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- EMDB-23980: Rhodopsin kinase (GRK1)-S5E/S488E/T489E in complex with rhodopsin... -

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Basic information

Entry
Database: EMDB / ID: EMD-23980
TitleRhodopsin kinase (GRK1)-S5E/S488E/T489E in complex with rhodopsin and Fab6
Map dataRhodopsin kinase (GRK1)-S5E/S488E/T489E in complex with rhodopsin and Fab6
Sample
  • Complex: Rhodopsin kinase (GRK1)-S5E/S488E/T489E in complex with rhodopsin stabilized by Fab6
    • Complex: Fab6 Heavy and Light Chains
      • Protein or peptide: Fab6 heavy chain
      • Protein or peptide: Fab6 light chain
    • Complex: Rhodopsin kinase, rhodopsin
      • Protein or peptide: Rhodopsin kinase GRK1
      • Protein or peptide: Rhodopsin
  • Ligand: SANGIVAMYCIN
  • Ligand: RETINAL
Function / homology
Function and homology information


rhodopsin kinase / rhodopsin kinase activity / regulation of opsin-mediated signaling pathway / Opsins / VxPx cargo-targeting to cilium / rod photoreceptor outer segment / rod bipolar cell differentiation / sperm head plasma membrane / podosome assembly / absorption of visible light ...rhodopsin kinase / rhodopsin kinase activity / regulation of opsin-mediated signaling pathway / 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 / regulation of signal transduction / sperm midpiece / visual perception / guanyl-nucleotide exchange factor activity / microtubule cytoskeleton organization / photoreceptor disc membrane / cell-cell junction / gene expression / protein autophosphorylation / G protein-coupled receptor signaling pathway / Golgi membrane / signal transduction / zinc ion binding / ATP binding / membrane / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
Rhodopsin kinase, catalytic domain / GPCR kinase / 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. / Regulator of G protein signaling domain / RGS, subdomain 2 ...Rhodopsin kinase, catalytic domain / GPCR kinase / 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. / Regulator of G protein signaling domain / RGS, subdomain 2 / RGS domain / RGS domain profile. / Regulator of G protein signalling domain / RGS domain superfamily / Extension to Ser/Thr-type protein kinases / AGC-kinase, C-terminal / AGC-kinase C-terminal domain profile. / 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) / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Rhodopsin / Rhodopsin kinase GRK1
Similarity search - Component
Biological speciesHomo sapiens (human) / Bos taurus (cattle) / Bovine (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsChen Q / Tesmer JJG
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL071818 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL122416 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA221289 United States
American Heart Association19POST34450193 United States
CitationJournal: Nature / Year: 2021
Title: Structures of rhodopsin in complex with G-protein-coupled receptor kinase 1.
Authors: Qiuyan Chen / Manolo Plasencia / Zhuang Li / Somnath Mukherjee / Dhabaleswar Patra / Chun-Liang Chen / Thomas Klose / Xin-Qiu Yao / Anthony A Kossiakoff / Leifu Chang / Philip C Andrews / John J G Tesmer /
Abstract: G-protein-coupled receptor (GPCR) kinases (GRKs) selectively phosphorylate activated GPCRs, thereby priming them for desensitization. Although it is unclear how GRKs recognize these receptors, a ...G-protein-coupled receptor (GPCR) kinases (GRKs) selectively phosphorylate activated GPCRs, thereby priming them for desensitization. Although it is unclear how GRKs recognize these receptors, a conserved region at the GRK N terminus is essential for this process. Here we report a series of cryo-electron microscopy single-particle reconstructions of light-activated rhodopsin (Rho*) bound to rhodopsin kinase (GRK1), wherein the N terminus of GRK1 forms a helix that docks into the open cytoplasmic cleft of Rho*. The helix also packs against the GRK1 kinase domain and stabilizes it in an active configuration. The complex is further stabilized by electrostatic interactions between basic residues that are conserved in most GPCRs and acidic residues that are conserved in GRKs. We did not observe any density for the regulator of G-protein signalling homology domain of GRK1 or the C terminus of rhodopsin. Crosslinking with mass spectrometry analysis confirmed these results and revealed dynamic behaviour in receptor-bound GRK1 that would allow the phosphorylation of multiple sites in the receptor tail. We have identified GRK1 residues whose mutation augments kinase activity and crosslinking with Rho*, as well as residues that are involved in activation by acidic phospholipids. From these data, we present a general model for how a small family of protein kinases can recognize and be activated by hundreds of different GPCRs.
History
DepositionMay 13, 2021-
Header (metadata) releaseJul 7, 2021-
Map releaseJul 7, 2021-
UpdateAug 25, 2021-
Current statusAug 25, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
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  • Surface level: 0.5
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  • Surface view with fitted model
  • Atomic models: PDB-7mtb
  • Surface level: 0.5
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7mtb
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23980.png

Downloads & links

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Map

FileDownload / File: emd_23980.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRhodopsin kinase (GRK1)-S5E/S488E/T489E in complex with rhodopsin and Fab6
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.5
Minimum - Maximum-2.4116635 - 4.22012
Average (Standard dev.)-0.0007927333 (±0.033688016)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 432.00003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z432.000432.000432.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-210-210-210
NX/NY/NZ420420420
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-2.4124.220-0.001

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Supplemental data

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Sample components

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Entire : Rhodopsin kinase (GRK1)-S5E/S488E/T489E in complex with rhodopsin...

