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- EMDB-53367: Cryo-EM structure of the binary MEGF8-MOSMO complex with nanobody 270 -

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Basic information

Entry
Database: EMDB / ID: EMD-53367
TitleCryo-EM structure of the binary MEGF8-MOSMO complex with nanobody 270
Map data
Sample
  • Complex: Human 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: 2.98 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 NR / Sarkar P / Lartey D / Gao L / Pardon E / Milenkovic L / Chalk R / Steyaert J / Bazan F / Rouse SL / ...Authors: Williams C / Nocka LM / Hedger G / Carrique L / Pusapati GV / Parashara P / Latorraca NR / Sarkar P / Lartey D / Gao L / Pardon E / Milenkovic L / Chalk R / Steyaert J / Bazan F / Rouse SL / Marqusee S / Kong JH / Siebold C / Rohatgi R
History
DepositionApr 9, 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_53367.png

Downloads & links

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Map

FileDownload / File: emd_53367.map.gz / Format: CCP4 / Size: 178 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 360 pix.
= 262.908 Å		0.73 Å/pix.
x 360 pix.
= 262.908 Å		0.73 Å/pix.
x 360 pix.
= 262.908 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: 0.306
Minimum - Maximum-1.1211053 - 1.8001544
Average (Standard dev.)0.0000664441 (±0.030110795)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 262.908 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_53367_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_53367_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_53367_half_map_2.map
Projections & Slices
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Sample components

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Entire : Human MEGF8-MOSMO binary complex bound to nanobody 270

EntireName: Human MEGF8-MOSMO binary complex bound to nanobody 270
Components
  • Complex: Human 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: Human MEGF8-MOSMO binary complex bound to nanobody 270

SupramoleculeName: Human MEGF8-MOSMO binary complex bound to nanobody 270
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: MEGF8-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 ...Details: MEGF8-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.
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: Binary complex with MEGF8 was obtained by co-expressing MEGF8 and MOSMO.
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: MEGF8

SupramoleculeName: MEGF8 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Details: MEGF8 (residues 2312-2645) was co-expressed with MOSMO, then purified with nanobody 270 on size-exclusion chromatography.
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: #2
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 human 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: Nanobody 270

MacromoleculeName: Nanobody 270 / type: protein_or_peptide / ID: 2 / Details: Nanobody 270 with 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 #3: 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: 3
Details: Human MEGF8 (residues 2312-2645) with C-terminal 1D4 epitope tag.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.349152 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 PPDPTAPAGT ETSQVAPA

UniProtKB: Multiple epidermal growth factor-like domains protein 8

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Macromolecule #4: PALMITIC ACID

MacromoleculeName: PALMITIC ACID / type: ligand / ID: 4
Details: Full-length human MOSMO with C-terminal 3C protease cleavage site-TwinStrep tags. Cys164 is palmitoylated (PLM).
Number of copies: 1 / Formula: PLM
Source (natural)Organism: Homo sapiens (human)
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: 10 / 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: 12157 / Average exposure time: 2.99 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: 1.4000000000000001 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: 2266859
Details: Template picker using 2D classes from a screening dataset collected on a different grid.
CTF correctionDetails: Patch CTF estimation was performed in cryoSPARC live pre-processing.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

9qqs


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: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Local refinement with a mask containing MEGF8, MOSMO and nanobody 270 was performed after non-uniform refinement, to mask out the micelle.
Number images used: 228513
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 3 / Avg.num./class: 146816
Details: Class probability filter (> 0.9) was performed after heterogenous refinement with three pre-defined volumes.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

9qqs


Chain - Source name: PDB / Chain - Initial model type: experimental 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: 143.4
Output model

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

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