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- EMDB-61446: Cryo-EM structure of neuropeptide FF receptor 2 in the ligand-fre... -

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Basic information

Entry
Database: EMDB / ID: EMD-61446
TitleCryo-EM structure of neuropeptide FF receptor 2 in the ligand-free state with BRIL fusion, anti-BRIL Fab, and nanobody
Map data
Sample
  • Complex: human neuropeptide FF receptor 2 in the apo state with BRIL fusion, anti-BRIL Fab, and nanobody
    • Protein or peptide: Isoform 2 of Neuropeptide FF receptor 2,Soluble cytochrome b562
    • Protein or peptide: Anti-BRIL fab heavy chain
    • Protein or peptide: Anti-fab nanobody
    • Protein or peptide: Anti-BRIL fab light chain
KeywordsGPCR / MEMBRANE PROTEIN
Function / homology
Function and homology information


opioid receptor binding / Orexin and neuropeptides FF and QRFP bind to their respective receptors / detection of abiotic stimulus / neuropeptide receptor activity / regulation of MAPK cascade / neuropeptide signaling pathway / cellular response to hormone stimulus / electron transport chain / G protein-coupled receptor activity / actin cytoskeleton ...opioid receptor binding / Orexin and neuropeptides FF and QRFP bind to their respective receptors / detection of abiotic stimulus / neuropeptide receptor activity / regulation of MAPK cascade / neuropeptide signaling pathway / cellular response to hormone stimulus / electron transport chain / G protein-coupled receptor activity / actin cytoskeleton / G alpha (q) signalling events / periplasmic space / electron transfer activity / G protein-coupled receptor signaling pathway / iron ion binding / heme binding / plasma membrane
Similarity search - Function
Neuropeptide FF receptor family / Neuropeptide FF receptor, type 2 / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / 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
Soluble cytochrome b562 / Neuropeptide FF receptor 2
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsKim J / Choi H-J
Funding support Korea, Republic Of, 1 items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea) Korea, Republic Of
CitationJournal: EMBO Rep / Year: 2025
Title: Structural insights into the selective recognition of RF-amide peptides by neuropeptide FF receptor 2.
Authors: Jeesoo Kim / Sooyoung Hong / Hajin Lee / Hyun Sik Lee / Chaehee Park / Jinuk Kim / Wonpil Im / Hee-Jung Choi /
Abstract: Neuropeptide FF Receptor 2 (NPFFR2), a G-protein-coupled receptor, plays a role in pain modulation and diet-induced thermogenesis. While NPFFR2 is strongly activated by neuropeptides FF (NPFFs), it ...Neuropeptide FF Receptor 2 (NPFFR2), a G-protein-coupled receptor, plays a role in pain modulation and diet-induced thermogenesis. While NPFFR2 is strongly activated by neuropeptides FF (NPFFs), it shows low activity in response to RF-amide-related peptides (RFRPs), despite the peptides belonging to a shared family. In contrast, NPFFR1, which shares high sequence similarity with NPFFR2, is activated by RFRPs and regulates reproductive hormone balance. The molecular basis for these receptor-specific interactions with their RF-amide peptides remains unclear. Here, we present cryo-electron microscopy structures of NPFFR2 in its active state bound to the agonist RF-amide peptide hNPSF, and in its ligand-free state. Structural analysis reveals that the C-terminal RF-amide moiety engages conserved residues in the transmembrane domain, while the N-terminal segment interacts in a receptor subtype-specific manner. Key selectivity-determining residues in NPFFR2 are also identified. A homology model of NPFFR1 bound to RFRP, supported by mutagenesis studies, further validates this selectivity mechanism. Additionally, structural comparison between the inactive and active states of NPFFR2 suggests a TM3-mediated activation mechanism. These findings provide insights into RF-amide peptide recognition by NPFF receptors.
History
DepositionSep 5, 2024-
Header (metadata) releaseApr 9, 2025-
Map releaseApr 9, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61446.png

Downloads & links

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Map

FileDownload / File: emd_61446.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 300 pix.
= 315. Å		1.05 Å/pix.
x 300 pix.
= 315. Å		1.05 Å/pix.
x 300 pix.
= 315. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-7.8731713 - 9.751092999999999
Average (Standard dev.)-0.0016184495 (±0.11522727)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 315.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: A composite map was made by applying separate...

