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- PDB-8p13: Cryo-EM structure of Rhodopsin-Gi bound with antibody fragments s... -

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Basic information

Entry
Database: PDB / ID: 8p13
TitleCryo-EM structure of Rhodopsin-Gi bound with antibody fragments scFv16 and Fab79, conformation 1
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • (Immunoglobin G ...) x 2
  • Rhodopsin
  • single-chain Fv 16
KeywordsSIGNALING PROTEIN / GPCR / Rhodopsin / G protein / Fab
Function / homology
Function and homology information


Opsins / VxPx cargo-targeting to cilium / opsin binding / sperm head plasma membrane / rod bipolar cell differentiation / absorption of visible light / The canonical retinoid cycle in rods (twilight vision) / G protein-coupled opsin signaling pathway / Olfactory Signaling Pathway / photoreceptor inner segment membrane ...Opsins / VxPx cargo-targeting to cilium / opsin binding / sperm head plasma membrane / rod bipolar cell differentiation / absorption of visible light / The canonical retinoid cycle in rods (twilight vision) / G protein-coupled opsin signaling pathway / Olfactory Signaling Pathway / photoreceptor inner segment membrane / Sensory perception of sweet, bitter, and umami (glutamate) taste / podosome assembly / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / 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 / phototransduction, visible light / 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 / Glucagon-type ligand receptors / G alpha (12/13) signalling events / 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 / Thrombin signalling through proteinase activated receptors (PARs) / Ca2+ pathway / G alpha (z) signalling events / G alpha (s) signalling events / Extra-nuclear estrogen signaling / G alpha (q) signalling events / photoreceptor outer segment membrane / G alpha (i) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / phototransduction / photoreceptor outer segment / G-protein alpha-subunit binding / response to light stimulus / T cell migration / Adenylate cyclase inhibitory pathway / positive regulation of protein localization to cell cortex / adenylate cyclase regulator activity / adenylate cyclase-inhibiting serotonin receptor signaling pathway / response to prostaglandin E / regulation of cAMP-mediated signaling / D2 dopamine receptor binding / G protein-coupled serotonin receptor binding / sperm midpiece / regulation of mitotic spindle organization / cellular response to forskolin / visual perception / guanyl-nucleotide exchange factor activity / positive regulation of cholesterol biosynthetic process / Regulation of insulin secretion / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / G protein-coupled receptor binding / G-protein beta/gamma-subunit complex binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / response to peptide hormone / microtubule cytoskeleton organization / ADP signalling through P2Y purinoceptor 12 / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / G alpha (z) signalling events / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / GDP binding / GPER1 signaling / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / cell-cell junction / heterotrimeric G-protein complex / signaling receptor complex adaptor activity / G protein activity / cell cortex / midbody / G alpha (i) signalling events / G alpha (s) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / gene expression
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 / 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. / WD domain, G-beta repeat / 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(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(i) subunit alpha-1
Similarity search - Component
Biological speciesBos taurus (cattle)
Homo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.2 Å
AuthorsPamula, F. / Tejero, O. / Muehle, J. / Thoma, R. / Schertler, G.F.X. / Marino, J. / Tsai, C.-J.
Funding support Switzerland, European Union, 4items
OrganizationGrant numberCountry
Swiss National Science Foundation173335 Switzerland
Swiss National Science Foundation153145 Switzerland
Swiss National Science Foundation192760 Switzerland
European Research Council (ERC)951644European Union
CitationJournal: To Be Published
Title: Characterization of two novel antibody fragments for obtaining cryo-EM structures of GPCR-G protein complexes
Authors: Pamula, F. / Tejero, O. / Muehle, J. / Thoma, R. / Schertler, G.F.X. / Marino, J. / Tsai, C.-J.
History
DepositionMay 11, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 20, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
R: Rhodopsin
A: Guanine nucleotide-binding protein G(i) subunit alpha-1
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein G(T) subunit gamma-T1
S: single-chain Fv 16
H: Immunoglobin G heavy chain FAB fragment
L: Immunoglobin G light chain


Theoretical massNumber of molelcules
Total (without water)210,3267
Polymers210,3267
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, The sample is purified by gel filtration showing a monodisperse peak.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 1 types, 1 molecules R

#1: Protein Rhodopsin


Mass: 39040.527 Da / Num. of mol.: 1 / Mutation: N2C, M257Y, D282C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Cell: rod photoreceptor cell / Gene: RHO / Cell (production host): embryonic / Cell line (production host): HEK293 / Organ (production host): Kidney / Production host: Homo sapiens (human) / Strain (production host): HEK293 / References: UniProt: P02699

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules ABG

#2: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 43182.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P63096
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37416.930 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: Eye / Tissue: retina / References: UniProt: P62871
#4: Protein Guanine nucleotide-binding protein G(T) subunit gamma-T1 / Transducin gamma chain


Mass: 8556.918 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: Eye / Tissue: retina / References: UniProt: P02698

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Antibody , 3 types, 3 molecules SHL

#5: Antibody single-chain Fv 16


Mass: 27784.896 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell: B cell / Production host: Trichoplusia ni (cabbage looper)
#6: Antibody Immunoglobin G heavy chain FAB fragment


Mass: 27971.973 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Cell line: hybridoma
#7: Antibody Immunoglobin G light chain


Mass: 26372.580 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Cell line: hybridoma

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Details

Has protein modificationY

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Rhodopsin-Gi-scFv16-Fab79 complexCOMPLEXbovine rhodopsin N2C/M257Y/D282Call0MULTIPLE SOURCES
2RhodopsinCOMPLEX#11RECOMBINANT
3Guanine nucleotide-binding protein G(i) subunit alpha-1COMPLEX#21RECOMBINANT
4Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 and Guanine nucleotide-binding protein G(T) subunit gamma-T1COMPLEX#3-#41NATURAL
5single-chain Fv 16COMPLEX#51RECOMBINANT
6Immunoglobin G heavy chain FAB fragment and Immunoglobin G light chainCOMPLEX#6-#71NATURAL
Molecular weightValue: 0.195 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Bos taurus (cattle)9913
33Homo sapiens (human)9606
44Bos taurus (cattle)9913
55Mus musculus (house mouse)10090
66Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Escherichia coli BL21(DE3) (bacteria)469008
35Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 7.5 / Details: 20 mM HEPES (pH 7.5), 100 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2100 mMsodium chlorideNaCl1
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
7UCSF Chimera1.16model fitting
9PHENIX1.20.1-4487model refinement
10cryoSPARC2.13initial Euler assignment
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7026029
3D reconstructionResolution: 5.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 98000 / Symmetry type: POINT
Atomic model buildingB value: 360 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: cross-correlation coefficient
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDChain residue rangeInitial refinement model-IDPdb chain residue rangeSource nameTypeDetails
16FUF

6fuf

R6FUFR1-32211-322PDBexperimental model
26QNO

6qno

A6QNOA1-4221-42PDBexperimental model
36QNO

6qno

A6QNOA181-3542181-354PDBexperimental model
4A43-180AlphaFoldin silico model
56QNO

6qno

B6QNOB2PDBexperimental model
66QNO

6qno

G6QNOG2PDBexperimental model
76CRK

6crk

S6CRKS4PDBexperimental modelchain D in 6CRK.pdb
8HAlphaFoldin silico model
9LAlphaFoldin silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00513292
ELECTRON MICROSCOPYf_angle_d1.07118030
ELECTRON MICROSCOPYf_dihedral_angle_d8.091815
ELECTRON MICROSCOPYf_chiral_restr0.0552016
ELECTRON MICROSCOPYf_plane_restr0.0072294

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