      • Protein or peptide: Gt-alpha/Gi1-alpha chimera
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
      • Protein or peptide: Guanine nucleotide-binding protein G(T) subunit gamma-T1
    • Complex: Rhodopsin
      • Protein or peptide: Rhodopsin
    • Complex: Nanobody
      • Protein or peptide: Camelid antibody VHH fragment
  • Ligand: RETINAL

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Supramolecule #1: Rhodopsin-Transducin-Nanobody Complex

SupramoleculeName: Rhodopsin-Transducin-Nanobody Complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Molecular weightTheoretical: 141.8 KDa

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Supramolecule #2: Transducin

SupramoleculeName: Transducin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Bos taurus (domestic cattle)

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Supramolecule #3: Rhodopsin

SupramoleculeName: Rhodopsin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Bos taurus (domestic cattle)

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Supramolecule #4: Nanobody

SupramoleculeName: Nanobody / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Lama glama (llama)

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Macromolecule #1: Gt-alpha/Gi1-alpha chimera

MacromoleculeName: Gt-alpha/Gi1-alpha chimera / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (domestic cattle)
Molecular weightTheoretical: 41.175715 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAHHHHHHAM GAGASAEEKH SRELEKKLKE DAEKDARTVK LLLLGAGESG KSTIVKQMKI IHQDGYSLEE CLEFIAIIYG NTLQSILAI VRAMTTLNIQ YGDSARQDDA RKLMHMADTI EEGTMPKEMS DIIQRLWKDS GIQACFDRAS EYQLNDSAGY Y LSDLERLV ...String:
MAHHHHHHAM GAGASAEEKH SRELEKKLKE DAEKDARTVK LLLLGAGESG KSTIVKQMKI IHQDGYSLEE CLEFIAIIYG NTLQSILAI VRAMTTLNIQ YGDSARQDDA RKLMHMADTI EEGTMPKEMS DIIQRLWKDS GIQACFDRAS EYQLNDSAGY Y LSDLERLV TPGYVPTEQD VLRSRVKTTG IIETQFSFKD LNFRMFDVGG QRDERRKWIH CFEGVTAIIF CVALSDYDMV LV EDNQTNR MQESMNLFKS ICNNKWFTDT SIILFLNKKD LFEEKIKKSP LTDYYPEYAG SNTYEEAGNY IKVQFLELNM ASD VKEIYS HMTCATDTQN VKFVFDAVTD IIIKENLKDC GLF

UniProtKB: Guanine nucleotide-binding protein G(t) subunit alpha-1

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Macromolecule #2: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (domestic cattle)
Molecular weightTheoretical: 37.430957 KDa
SequenceString: MSELDQLRQE AEQLKNQIRD ARKACADATL SQITNNIDPV GRIQMRTRRT LRGHLAKIYA MHWGTDSRLL LSASQDGKLI IWDSYTTNK VHAIPLRSSW VMTCAYAPSG NYVACGGLDN ICSIYNLKTR EGNVRVSREL AGHTGYLSCC RFLDDNQIVT S SGDTTCAL ...String:
MSELDQLRQE AEQLKNQIRD ARKACADATL SQITNNIDPV GRIQMRTRRT LRGHLAKIYA MHWGTDSRLL LSASQDGKLI IWDSYTTNK VHAIPLRSSW VMTCAYAPSG NYVACGGLDN ICSIYNLKTR EGNVRVSREL AGHTGYLSCC RFLDDNQIVT S SGDTTCAL WDIETGQQTT TFTGHTGDVM SLSLAPDTRL FVSGACDASA KLWDVREGMC RQTFTGHESD INAICFFPNG NA FATGSDD ATCRLFDLRA DQELMTYSHD NIICGITSVS FSKSGRLLLA GYDDFNCNVW DALKADRAGV LAGHDNRVSC LGV TDDGMA VATGSWDSFL KIWN

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

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Macromolecule #3: Guanine nucleotide-binding protein G(T) subunit gamma-T1

MacromoleculeName: Guanine nucleotide-binding protein G(T) subunit gamma-T1
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (domestic cattle)
Molecular weightTheoretical: 9.337784 KDa
SequenceString:
MPVINIEDPV INIEDLTEKD KLKMEVDQLK KEVTLERMLV SKCCEEFRDY VEERSGEDPL VKGIPEDKNP FKELKGGCVI S

UniProtKB: Guanine nucleotide-binding protein G(T) subunit gamma-T1

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Macromolecule #4: Camelid antibody VHH fragment

MacromoleculeName: Camelid antibody VHH fragment / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 15.05268 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQPGGSLRL SCAASGFTFS NYKMNWVRQA PGKGLQWVSD ISQSGASISY TGSVKGRFTI SRDDAKNTLY LQMNSLKPA DTAVYYCARC PAPFTRDCFD VTSTAYAYRG QGTQVTVSSH HHHHHEPEA

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Macromolecule #5: Rhodopsin

MacromoleculeName: Rhodopsin / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (domestic cattle)
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

UniProtKB: Rhodopsin

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Macromolecule #6: RETINAL

MacromoleculeName: RETINAL / type: ligand / ID: 6 / Number of copies: 1 / Formula: RET
Molecular weightTheoretical: 284.436 Da
Chemical component information

ChemComp-RET:
RETINAL

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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

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 80 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III
Details: Blot for 1 second before plunging; avoid light as much as possible..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 3837 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Number images used: 309116
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementProtocol: BACKBONE TRACE
Output model

PDB-6oya:
Structure of the Rhodopsin-Transducin-Nanobody Complex

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