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- PDB-9qty: Cryo-EM structure of the binary MEGF8-MOSMO complex with nanobody 270 -

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Basic information

Entry
Database: PDB / ID: 9qty
TitleCryo-EM structure of the binary MEGF8-MOSMO complex with nanobody 270
Components
  • Isoform 2 of Multiple epidermal growth factor-like domains protein 8
  • Modulator of smoothened protein
  • Nanobody 270
KeywordsMEMBRANE PROTEIN / E3 Ubiquitin Ligase / Hedgehog Signaling / Single-pass Membrane Protein / Membrane Protein Complex / Smoothened / Tetraspanin / Cell Surface Receptor / Primary Cilium / Morphogen / Signal Transduction / Human / Carpenter Syndrome / Cancer / Nanobody / Palmitoylation / GDN
Function / homology
Function and homology information


epiboly involved in gastrulation with mouth forming second / determination of heart left/right asymmetry / determination of digestive tract left/right asymmetry / craniofacial suture morphogenesis / embryonic heart tube left/right pattern formation / left/right pattern formation / fasciculation of sensory neuron axon / cell migration involved in gastrulation / pharyngeal arch artery morphogenesis / embryonic skeletal system morphogenesis ...epiboly involved in gastrulation with mouth forming second / determination of heart left/right asymmetry / determination of digestive tract left/right asymmetry / craniofacial suture morphogenesis / embryonic heart tube left/right pattern formation / left/right pattern formation / fasciculation of sensory neuron axon / cell migration involved in gastrulation / pharyngeal arch artery morphogenesis / embryonic skeletal system morphogenesis / embryonic skeletal system development / positive regulation of axon extension involved in axon guidance / limb morphogenesis / embryonic heart tube morphogenesis / embryonic brain development / coronary vasculature development / embryonic limb morphogenesis / regulation of neuron differentiation / embryonic digit morphogenesis / aorta development / smoothened signaling pathway / ciliary membrane / BMP signaling pathway / ubiquitin ligase complex / lung development / negative regulation of smoothened signaling pathway / regulation of protein stability / intracellular protein localization / heart development / regulation of gene expression / protein-containing complex assembly / gene expression / in utero embryonic development / cell differentiation / protein ubiquitination / calcium ion binding / Golgi apparatus / extracellular exosome / membrane / nucleus / plasma membrane
Similarity search - Function
: / Attractin/LZTR1 beta-propeller / Modulator of smoothened protein / Attenuator of Hedgehog / EGF domain / : / Laminin/attractin EGF domain / EGF domain / Laminin-type EGF-like (LE) domain profile. / Laminin-type EGF-like (LE) domain signature. ...: / Attractin/LZTR1 beta-propeller / Modulator of smoothened protein / Attenuator of Hedgehog / EGF domain / : / Laminin/attractin EGF domain / EGF domain / Laminin-type EGF-like (LE) domain profile. / Laminin-type EGF-like (LE) domain signature. / Laminin-type epidermal growth factor-like domai / Laminin EGF domain / Laminin-type EGF domain / Plexin repeat / Plexin repeat / CUB domain / Domain first found in C1r, C1s, uEGF, and bone morphogenetic protein. / CUB domain / Spermadhesin, CUB domain superfamily / CUB domain profile. / : / Calcium-binding EGF domain / Kelch-type beta propeller / PSI domain / domain found in Plexins, Semaphorins and Integrins / EGF-type aspartate/asparagine hydroxylation site / EGF-like calcium-binding, conserved site / Calcium-binding EGF-like domain signature. / Aspartic acid and asparagine hydroxylation site. / EGF-like calcium-binding domain / Calcium-binding EGF-like domain / Epidermal growth factor-like domain. / EGF-like domain profile. / EGF-like domain signature 1. / EGF-like domain signature 2. / EGF-like domain
