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- EMDB-53313: Structure of the MEGF8-MOSMO complex with nanobody 270 (Focused r... -

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Basic information

Entry
Database: EMDB / ID: EMD-53313
TitleStructure of the MEGF8-MOSMO complex with nanobody 270 (Focused refinement)
Map data
Sample
  • Complex: Focused refinement of the MEGF8-MOSMO binary complex bound to nanobody 270
    • Complex: MOSMO
      • Protein or peptide: Modulator of smoothened protein
    • Complex: MEGF8
      • Protein or peptide: Isoform 2 of Multiple epidermal growth factor-like domains protein 8
    • Complex: Nanobody 270
      • Protein or peptide: Nanobody 270
  • Ligand: PALMITIC ACID
  • Ligand: (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]
  • Ligand: water
KeywordsE3 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 / MEMBRANE PROTEIN
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
Multiple epidermal growth factor-like domains protein 8 / Modulator of smoothened protein
Similarity search - Component
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.65 Å
AuthorsWilliams C / Carrique L / Pardon E / Nocka LM / Hedger G / Pusapati GV / Parashara P / Latorraca NR / Sarkar P / Lartey D ...Williams C / Carrique L / Pardon E / Nocka LM / Hedger G / Pusapati GV / Parashara P / Latorraca NR / Sarkar P / Lartey D / Gao L / Milenkovic L / Chalk R / Steyaert J / Bazan F / Rouse SL / Marqusee S / Kong JH / Rohatgi R / Siebold C
Funding support United Kingdom, 5 items
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 LM / Hedger G / Carrique L / Pusapati GV / Parashara P / Latorraca N / Sarkar P / Lartey D / Gao L / Pardon E / Milenkovic L / Steyaert J / Chalk R / Bazan F / Rouse SL / ...Authors: Williams C / Nocka LM / Hedger G / Carrique L / Pusapati GV / Parashara P / Latorraca N / Sarkar P / Lartey D / Gao L / Pardon E / Milenkovic L / Steyaert J / Chalk R / Bazan F / Rouse SL / Marqusee S / Kong JH / Siebold C / Rohatgi R
History
DepositionApr 2, 2025-
Header (metadata) releaseMay 27, 2026-
Map releaseMay 27, 2026-
UpdateMay 27, 2026-
Current statusMay 27, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53313.png

Downloads & links

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Map

FileDownload / File: emd_53313.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 480 pix.
= 350.544 Å		0.73 Å/pix.
x 480 pix.
= 350.544 Å		0.73 Å/pix.
x 480 pix.
= 350.544 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.7303 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.386
Minimum - Maximum-1.6192799 - 2.0431163
Average (Standard dev.)-0.00024900888 (±0.028207727)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 350.544 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_53313_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_53313_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_53313_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Focused refinement of the MEGF8-MOSMO binary complex bound to nan...

EntireName: Focused refinement of the MEGF8-MOSMO binary complex bound to nanobody 270
Components
  • Complex: Focused refinement of the MEGF8-MOSMO binary complex bound to nanobody 270
    • Complex: MOSMO
      • Protein or peptide: Modulator of smoothened protein
    • Complex: MEGF8
      • Protein or peptide: Isoform 2 of Multiple epidermal growth factor-like domains protein 8
    • Complex: Nanobody 270
      • Protein or peptide: Nanobody 270
  • Ligand: PALMITIC ACID
  • Ligand: (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]
  • Ligand: water

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Supramolecule #1: Focused refinement of the MEGF8-MOSMO binary complex bound to nan...

SupramoleculeName: Focused refinement of the MEGF8-MOSMO binary complex bound to nanobody 270
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: MMM 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 ...Details: MMM 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.
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: MOSMO

SupramoleculeName: MOSMO / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: Ternary complex with MEGF8 and MGRN1 was obtained by co-expressing MEGF8, MOSMO and MGRN1 to form the MMM complex.
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: MEGF8

SupramoleculeName: MEGF8 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Details: MEGF8 was co-expressed with MOSMO and MGRN1, then the complex was purified on size-exclusion chromatography with nanobody 270.
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: Nanobody 270

SupramoleculeName: Nanobody 270 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Lama glama (llama)

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Macromolecule #1: Modulator of smoothened protein

MacromoleculeName: Modulator of smoothened protein / type: protein_or_peptide / ID: 1
Details: Full-length MOSMO with C-terminal 3C protease cleavage site-TwinStrep tags. Cys164 is palmitoylated (PLM).
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.594246 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDKLTIISGC LFLAADIFAI ASIANPDWIN TGESAGALTV GLVRQCQTIH GRDRTCIPPR LPPEWVTTLF FIIMGIISLT VTCGLLVAS HWRREATKYA RWIAFTGMIL FCMAALIFPI GFYINEVGGQ PYKLPNNTVV GSSYVLFVLS IFFTIVGLLF A GKVCLPGG ...String:
MDKLTIISGC LFLAADIFAI ASIANPDWIN TGESAGALTV GLVRQCQTIH GRDRTCIPPR LPPEWVTTLF FIIMGIISLT VTCGLLVAS HWRREATKYA RWIAFTGMIL FCMAALIFPI GFYINEVGGQ PYKLPNNTVV GSSYVLFVLS IFFTIVGLLF A GKVCLPGG TLEVLFQGPG GSGSAWSHPQ FEKGGGSGGG SGGSAWSHPQ FEK

