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- PDB-9qsh: Cryo-EM structure of the MMM ubiquitin ligase complex with nanobo... -

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Basic information

Entry
Database: PDB / ID: 9qsh
TitleCryo-EM structure of the MMM ubiquitin ligase complex with nanobody 992 (Composite map)
Components
  • Isoform 2 of Multiple epidermal growth factor-like domains protein 8
  • Isoform 4 of E3 ubiquitin-protein ligase MGRN1
  • Modulator of smoothened protein
  • Nanobody 992
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 / negative regulation of melanin biosynthetic process / 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 ...epiboly involved in gastrulation with mouth forming second / negative regulation of melanin biosynthetic process / 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 / positive regulation of eating behavior / embryonic skeletal system morphogenesis / embryonic skeletal system development / positive regulation of axon extension involved in axon guidance / limb morphogenesis / negative regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / embryonic heart tube morphogenesis / embryonic brain development / coronary vasculature development / embryonic limb morphogenesis / regulation of neuron differentiation / embryonic digit morphogenesis / negative regulation of G protein-coupled receptor signaling pathway / aorta development / smoothened signaling pathway / ciliary membrane / endosome to lysosome transport / BMP signaling pathway / protein monoubiquitination / ubiquitin ligase complex / lung development / negative regulation of smoothened signaling pathway / protein sequestering activity / regulation of protein stability / RING-type E3 ubiquitin transferase / protein polyubiquitination / ubiquitin-protein transferase activity / intracellular protein localization / ubiquitin protein ligase activity / Antigen processing: Ubiquitination & Proteasome degradation / heart development / regulation of gene expression / protein-containing complex assembly / gene expression / in utero embryonic development / early endosome / cell differentiation / protein ubiquitination / calcium ion binding / Golgi apparatus / endoplasmic reticulum / extracellular exosome / zinc ion binding / membrane / nucleus / plasma membrane / cytoplasm / cytosol
Similarity search - Function
E3 ubiquitin-protein ligase MGRN1/RNF157-like / : / RNF157-like N-terminal domain / : / Attractin/LZTR1 beta-propeller / Modulator of smoothened protein / Attenuator of Hedgehog / EGF domain / : / Laminin/attractin EGF domain ...E3 ubiquitin-protein ligase MGRN1/RNF157-like / : / RNF157-like N-terminal domain / : / 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 / Zinc finger, C3HC4 type (RING finger) / 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. / Ring finger / EGF-like domain signature 1. / EGF-like domain signature 2. / EGF-like domain / Zinc finger RING-type profile. / Zinc finger, RING-type / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
: / PALMITIC ACID / E3 ubiquitin-protein ligase MGRN1 / 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: 3.5 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. / 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 4, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 27, 2026Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jun 10, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.1Jun 10, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Isoform 4 of E3 ubiquitin-protein ligase MGRN1
A: Modulator of smoothened protein
B: Isoform 2 of Multiple epidermal growth factor-like domains protein 8
C: Nanobody 992
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,0518
Polymers146,2354
Non-polymers8164
Water1086
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 , 3 types, 3 molecules DAB

#1: Protein Isoform 4 of E3 ubiquitin-protein ligase MGRN1 / Mahogunin RING finger protein 1 / RING finger protein 156 / RING-type E3 ubiquitin transferase MGRN1


Mass: 59454.988 Da / Num. of mol.: 1 / Mutation: R317E
Source method: isolated from a genetically manipulated source
Details: Full-length human MGRN1 R317E with C-terminal His-6 tag.
Source: (gene. exp.) Homo sapiens (human) / Gene: MGRN1, KIAA0544, RNF156 / Plasmid: pHR-CMV-TetO2 / Cell (production host): Endothelial / Organ (production host): Kidney / Production host: Homo sapiens (human) / Tissue (production host): Embryonic kidney
References: UniProt: O60291, RING-type E3 ubiquitin transferase
#2: 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: 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: 50352.977 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Human MEGF8 (residues 2312-2778) with C-terminal 1D4 epitope tag.
Source: (gene. exp.) Homo sapiens (human) / 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

#4: Antibody Nanobody 992


Mass: 13833.139 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Nanobody 992 with C-terminal His6-EPEA tags. / 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 , 4 types, 10 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-PLM / PALMITIC ACID


Mass: 256.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H32O2 / Feature type: SUBJECT OF INVESTIGATION
#7: 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
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / 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-MGRN1 (MMM) ternary complex bound to nanobody 992COMPLEXMMM 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 992, from E. coli WK6 cells, was added to MMM complex and the MMM-nanobody 992 complex formed on size-exclusion chromatography.#1-#40RECOMBINANT
2MOSMOCOMPLEXTernary complex with MEGF8 and MGRN1 was obtained by co-expressing MEGF8, MOSMO and MGRN1 to form the MMM complex.#21RECOMBINANT
3MEGF8COMPLEXMEGF8 was co-expressed with MOSMO and MGRN1, then the complex was purified on size-exclusion chromatography with nanobody 992.#31RECOMBINANT
4Nanobody 992COMPLEX#41RECOMBINANT
5MGRN1COMPLEXMGRN1 was co-expressed with MEGF8 and MOSMO to form the MMM complex.#11RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
41NO
51NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Lama glama (llama)9844
65Homo sapiens (human)9606
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
65Homo sapiens (human)9606Embryonic kidneypHR-CMV-TetO2
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: 11391 / 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
IDNameVersionCategoryDetails (eV)
1PHENIXmodel refinement
3cryoSPARC4.3.1particle selectionBlob picker, template picking, Topaz
7cryoSPARC4.5.13D reconstructionLocal refinement
CTF correctionDetails: Patch CTF estimation was performed in cryoSPARC live pre-processing.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1959769
Details: Blob picker initially was used to pick particles containing a detergent micelle.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: OTHER / Num. of particles: 187459
Details: Using map alone (d99), resolution was estimated in Phenix real-space refinement of the composite map.
Symmetry type: POINT
Atomic model buildingProtocol: 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 building
IDPDB-ID 3D fitting-IDAccession codeDetails (eV)Source nameType
19QRU

9qru

19QRUThe initial model consisted of chains A, B, D, F, G, H for PDB entry 9QRU.PDBexperimental model
21Nanobody 992AlphaFoldin silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047450
ELECTRON MICROSCOPYf_angle_d0.50410126
ELECTRON MICROSCOPYf_dihedral_angle_d4.4441101
ELECTRON MICROSCOPYf_chiral_restr0.0441131
ELECTRON MICROSCOPYf_plane_restr0.0041306

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