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- PDB-9qqs: Structure of the MEGF8-MOSMO complex with nanobody 270 (Focused r... -

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Basic information

Entry
Database: PDB / ID: 9qqs
TitleStructure of the MEGF8-MOSMO complex with nanobody 270 (Focused refinement)
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: 2.65 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 2, 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
B: Isoform 2 of Multiple epidermal growth factor-like domains protein 8
C: Nanobody 270
hetero molecules


Theoretical massNumber of molelcules
Total (without water)88,0115
Polymers87,3263
Non-polymers6852
Water57632
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 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
#2: 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: 50352.977 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: MEGF8 (residues 2312-2778) with C-terminal 1D4 epitope tag.
Source: (gene. exp.) Homo sapiens (human) / Cell line: HEK293S GnTI- TetR / Gene: MEGF8, C19orf49, EGFL4, KIAA0817 / 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: Q7Z7M0

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Antibody , 1 types, 1 molecules C

#3: Antibody Nanobody 270


Mass: 14378.924 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: 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, 34 molecules

#4: Chemical ChemComp-PLM / PALMITIC ACID


Mass: 256.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H32O2 / 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 / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 32 / 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
1Focused refinement of the MEGF8-MOSMO binary complex bound to nanobody 270COMPLEXMMM complex was obtained by co-expression of MEGF8, MOSMO and MGRN1 components in HEK293S GnTI- TetR cells. The MMM complex was purified by tandem affinity purification. Purified nanobody 270, from E. coli WK6 cells, was added to MMM complex and the MMM-nanobody 270 complex formed on size-exclusion chromatography. A focused refinement of extracellular and transmembrane regions of the MMM complex was performed to obtain this MEGF8-MOSMO map.#1-#30RECOMBINANT
2MOSMOCOMPLEXTernary complex with MEGF8 and MGRN1 was obtained by co-expressing MEGF8, MOSMO and MGRN1 to form the MMM complex.#11RECOMBINANT
3MEGF8COMPLEXMEGF8 was co-expressed with MOSMO and MGRN1, then the complex was purified on size-exclusion chromatography with nanobody 270.#21RECOMBINANT
4Nanobody 270COMPLEX#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11NO
21NO
3163 kDa/nmNO
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: 1600 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: 3.1 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: 12956 / 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 software
IDNameCategory
1PHENIXmodel refinement
3cryoSPARCparticle selection
7cryoSPARC3D reconstruction
CTF correctionDetails: Patch CTF estimation was performed in cryoSPARC live pre-processing.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4203652
Details: 2D classes from a Glacios screening dataset facilitated template particle picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142461
Details: Particle subtraction followed by local refinement in cryoSPARC was used for the final reconstruction.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 79.9 / 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 buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0033995
ELECTRON MICROSCOPYf_angle_d0.4835439
ELECTRON MICROSCOPYf_dihedral_angle_d13.0731494
ELECTRON MICROSCOPYf_chiral_restr0.042622
ELECTRON MICROSCOPYf_plane_restr0.005679

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