Similarity search - Domain/homology
: / PALMITIC ACID / Multiple epidermal growth factor-like domains protein 8 / Modulator of smoothened protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsWilliams, C. / Carrique, L. / Pardon, E. / Nocka, L.M. / Hedger, G. / Pusapati, G.V. / Parashara, P. / Latorraca, N.R. / Sarkar, P. / Lartey, D. ...Williams, C. / Carrique, L. / Pardon, E. / Nocka, L.M. / Hedger, G. / Pusapati, G.V. / Parashara, P. / Latorraca, N.R. / Sarkar, P. / Lartey, D. / Gao, L. / Milenkovic, L. / Chalk, R. / Steyaert, J. / Bazan, F. / Rouse, S.L. / Marqusee, S. / Kong, J.H. / Rohatgi, R. / Siebold, C.
Funding support United Kingdom, 5items
OrganizationGrant numberCountry
Cancer Research UKDRCRPG-May23/100002 United Kingdom
Cancer Research UKC20724/A26752 United Kingdom
Wellcome Trust218482/Z/19/Z United Kingdom
Wellcome Trust060208/Z/00/Z United Kingdom
Wellcome Trust093305/Z/10/Z United Kingdom
CitationJournal: Mol Cell / Year: 2026
Title: Design principles of a membrane-spanning ubiquitin ligase.
Authors: Carys Williams / Laura M Nocka / George Hedger / Pragya Parashara / Els Pardon / Naomi R Latorraca / Ganesh V Pusapati / Parijat Sarkar / Dorothy Lartey / Lei Gao / Ljiljana Milenkovic / Rod ...Authors: Carys Williams / Laura M Nocka / George Hedger / Pragya Parashara / Els Pardon / Naomi R Latorraca / Ganesh V Pusapati / Parijat Sarkar / Dorothy Lartey / Lei Gao / Ljiljana Milenkovic / Rod Chalk / Jan Steyaert / Susan Marqusee / Loïc Carrique / J Fernando Bazan / Sarah L Rouse / Jennifer H Kong / Christian Siebold / Rajat Rohatgi /
Abstract: Receptor-type E3 ubiquitin ligases enable extracellular signals to control ubiquitylation in the cytoplasm, playing widespread roles in development, metabolism, and immunity. Using cryoelectron ...Receptor-type E3 ubiquitin ligases enable extracellular signals to control ubiquitylation in the cytoplasm, playing widespread roles in development, metabolism, and immunity. Using cryoelectron microscopy, integrated with biophysical and functional studies, we visualized a human E3 complex composed of two transmembrane proteins, MEGF8 and MOSMO, and the intracellular RING-family protein MGRN1. This MEGF8-MOSMO-MGRN1 (MMM) complex attenuates Hedgehog signaling by ubiquitylating Smoothened (SMO), a G-protein-coupled receptor (GPCR) that transduces morphogen signals. A long helix in the MMM complex engages SMO using an intramembrane degron and extends into the cytoplasm to suspend an activated and precisely oriented RING domain below the plasma membrane. This architecture enables ubiquitylation of the cytoplasmic surface of SMO, reducing SMO abundance at primary cilia. Our structure provides insights into MEGF8 mutations, which cause multi-organ birth defects, and defines a paradigm for how transmembrane E3 ligases control the cell surface abundance of GPCRs and other signaling receptors.
History
DepositionApr 9, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 27, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Modulator of smoothened protein
C: Nanobody 270
B: Isoform 2 of Multiple epidermal growth factor-like domains protein 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,0075
Polymers74,3223
Non-polymers6852
Water18010
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Modulator of smoothened protein / Attenuator of hedgehog


