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- EMDB-47154: Cryo-EM structure of GPR119-Gs-Nb35 complex with endogenous agoni... -

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Basic information

Entry
Database: EMDB / ID: EMD-47154
TitleCryo-EM structure of GPR119-Gs-Nb35 complex with endogenous agonist oleoylethanolamide (OEA) (consensus map)
Map dataOriginal
Sample
  • Complex: OEA-GPR119-Gs-Nb35 complex
    • Complex: Glucose-dependent insulinotropic receptor GPR119
      • Protein or peptide: Soluble cytochrome b562,Glucose-dependent insulinotropic receptor GPR119,LgBiT
    • Complex: Heterotrimeric Gs protein
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1,HiBiT
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
      • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • Complex: Nanobody35
      • Protein or peptide: Nanobody35
KeywordsCryoEM / GPCR / Orphan / G protein complex / Active / Agonist / MEMBRANE PROTEIN / Diabetes / Native Mass Spectrometry
Biological speciesEscherichia coli (E. coli) / Homo sapiens (human) / Lama glama (llama) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsKim D-G / Cherezov V
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM127086 United States
CitationJournal: Structure / Year: 2024
Title: Native mass spectrometry prescreening of G protein-coupled receptor complexes for cryo-EM structure determination.
Authors: Donggyun Kim / Weijing Liu / Rosa Viner / Vadim Cherezov /
Abstract: G protein-coupled receptors (GPCRs) are essential transmembrane proteins playing key roles in human health and disease. Understanding their atomic-level molecular structure and conformational states ...G protein-coupled receptors (GPCRs) are essential transmembrane proteins playing key roles in human health and disease. Understanding their atomic-level molecular structure and conformational states is imperative for advancing drug development. Recent breakthroughs in single-particle cryogenic electron microscopy (cryo-EM) have propelled the structural biology of GPCRs into a new era. Nevertheless, the preparation of suitable GPCR samples and their complexes for cryo-EM analysis remains challenging due to their poor stability and highly dynamic nature. Here, we present our online buffer exchange-native MS method combined with Direct Mass Technology (OBE-nMS+DMT) which facilitates high-throughput analysis and guides sample preparation. We applied this method to optimize the GPR119-G complex sample prior to cryo-EM analysis, leading to a 3.51 Å resolution structure from only 396 movies collected on a 200 kV Glacios. This study suggests that the OBE-nMS+DMT method emerges as a powerful tool for prescreening sample conditions in cryo-EM studies of GPCRs and other membrane protein complexes.
History
DepositionSep 30, 2024-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateMar 5, 2025-
Current statusMar 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47154.png

Downloads & links

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Map

FileDownload / File: emd_47154.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOriginal
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.92 Å/pix.
x 324 pix.
= 298.08 Å		0.92 Å/pix.
x 324 pix.
= 298.08 Å		0.92 Å/pix.
x 324 pix.
= 298.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.92 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.4327295 - 0.74421775
Average (Standard dev.)-0.00003778919 (±0.015590045)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 298.08002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half B

Fileemd_47154_half_map_1.map
AnnotationHalf_B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half A

Fileemd_47154_half_map_2.map
AnnotationHalf_A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : OEA-GPR119-Gs-Nb35 complex

EntireName: OEA-GPR119-Gs-Nb35 complex
Components
  • Complex: OEA-GPR119-Gs-Nb35 complex
    • Complex: Glucose-dependent insulinotropic receptor GPR119
      • Protein or peptide: Soluble cytochrome b562,Glucose-dependent insulinotropic receptor GPR119,LgBiT
    • Complex: Heterotrimeric Gs protein
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1,HiBiT
      • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
      • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • Complex: Nanobody35
      • Protein or peptide: Nanobody35

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Supramolecule #1: OEA-GPR119-Gs-Nb35 complex

SupramoleculeName: OEA-GPR119-Gs-Nb35 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: consensus map
Molecular weightTheoretical: 177 KDa

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Supramolecule #2: Glucose-dependent insulinotropic receptor GPR119

SupramoleculeName: Glucose-dependent insulinotropic receptor GPR119 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: Heterotrimeric Gs protein

SupramoleculeName: Heterotrimeric Gs protein / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#2, #4
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: Nanobody35

SupramoleculeName: Nanobody35 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Lama glama (llama)

