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- EMDB-26590: CryoEM Structure of Inactive H2R Bound to Famotidine, Nb6M, and NabFab -

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Basic information

Entry
Database: EMDB / ID: EMD-26590
TitleCryoEM Structure of Inactive H2R Bound to Famotidine, Nb6M, and NabFab
Map dataFocused refinement map on receptor-nanobody
Sample
  • Complex: Complex of H2R, Nb6M, and NabFab
    • Complex: H2R
      • Protein or peptide: Histamine H2 receptor
    • Complex: Nb6M
      • Protein or peptide: NabFab LC
    • Complex: NabFab
      • Protein or peptide: NabFab HC
    • Protein or peptide: Nanobody 6M
  • Ligand: famotidine
KeywordsAntagonist / Complex / MEMBRANE PROTEIN
Function / homology
Function and homology information


adenylate cyclase-inhibiting opioid receptor signaling pathway / dynorphin receptor activity / regulation of saliva secretion / gastric acid secretion / Histamine receptors / histamine receptor activity / neurotransmitter receptor activity / neuropeptide binding / positive regulation of vasoconstriction / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger ...adenylate cyclase-inhibiting opioid receptor signaling pathway / dynorphin receptor activity / regulation of saliva secretion / gastric acid secretion / Histamine receptors / histamine receptor activity / neurotransmitter receptor activity / neuropeptide binding / positive regulation of vasoconstriction / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / neuropeptide signaling pathway / sensory perception of pain / locomotory behavior / G alpha (s) signalling events / defense response to virus / chemical synaptic transmission / neuron projection / immune response / dendrite / synapse / nucleoplasm / plasma membrane / cytosol
Similarity search - Function
Histamine H2 receptor / Kappa opioid receptor / Opioid receptor / 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)
Similarity search - Domain/homology
Kappa-type opioid receptor / Histamine H2 receptor
Similarity search - Component
Biological speciesHomo sapiens (human) / Lama glama (llama) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsRobertson MJ / Skiniotis G
Funding support United States, 1 items
OrganizationGrant numberCountry
The G. Harold and Leila Y. Mathers Foundation United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Structure determination of inactive-state GPCRs with a universal nanobody.
Authors: Michael J Robertson / Makaía M Papasergi-Scott / Feng He / Alpay B Seven / Justin G Meyerowitz / Ouliana Panova / Maria Claudia Peroto / Tao Che / Georgios Skiniotis /
Abstract: Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. Despite ...Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. Despite representing one of the largest classes of therapeutic targets, most inactive-state G protein-coupled receptors (GPCRs) have remained inaccessible for cryo-EM because their small size and membrane-embedded nature impedes projection alignment for high-resolution map reconstructions. Here we demonstrate that the same single-chain camelid antibody (nanobody) recognizing a grafted intracellular loop can be used to obtain cryo-EM structures of inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained structures of neurotensin 1 receptor bound to antagonist SR48692, μ-opioid receptor bound to alvimopan, apo somatostatin receptor 2 and histamine receptor 2 bound to famotidine. We expect this rapid, straightforward approach to facilitate the broad exploration of GPCR inactive states without the need for extensive engineering and crystallization.
History
DepositionApr 3, 2022-
Header (metadata) releaseJun 29, 2022-
Map releaseJun 29, 2022-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26590.png

Downloads & links

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Map

FileDownload / File: emd_26590.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocused refinement map on receptor-nanobody
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 380 pix.
= 329.726 Å		0.87 Å/pix.
x 380 pix.
= 329.726 Å		0.87 Å/pix.
x 380 pix.
= 329.726 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8677 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.6005778 - 2.1898432
Average (Standard dev.)-0.0001566174 (±0.019372337)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 329.72598 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_26590_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_26590_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_26590_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of H2R, Nb6M, and NabFab

EntireName: Complex of H2R, Nb6M, and NabFab
Components
  • Complex: Complex of H2R, Nb6M, and NabFab
    • Complex: H2R
      • Protein or peptide: Histamine H2 receptor
    • Complex: Nb6M
      • Protein or peptide: NabFab LC
    • Complex: NabFab
      • Protein or peptide: NabFab HC
    • Protein or peptide: Nanobody 6M
  • Ligand: famotidine

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Supramolecule #1: Complex of H2R, Nb6M, and NabFab

SupramoleculeName: Complex of H2R, Nb6M, and NabFab / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: H2R

SupramoleculeName: H2R / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Nb6M

SupramoleculeName: Nb6M / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Lama glama (llama)

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Supramolecule #4: NabFab

SupramoleculeName: NabFab / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Histamine H2 receptor

MacromoleculeName: Histamine H2 receptor / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.770184 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DYKDDDDAMG QPGNGSAFLL APNRSHAPDH DVENLYFQGM APNGTASSFC LDSTACKITI TVVLAVLILI TVAGNVVVCL AVGLNRRLR NLTNCFIVSL AITDLLLGLL VLPFSAIYQL SCKWSFGKVF CNIYTSLDVM LCTASILNLF MISLDRYCAV M DPLRYPVL ...String:
DYKDDDDAMG QPGNGSAFLL APNRSHAPDH DVENLYFQGM APNGTASSFC LDSTACKITI TVVLAVLILI TVAGNVVVCL AVGLNRRLR NLTNCFIVSL AITDLLLGLL VLPFSAIYQL SCKWSFGKVF CNIYTSLDVM LCTASILNLF MISLDRYCAV M DPLRYPVL VTPVRVAISL VLIWVISITL SFLSIHLGWN SRNETSKGNH TTSKCKVQVN EVYGLVDGLV TFYLPLLIMC VC YTLMILR LKSVRLLSGS REKDRNLRRI TRLVLVVVAV FVICWFPYFT AFVYRGLRGD DAINEVLEAI VLWLGYANSA LNP ILYAAL NRDFRTGYQQ LFCCRLANRN SHKTSLRSNA SQLSRTQSRE PRQQEEKPLK LQVWSGTEVT APQGATDRLE VLFQ

UniProtKB: Histamine H2 receptor, Kappa-type opioid receptor, Histamine H2 receptor

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Macromolecule #2: Nanobody 6M

MacromoleculeName: Nanobody 6M / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.418919 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QRQLVESGGG LVQPGGSLRL SCAASGTIFR LYDMGWFRQA PGKEREGVAS ITSGGSTKYA DSVKGRFTIS RDNAKNTVYL QMNSLEPED TAVYYCNAEY RTGIWEELLD GWGKGTPVTV SSHHHHHHEP EA

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Macromolecule #3: NabFab HC

MacromoleculeName: NabFab HC / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 25.684463 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NFSYYSIHWV RQAPGKGLEW VAYISSSSSY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARGYQYWQYH ASWYWNGGLD YWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL V KDYFPEPV ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NFSYYSIHWV RQAPGKGLEW VAYISSSSSY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARGYQYWQYH ASWYWNGGLD YWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL V KDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH T

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Macromolecule #4: NabFab LC

MacromoleculeName: NabFab LC / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.258783 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #5: famotidine

MacromoleculeName: famotidine / type: ligand / ID: 5 / Number of copies: 1 / Formula: FO9
Molecular weightTheoretical: 339.461 Da
Chemical component information

ChemComp-FO9:
famotidine

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 58.58 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 365068
Initial angle assignmentType: OTHER
Final angle assignmentType: ANGULAR RECONSTITUTION

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