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- PDB-9qs6: Cryo-EM structure of the helix-stabilized MMM ubiquitin ligase co... -

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Basic information

Entry
Database: PDB / ID: 9qs6
TitleCryo-EM structure of the helix-stabilized MMM ubiquitin ligase complex with nanobody 270 (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 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 / negative regulation of melanin biosynthetic process / 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 ...epiboly involved in gastrulation with mouth forming second / negative regulation of melanin biosynthetic process / 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 / 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 brain development / embryonic heart tube morphogenesis / coronary vasculature development / embryonic limb morphogenesis / regulation of neuron differentiation / negative regulation of G protein-coupled receptor signaling pathway / embryonic digit morphogenesis / aorta development / ciliary membrane / smoothened signaling pathway / 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 / endoplasmic reticulum / Golgi apparatus / 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
: / 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: 3.3 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. / 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. / Steyaert, J. / ...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. / Steyaert, J. / Bazan, F. / Rouse, S. / Marqusee, S. / Kong, J.H. / Siebold, C. / Rohatgi, R.
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

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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
C: Nanobody 270
B: Isoform 2 of Multiple epidermal growth factor-like domains protein 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,5817
Polymers147,0214
Non-polymers5593
Water34219
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 with C-terminal His-6 tag. / Source: (gene. exp.) Homo sapiens (human) / Gene: MGRN1, KIAA0544, RNF156 / 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: 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 site-TwinStrep tags.
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
#4: 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: 50593.105 Da / Num. of mol.: 1
Mutation: A2605E, K2606E, Q2607E, A2608E, L2609K, D2610K, Q2611K, Q2613E, Q2615E, R2616E, H2618R, L2619R, Q2620R, M2622E, T2623E
Source method: isolated from a genetically manipulated source
Details: Cytoplasmic helix of human MEGF8 (residues 2605-2624) were replaced with E/RK repeats (EEEEKKKREEEERRRREEEK). Human MEGF8 has a C-terminal 1D4 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

#3: 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 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 , 3 types, 22 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-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
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 19 / 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
1Helix-stabilized human MEGF8-MOSMO-MGRN1 (MMM) ternary 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.#1-#40RECOMBINANT
2MOSMOCOMPLEXTernary complex with MEGF8 and MGRN1 was obtained by co-expressing helix-stabilized MEGF8, MOSMO and MGRN1 to form the MMM complex.#21RECOMBINANT
3Helix-stabilized MEGF8COMPLEXCytoplasmic helix of MEGF8 (residues 2605-2624) were replaced with E/RK repeats (EEEEKKKREEEERRRREEEK). MEGF8 was co-expressed with MOSMO and MGRN1, then the complex was purified on size-exclusion chromatography with nanobody 270.#41RECOMBINANT
4Nanobody 270COMPLEX#31RECOMBINANT
5MGRN1COMPLEXMGRN1 was co-expressed with helix-stabilized 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: 3.07 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: 20432 / 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: 5097418
Details: Particles initially picked from 2D templates (imported from EMD-53323 dataset).
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: OTHER / Num. of particles: 191977
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

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeDetails (eV)Initial refinement model-ID
19QRU

9qru

9QRUChain B (residues 2615-2749) and chains D, G, H.1
29QS3

9qs3
PDB Unreleased entry

9QS3Chain B (residues 2374-2614), and chains A, C, E, F.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027516
ELECTRON MICROSCOPYf_angle_d0.43610221
ELECTRON MICROSCOPYf_dihedral_angle_d3.2661100
ELECTRON MICROSCOPYf_chiral_restr0.0421140
ELECTRON MICROSCOPYf_plane_restr0.0031319

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