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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 digestive tract left/right asymmetry / determination of heart 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 digestive tract left/right asymmetry / determination of heart 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 brain development / embryonic heart tube morphogenesis / coronary vasculature development / embryonic limb morphogenesis / regulation of neuron differentiation / embryonic digit morphogenesis / aorta development / ciliary membrane / smoothened signaling pathway / 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: Williams, C. / Nocka, L.M. / Hedger, G. / Carrique, L. / Pusapati, G.V. / Parashara, P. / Latorraca, N.R. / Sarkar, P. / Lartey, D. / Gao, L. / Pardon, E. / Milenkovic, L. / Chalk, R. / ...Authors: Williams, C. / Nocka, L.M. / Hedger, G. / Carrique, L. / Pusapati, G.V. / Parashara, P. / Latorraca, N.R. / Sarkar, P. / Lartey, D. / Gao, L. / Pardon, E. / Milenkovic, L. / Chalk, R. / Steyaert, J. / Bazan, F. / Rouse, S.L. / Marqusee, S. / Kong, J.H. / Siebold, C. / Rohatgi, R.
History
DepositionApr 9, 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: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / 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: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0May 27, 2026Data content type: Primary map / Data content type: Primary map / Provider: 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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