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

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1,HiBiT
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHHHH HLEVLFQGPG SSGSELDQLR QEAEQLKNQI RDARKACADA TLSQITNNID PVGRIQMRTR RTLRGHLAKI YAMHWGTDSR LLVSASQDGK LIIWDSYTTN KVHAIPLRSS WVMTCAYAPS GNYVACGGLD NICSIYNLKT REGNVRVSRE LAGHTGYLSC ...String:
MHHHHHHHHH HLEVLFQGPG SSGSELDQLR QEAEQLKNQI RDARKACADA TLSQITNNID PVGRIQMRTR RTLRGHLAKI YAMHWGTDSR LLVSASQDGK LIIWDSYTTN KVHAIPLRSS WVMTCAYAPS GNYVACGGLD NICSIYNLKT REGNVRVSRE LAGHTGYLSC CRFLDDNQIV TSSGDTTCAL WDIETGQQTT TFTGHTGDVM SLSLAPDTRL FVSGACDASA KLWDVREGMC RQTFTGHESD INAICFFPNG NAFATGSDDA TCRLFDLRAD QELMTYSHDN IICGITSVSF SKSGRLLLAG YDDFNCNVWD ALKADRAGVL AGHDNRVSCL GVTDDGMAVA TGSWDSFLKI WNGSSGGGGS GGGGSSGVSG WRLFKKIS

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Macromolecule #2: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MASNNTASIA QARKLVEQLK MEANIDRIKV SKAAADLMAY CEAHAKEDPL LTPVPASENP FREKKFFCAI L

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Macromolecule #3: Nanobody35

MacromoleculeName: Nanobody35 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
QVQLQESGGG LVQPGGSLRL SCAASGFTFS NYKMNWVRQA PGKGLEWVSD ISQSGASISY TGSVKGRFTI SRDNAKNTLY LQMNSLKPED TAVYYCARCP APFTRDCFDV TSTTYAYRGQ GTQVTVSSHH HHHHEPEA

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Macromolecule #4: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

MacromoleculeName: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKNTIVKQM RILHVNGFNG EGGEEDPQAA RSNSDGEKAT KVQDIKNNLK EAIETIVAAM SNLVPPVELA NPENQFRVDY ILSVMNVPDF DFPPEFYEHA KALWEDEGVR ACYERSNEYQ ...String:
MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKNTIVKQM RILHVNGFNG EGGEEDPQAA RSNSDGEKAT KVQDIKNNLK EAIETIVAAM SNLVPPVELA NPENQFRVDY ILSVMNVPDF DFPPEFYEHA KALWEDEGVR ACYERSNEYQ LIDCAQYFLD KIDVIKQADY VPSDQDLLRC RVLTSGIFET KFQVDKVNFH MFDVGAQRDE RRKWIQCFND VTAIIFVVAS SSYNMVIRED NQTNRLQAAL KLFDSIWNNK WLRDTSVILF LNKQDLLAEK VLAGKSKIED YFPEFARYTT PEDATPEPGE DPRVTRAKYF IRDEFLRIST ASGDGRHYCY PHFTCAVDTE NIRRVFNDCR DIIQRMHLRQ YELL

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Macromolecule #5: Soluble cytochrome b562,Glucose-dependent insulinotropic receptor...

MacromoleculeName: Soluble cytochrome b562,Glucose-dependent insulinotropic receptor GPR119,LgBiT
type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: PGADLEDNWE TLNDNLKVIE KADNAAQVKD ALTKMRAAAL DAQKATPPKL EDKSPDSPEM KDFRHGFDIL VGQIDDALKL ANEGKVKEAQ AAAEQLKTTR NAYIQKYLGS PIMESSFSFG VILAVLASLI IATNTLVAVA VLLLIHKNDG VSLCFTLNLA VADTLIGVAI ...String:
PGADLEDNWE TLNDNLKVIE KADNAAQVKD ALTKMRAAAL DAQKATPPKL EDKSPDSPEM KDFRHGFDIL VGQIDDALKL ANEGKVKEAQ AAAEQLKTTR NAYIQKYLGS PIMESSFSFG VILAVLASLI IATNTLVAVA VLLLIHKNDG VSLCFTLNLA VADTLIGVAI SGLLTDQLSS PSRPTQKTLC SLRMAFVTSS AAASVLTVML ITFDRYLAIK QPFRYLKIMS GFVAGACIAG LWLVSYLIGF LPLGIPMFQQ TAYKGQCSFF AVFHPHFVLT LSCVGFFPAM LLFVFFYCDM LKIASMHSQQ IRKMEHAGAM AGGYRSPRTP SDFKALRTVS VLIGSFALSW TPFLITGIVQ VACQECHLYL VLERYLWLLG VGNSLLNPLI YAYWQKEVRL QLYHMALGVK KVLTSFLLFL SARNCGPERP RESSCHIVTI SSSEFDGVFT LEDFVGDWEQ TAAYNLDQVL EQGGVSSLLQ NLAVSVTPIQ RIVRSGENAL KIDIHVIIPY EGLSADQMAQ IEEVFKVVYP VDDHHFKVIL PYGTLVIDGV TPNMLNYFGR PYEGIAVFDG KKITVTGTLW NGNKIIDERL ITPDGSMLFR VTINSGGSLE VLFQG

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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

Concentration3 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 396 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 150000

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 37758
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7wcm


Chain - Source name: PDB / Chain - Initial model type: experimental model

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