Mass: 22594.246 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Full-length human MOSMO with C-terminal 3C protease cleavage site-TwinStrep tags. Cys164 is palmitoylated (PLM).
Source: (gene. exp.) Homo sapiens (human) / Gene: MOSMO, ATTHOG, C16orf52 / Plasmid: pHR-CMV-TetO2 / Cell (production host): Endothelial / Cell line (production host): HEK293S GnTI- TetR / Organ (production host): Kidney / Production host: Homo sapiens (human) / Tissue (production host): Embryonic kidney / References: UniProt: Q8NHV5
#3: Protein Isoform 2 of Multiple epidermal growth factor-like domains protein 8 / Multiple EGF-like domains protein 8 / Epidermal growth factor-like protein 4 / EGF-like protein 4


Mass: 37349.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Human MEGF8 (residues 2312-2645) with C-terminal 1D4 epitope tag.
Source: (gene. exp.) Homo sapiens (human) / Gene: MEGF8, C19orf49, EGFL4, KIAA0817 / Production host: Homo sapiens (human) / References: UniProt: Q7Z7M0

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Antibody , 1 types, 1 molecules C

#2: Antibody Nanobody 270


Mass: 14378.924 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Nanobody 270 with C-terminal His6-EPEA tag. / Source: (gene. exp.) Lama glama (llama) / Plasmid: pMESy4 / Production host: Escherichia coli K-12 (bacteria) / Strain (production host): WK6

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Non-polymers , 3 types, 12 molecules

#4: Chemical ChemComp-PLM / PALMITIC ACID / Attenuator of hedgehog


Mass: 256.424 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C16H32O2
Details: Full-length human MOSMO with C-terminal 3C protease cleavage site-TwinStrep tags. Cys164 is palmitoylated (PLM).
Source: (gene. exp.) Homo sapiens (human) / Gene: MOSMO, ATTHOG, C16orf52 / Plasmid: pHR-CMV-TetO2 / Cell (production host): Endothelial / Cell line (production host): HEK293S GnTI- TetR / Organ (production host): Kidney / Production host: Homo sapiens (human) / Tissue (production host): Embryonic kidney / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-A1JAX / (1~{S},2~{S},4~{S},5'~{R},6~{R},7~{S},8~{R},9~{S},12~{S},13~{R},16~{S})-16-methoxy-5',7,9,13-tetramethyl-spiro[5-oxapentacyclo[10.8.0.0^{2,9}.0^{4,8}.0^{13,18}]icos-18-ene-6,2'-oxane]


Mass: 428.647 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C28H44O3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetails (eV)Entity IDParent-IDSource
1Human MEGF8-MOSMO binary complex bound to nanobody 270COMPLEXMEGF8-MOSMO complex was obtained by co-expression of MEGF8 (residues 2312-2645) and MOSMO components in HEK293S GnTI- TetR cells. Purified nanobody 270, from E. coli WK6 cells, was added to MEGF8-MOSMO and the ternary complex formed on size-exclusion chromatography.#1-#30RECOMBINANT
2MOSMOCOMPLEXBinary complex with MEGF8 was obtained by co-expressing MEGF8 and MOSMO.#11RECOMBINANT
3MEGF8COMPLEXMEGF8 (residues 2312-2645) was co-expressed with MOSMO, then purified with nanobody 270 on size-exclusion chromatography.#31RECOMBINANT
4Nanobody 270COMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
41NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
21Homo sapiens (human)9606Embryonic kidneypHR-CMV-TetO2
32Homo sapiens (human)9606Embryonic kidneypHR-CMV-TetO2
43Homo sapiens (human)9606Embryonic kidneypHR-CMV-TetO2
54Escherichia coli K-12 (bacteria)83333WK6pMESy4
Buffer solutionpH: 7.5
Details: 20 mM HEPES pH 7.5, 150 mM NaCl, 2% (v/v) glycerol, 2 mM CaCl2, 0.02% (w/v) GDN
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMHEPESC8H19NO4S1
32 % v/vglycerolC3H8O31
42 mMcalcium chlorideCaCl21
50.02 % w/vglycyl-dodecyl-beta-D-maltoside (GDN)C19H36O101
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.99 sec. / Electron dose: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12157 / Details: Images were collected in counted mode.
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM softwareName: PHENIX / Version: 1.21rc1_5156: / Category: model refinement
CTF correctionDetails: Patch CTF estimation was performed in cryoSPARC live pre-processing.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2266859
Details: Template picker using 2D classes from a screening dataset collected on a different grid.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 228513
Details: Local refinement with a mask containing MEGF8, MOSMO and nanobody 270 was performed after non-uniform refinement, to mask out the micelle.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 143.4 / Protocol: RIGID BODY FIT / Space: REAL
Details: Initial local fitting was done using ChimeraX then manual model building in Coot was interspersed with real space refinement in Phenix.
Atomic model buildingPDB-ID: 9QQS

9qqs


Accession code: 9QQS / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0023944
ELECTRON MICROSCOPYf_angle_d0.4925370
ELECTRON MICROSCOPYf_dihedral_angle_d6.462548
ELECTRON MICROSCOPYf_chiral_restr0.042617
ELECTRON MICROSCOPYf_plane_restr0.004668

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