Fileemd_61446_additional_1.map
AnnotationA composite map was made by applying separate masks for local refinement of the GPCR and BRIL-Fab-nanobody, then merging them using vop maximum.
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_61446_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_61446_half_map_2.map
Projections & Slices
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Sample components

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Entire : human neuropeptide FF receptor 2 in the apo state with BRIL fusio...

EntireName: human neuropeptide FF receptor 2 in the apo state with BRIL fusion, anti-BRIL Fab, and nanobody
Components
  • Complex: human neuropeptide FF receptor 2 in the apo state with BRIL fusion, anti-BRIL Fab, and nanobody
    • Protein or peptide: Isoform 2 of Neuropeptide FF receptor 2,Soluble cytochrome b562
    • Protein or peptide: Anti-BRIL fab heavy chain
    • Protein or peptide: Anti-fab nanobody
    • Protein or peptide: Anti-BRIL fab light chain

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Supramolecule #1: human neuropeptide FF receptor 2 in the apo state with BRIL fusio...

SupramoleculeName: human neuropeptide FF receptor 2 in the apo state with BRIL fusion, anti-BRIL Fab, and nanobody
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 120 KDa

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Macromolecule #1: Isoform 2 of Neuropeptide FF receptor 2,Soluble cytochrome b562

MacromoleculeName: Isoform 2 of Neuropeptide FF receptor 2,Soluble cytochrome b562
type: protein_or_peptide / ID: 1 / Details: 246 to 363 is soluble cytochrome b562 with linker / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 60.471199 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DYKDDDDASS ENWHPIWNVN DTKHHLYSDI NITYVNYYLH QPQVAAIFII SYFLIFFLCM MGNTVVCFIV MRNKHMHTVT NLFILNLAI SDLLVGIFCM PITLLDNIIA GWPFGNTMCK ISGLVQGISV AASVFTLVAI AVDRFQCVVY PFKPKLTIKT A FVIIMIIW ...String:
DYKDDDDASS ENWHPIWNVN DTKHHLYSDI NITYVNYYLH QPQVAAIFII SYFLIFFLCM MGNTVVCFIV MRNKHMHTVT NLFILNLAI SDLLVGIFCM PITLLDNIIA GWPFGNTMCK ISGLVQGISV AASVFTLVAI AVDRFQCVVY PFKPKLTIKT A FVIIMIIW VLAITIMSPS AVMLHVQEEK YYRVRLNSQN KTSPVYWCRE DWPNQEMRKI YTTVLFANIY LAPLSLIVIM YG RIGISAR RQLADLEDNW ETLNDNLKVI EKADNAAQVK DALTKMRAAA LDAQKATPPK LEDKSPDSPE MKDFRHGFDI LVG QIDDAL KLANEGKVKE AQAAAEQLKT TRNAYIQKYL ERARSTLRKK QKIIKMLLIV ALLFILSWLP LWTLMMLSDY ADLS PNELQ IINIYIYPFA HWLAFGNSSV NPIIYGFFNE NFRRGFQEAF QLQLCQKRAK PMEAYALKAK SHVLINTSNQ LVQES TFQN PHGETLLYRK SAEKPQQELV MEELKETTNS SEIHMGLEVL FQ

UniProtKB: Neuropeptide FF receptor 2, Soluble cytochrome b562, Neuropeptide FF receptor 2

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Macromolecule #2: Anti-BRIL fab heavy chain

MacromoleculeName: Anti-BRIL fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.321084 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK S

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Macromolecule #3: Anti-fab nanobody

MacromoleculeName: Anti-fab nanobody / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.461796 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSQVQLQESG GGLVQPGGSL RLSCAASGRT ISRYAMSWFR QAPGKEREFV AVARRSGDGA FYADSVQGRF TVSRDDAKNT VYLQMNSLK PEDTAVYYCA IDSDTFYSGS YDYWGQGTQV TVSSLEHHHH HH

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Macromolecule #4: Anti-BRIL fab light chain

MacromoleculeName: Anti-BRIL fab light chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.483062 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYLYYSLVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYLYYSLVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGE

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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

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7tuy

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 324660
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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