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- EMDB-43253: Cryo-EM structure of GPR119-Gs-Nb35 complex with small molecule a... -

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Basic information

Entry
Database: EMDB / ID: EMD-43253
TitleCryo-EM structure of GPR119-Gs-Nb35 complex with small molecule agonist MBX-2982 (Receptor-focused map)
Map data
Sample
  • Complex: GPR119-Gs-Nb35 complex
    • Complex: Glucose-dependent insulinotropic receptor
      • Protein or peptide: Soluble cytochrome b562,Glucose-dependent insulinotropic receptor,LgBiT
    • Complex: G-protein complex
      • 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: 3.61 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
DepositionJan 3, 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_43253.png

Downloads & links

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Map

FileDownload / File: emd_43253.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.92 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.17
Minimum - Maximum-0.8758604 - 1.3089254
Average (Standard dev.)0.00040961392 (±0.013710598)
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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Mask #1

Fileemd_43253_msk_1.map
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Additional map: original map

Fileemd_43253_additional_1.map
Annotationoriginal map
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Half map: #2

Fileemd_43253_half_map_1.map
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Half map: #1

Fileemd_43253_half_map_2.map
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Sample components

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Entire : GPR119-Gs-Nb35 complex

EntireName: GPR119-Gs-Nb35 complex
Components
  • Complex: GPR119-Gs-Nb35 complex
    • Complex: Glucose-dependent insulinotropic receptor
      • Protein or peptide: Soluble cytochrome b562,Glucose-dependent insulinotropic receptor,LgBiT
    • Complex: G-protein complex
      • 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: GPR119-Gs-Nb35 complex

SupramoleculeName: GPR119-Gs-Nb35 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: GPR119 receptor-focused map

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Supramolecule #2: Glucose-dependent insulinotropic receptor

SupramoleculeName: Glucose-dependent insulinotropic receptor / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 67 KDa

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Supramolecule #3: G-protein complex

SupramoleculeName: G-protein complex / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #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: #5
Source (natural)Organism: Lama glama (llama)

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Macromolecule #1: Soluble cytochrome b562,Glucose-dependent insulinotropic receptor...

MacromoleculeName: Soluble cytochrome b562,Glucose-dependent insulinotropic receptor,LgBiT
type: protein_or_peptide / ID: 1 / 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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Macromolecule #2: 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: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MHHHHHHHHH HLEVLFQGPG SSGSELDQLR QEAEQLKNQI RDARKACADA TLSQITNNID PVGRIQMRTR RTLRGHLAKI YAMHWGTDS RLLVSASQDG KLIIWDSYTT NKVHAIPLRS SWVMTCAYAP SGNYVACGGL DNICSIYNLK TREGNVRVSR E LAGHTGYL ...String:
MHHHHHHHHH HLEVLFQGPG SSGSELDQLR QEAEQLKNQI RDARKACADA TLSQITNNID PVGRIQMRTR RTLRGHLAKI YAMHWGTDS RLLVSASQDG KLIIWDSYTT NKVHAIPLRS SWVMTCAYAP SGNYVACGGL DNICSIYNLK TREGNVRVSR E LAGHTGYL SCCRFLDDNQ IVTSSGDTTC ALWDIETGQQ TTTFTGHTGD VMSLSLAPDT RLFVSGACDA SAKLWDVREG MC RQTFTGH ESDINAICFF PNGNAFATGS DDATCRLFDL RADQELMTYS HDNIICGITS VSFSKSGRLL LAGYDDFNCN VWD ALKADR AGVLAGHDNR VSCLGVTDDG MAVATGSWDS FLKIWNGSSG GGGSGGGGSS GVSGWRLFKK IS

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Macromolecule #3: 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: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
MASNNTASIA QARKLVEQLK MEANIDRIKV SKAAADLMAY CEAHAKEDPL LTPVPASENP FREKKFFCAI L

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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)
SequenceString: MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKNTIVKQM RILHVNGFNG EGGEEDPQAA RSNSDGEKA TKVQDIKNNL KEAIETIVAA MSNLVPPVEL ANPENQFRVD YILSVMNVPD FDFPPEFYEH AKALWEDEGV R ACYERSNE ...String:
MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKNTIVKQM RILHVNGFNG EGGEEDPQAA RSNSDGEKA TKVQDIKNNL KEAIETIVAA MSNLVPPVEL ANPENQFRVD YILSVMNVPD FDFPPEFYEH AKALWEDEGV R ACYERSNE YQLIDCAQYF LDKIDVIKQA DYVPSDQDLL RCRVLTSGIF ETKFQVDKVN FHMFDVGAQR DERRKWIQCF ND VTAIIFV VASSSYNMVI REDNQTNRLQ AALKLFDSIW NNKWLRDTSV ILFLNKQDLL AEKVLAGKSK IEDYFPEFAR YTT PEDATP EPGEDPRVTR AKYFIRDEFL RISTASGDGR HYCYPHFTCA VDTENIRRVF NDCRDIIQRM HLRQYELL

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Macromolecule #5: Nanobody35

MacromoleculeName: Nanobody35 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
SequenceString:
QVQLQESGGG LVQPGGSLRL SCAASGFTFS NYKMNWVRQA PGKGLEWVSD ISQSGASISY TGSVKGRFTI SRDNAKNTLY LQMNSLKPE DTAVYYCARC PAPFTRDCFD VTSTTYAYRG QGTQVTVSSH HHHHHEPEA

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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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.61 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 57653
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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