EntireName: Rhodopsin kinase (GRK1)-S5E/S488E/T489E in complex with rhodopsin stabilized by Fab6
Components
  • Complex: Rhodopsin kinase (GRK1)-S5E/S488E/T489E in complex with rhodopsin stabilized by Fab6
    • Complex: Fab6 Heavy and Light Chains
      • Protein or peptide: Fab6 heavy chain
      • Protein or peptide: Fab6 light chain
    • Complex: Rhodopsin kinase, rhodopsin
      • Protein or peptide: Rhodopsin kinase GRK1
      • Protein or peptide: Rhodopsin
  • Ligand: SANGIVAMYCIN
  • Ligand: RETINAL

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Supramolecule #1: Rhodopsin kinase (GRK1)-S5E/S488E/T489E in complex with rhodopsin...

SupramoleculeName: Rhodopsin kinase (GRK1)-S5E/S488E/T489E in complex with rhodopsin stabilized by Fab6
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4

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Supramolecule #2: Fab6 Heavy and Light Chains

SupramoleculeName: Fab6 Heavy and Light Chains / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Rhodopsin kinase, rhodopsin

SupramoleculeName: Rhodopsin kinase, rhodopsin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1, #4
Source (natural)Organism: Bos taurus (cattle)

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Macromolecule #1: Rhodopsin kinase GRK1

MacromoleculeName: Rhodopsin kinase GRK1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: rhodopsin kinase
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 61.542012 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDFGELETVV ANSAFIAARG SFDASSGPAS RDRKYLARLK LPPLSKCEAL RESLDLGFEG MCLEQPIGKR LFQQFLRTHE QHGPALQLW KDIEDYDTAD DALRPQKAQA LRAAYLEPQA QLFCSFLDAE TVARARAGAG DGLFQPLLRA VLAHLGQAPF Q EFLDSLYF ...String:
MDFGELETVV ANSAFIAARG SFDASSGPAS RDRKYLARLK LPPLSKCEAL RESLDLGFEG MCLEQPIGKR LFQQFLRTHE QHGPALQLW KDIEDYDTAD DALRPQKAQA LRAAYLEPQA QLFCSFLDAE TVARARAGAG DGLFQPLLRA VLAHLGQAPF Q EFLDSLYF LRFLQWKWLE AQPMGEDWFL DFRVLGRGGF GEVFACQMKA TGKLYACKKL NKKRLKKRKG YQGAMVEKKI LA KVHSRFI VSLAYAFETK TDLCLVMTIM NGGDIRYHIY NVDEDNPGFQ EPRAIFYTAQ IVSGLEHLHQ RNIIYRDLKP ENV LLDDDG NVRISDLGLA VELKAGQTKT KGYAGTPGFM APELLLGEEY DFSVDYFALG VTLYEMIAAR GPFRARGEKV ENKE LKQRV LEQAVTYPDK FSPASKDFCE ALLQKDPEKR LGFRDGSCDG LRTHPLFRDI SWRQLEAGML TPPFVPDSRT VYAKN IQDV GAFEEVKGVA FEKADTEFFQ EFASGTCPIP WQEEMIETGV FGDLNVWRPD GVDHHHHHH

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Macromolecule #2: Fab6 heavy chain

MacromoleculeName: Fab6 heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.170939 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NFSSYSIHWV RQAPGKGLEW VAYISSYYGS TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARHEYGWWND RWGLDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF P EPVTVSWN ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NFSSYSIHWV RQAPGKGLEW VAYISSYYGS TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARHEYGWWND RWGLDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF P EPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHT

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Macromolecule #3: Fab6 light chain

MacromoleculeName: Fab6 light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.509039 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSWYGPFT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSWYGPFT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

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Macromolecule #4: Rhodopsin

MacromoleculeName: Rhodopsin / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bovine (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

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Macromolecule #5: SANGIVAMYCIN

MacromoleculeName: SANGIVAMYCIN / type: ligand / ID: 5 / Number of copies: 1 / Formula: SGV
Molecular weightTheoretical: 309.278 Da
Chemical component information

ChemComp-SGV:
SANGIVAMYCIN / inhibitor*YM / Sangivamycin

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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 / 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: 8
VitrificationCryogen name: ETHANE

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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 K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 250547
FSC plot (resolution estimation)

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