UniProtKB: Modulator of smoothened protein

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Macromolecule #2: Isoform 2 of Multiple epidermal growth factor-like domains protein 8

MacromoleculeName: Isoform 2 of Multiple epidermal growth factor-like domains protein 8
type: protein_or_peptide / ID: 2
Details: MEGF8 (residues 2312-2778) with C-terminal 1D4 epitope tag.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 50.352977 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: KCQCNGHADT CNEQDGTGCP CQNNTETGTC QGSSPSDRRD CYKYQCAKCR ESFHGSPLGG QQCYRLISVE QECCLDPTSQ TNCFHEPKR RALGPGRTVL FGVQPKFTNV DIRLTLDVTF GAVDLYVSTS YDTFVVRVAP DTGVHTVHIQ PPPAPPPPPP P ADGGPRGA ...String:
KCQCNGHADT CNEQDGTGCP CQNNTETGTC QGSSPSDRRD CYKYQCAKCR ESFHGSPLGG QQCYRLISVE QECCLDPTSQ TNCFHEPKR RALGPGRTVL FGVQPKFTNV DIRLTLDVTF GAVDLYVSTS YDTFVVRVAP DTGVHTVHIQ PPPAPPPPPP P ADGGPRGA GDPGGAGASS GPGAPAEPRV REVWPRGLIT YVTVTEPSAV LVVRGVRDRL VITYPHEHHA LKSSRFYLLL LG VGDPSGP GANGSADSQG LLFFRQDQAH IDLFVFFSVF FSCFFLFLSL CVLLWKAKQA LDQRQEQRRH LQEMTKMASR PFA KVTVCF PPDPTAPASA WKPAGLPPPA FRRSEPFLAP LLLTGAGGPW GPMGGGCCPP AIPATTAGLR AGPITLEPTE DGMA GVATL LLQLPGGPHA PNGACLGSAL VTLRHRLHEY CGGGGGAGGS GHGTGAGRKG LLSQDNLTSM SLGTETSQVA PA

UniProtKB: Multiple epidermal growth factor-like domains protein 8

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Macromolecule #3: Nanobody 270

MacromoleculeName: Nanobody 270 / type: protein_or_peptide / ID: 3 / Details: C-terminal His6-EPEA tag. / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.378924 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString:
QVQLVESGGG LVQAGGSLRL SCAASGSIFS YDDMGWYRQA PGKQRELVAT FTNVGSTNYV DSVKGRFTIS RDNAKNTVYL QMNSLKPED TAVYYCHAYT VRRFQGMEYW GKGTQVTVSS HHHHHHEPEA

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Macromolecule #4: PALMITIC ACID

MacromoleculeName: PALMITIC ACID / type: ligand / ID: 4 / Number of copies: 1 / Formula: PLM
Molecular weightTheoretical: 256.424 Da
Chemical component information

ChemComp-PLM:
PALMITIC ACID

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Macromolecule #5: (1~{S},2~{S},4~{S},5'~{R},6~{R},7~{S},8~{R},9~{S},12~{S},13~{R},1...

MacromoleculeName: (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]
type: ligand / ID: 5 / Number of copies: 1 / Formula: A1JAX
Molecular weightTheoretical: 428.647 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 32 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMC8H19NO4SHEPES
2.0 % v/vC3H8O3glycerol
2.0 mMCaCl2calcium chloride
0.02 % w/vC19H36O10glycyl-dodecyl-beta-D-maltoside (GDN)

Details: 20 mM HEPES pH 7.5, 150 mM NaCl, 2% (v/v) glycerol, 2 mM CaCl2, 0.02% (w/v) GDN
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Number grids imaged: 1 / Number real images: 12956 / Average exposure time: 3.1 sec. / Average electron dose: 50.0 e/Å2 / Details: Images were collected in counted mode.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4203652
Details: 2D classes from a Glacios screening dataset facilitated template particle picking
CTF correctionDetails: Patch CTF estimation was performed in cryoSPARC live pre-processing.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: AlphaFold2 was used to generate a model of the MEGF8-MOSMO complex, and for nanobody 270.
Details: Initial model was docked in the map using fit-in-map tool in ChimeraX.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.65 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC
Details: Particle subtraction followed by local refinement in cryoSPARC was used for the final reconstruction.
Number images used: 142461
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 4 / Avg.num./class: 128000
Details: 3D classification (without alignment) was performed with a mask around the transmembrane and extracellular domains of MEGF8, MOSMO and nanobody 270.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsInitial local fitting was done using ChimeraX then manual model building in Coot was interspersed with real space refinement in Phenix.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 79.9
Output model

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

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