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- EMDB-53328: Focused refinement of the MGRN1 ubiquitin ligase in complex with ... -

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Entry
Database: EMDB / ID: EMD-53328
TitleFocused refinement of the MGRN1 ubiquitin ligase in complex with helix-stabilized MEGF8, MOSMO and nanobody 270
Map data
Sample
  • Complex: Helix-stabilized human MEGF8-MOSMO-MGRN1 (MMM) ternary complex bound to nanobody 270
    • Complex: MOSMO
      • Protein or peptide: Human Modulator of Smoothened (MOSMO)
    • Complex: Helix-stabilized MEGF8
      • Protein or peptide: Human Multiple Epidermal Growth Factor-like Domains Protein 8 (MEGF8) with stabilized cytoplasmic helix
    • Complex: Nanobody 270
      • Protein or peptide: Nanobody 270
    • Complex: MGRN1
      • Protein or peptide: Human Mahogunin Ring Finger 1 (MGRN1) E3 ligase
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


left/right pattern formation / embryonic skeletal system development / embryonic limb morphogenesis / regulation of neuron differentiation / ciliary membrane / smoothened signaling pathway / lung development / negative regulation of smoothened signaling pathway / regulation of protein stability / RING-type E3 ubiquitin transferase ...left/right pattern formation / embryonic skeletal system development / embryonic limb morphogenesis / regulation of neuron differentiation / ciliary membrane / smoothened signaling pathway / lung development / negative regulation of smoothened signaling pathway / regulation of protein stability / RING-type E3 ubiquitin transferase / intracellular protein localization / heart development / gene expression / in utero embryonic development / cell differentiation / Golgi apparatus / plasma membrane
Similarity search - Function
Modulator of smoothened protein / Attenuator of Hedgehog
Similarity search - Domain/homology
Isoform 4 of E3 ubiquitin-protein ligase MGRN1 / Isoform 2 of 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: 4.41 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 4, 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_53328.png

Downloads & links

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Map

FileDownload / File: emd_53328.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: 0.07
Minimum - Maximum-0.6501897 - 0.60944706
Average (Standard dev.)-0.00036275343 (±0.0067176227)
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_53328_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_53328_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_53328_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : Helix-stabilized human MEGF8-MOSMO-MGRN1 (MMM) ternary complex bo...

EntireName: Helix-stabilized human MEGF8-MOSMO-MGRN1 (MMM) ternary complex bound to nanobody 270
Components
  • Complex: Helix-stabilized human MEGF8-MOSMO-MGRN1 (MMM) ternary complex bound to nanobody 270
    • Complex: MOSMO
      • Protein or peptide: Human Modulator of Smoothened (MOSMO)
    • Complex: Helix-stabilized MEGF8
      • Protein or peptide: Human Multiple Epidermal Growth Factor-like Domains Protein 8 (MEGF8) with stabilized cytoplasmic helix
    • Complex: Nanobody 270
      • Protein or peptide: Nanobody 270
    • Complex: MGRN1
      • Protein or peptide: Human Mahogunin Ring Finger 1 (MGRN1) E3 ligase

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Supramolecule #1: Helix-stabilized human MEGF8-MOSMO-MGRN1 (MMM) ternary complex bo...

SupramoleculeName: Helix-stabilized human MEGF8-MOSMO-MGRN1 (MMM) ternary complex bound to nanobody 270
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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 MGRN1 and MEGF8 intracellular domain (ICD), residues 2615-2749, was performed to obtain this map.
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: MOSMO

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

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Supramolecule #3: Helix-stabilized MEGF8

SupramoleculeName: Helix-stabilized MEGF8 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Details: Cytoplasmic 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- ...Details: Cytoplasmic 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.
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: #4
Source (natural)Organism: Lama glama (llama)

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Supramolecule #5: MGRN1

SupramoleculeName: MGRN1 / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #1
Details: MGRN1 was co-expressed with helix-stabilized MEGF8 and MOSMO to form the MMM complex.
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Human Mahogunin Ring Finger 1 (MGRN1) E3 ligase

MacromoleculeName: Human Mahogunin Ring Finger 1 (MGRN1) E3 ligase / type: protein_or_peptide / ID: 1 / Details: Full-length human MGRN1 with C-terminal His-6 tag. / Enantiomer: LEVO / EC number: RING-type E3 ubiquitin transferase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGSILSRRIA GVEDIDIQAN SAYRYPPKSG NYFASHFFMG GEKFDTPHPE GYLFGENMDL NFLGSRPVQF PYVTPAPHEP VKTLRSLVN IRKDSLRLVR YKDDADSPTE DGDKPRVLYS LEFTFDADAR VAITIYCQAS EEFLNGRAVY SPKSPSLQSE T VHYKRGVS ...String:
MGSILSRRIA GVEDIDIQAN SAYRYPPKSG NYFASHFFMG GEKFDTPHPE GYLFGENMDL NFLGSRPVQF PYVTPAPHEP VKTLRSLVN IRKDSLRLVR YKDDADSPTE DGDKPRVLYS LEFTFDADAR VAITIYCQAS EEFLNGRAVY SPKSPSLQSE T VHYKRGVS QQFSLPSFKI DFSEWKDDEL NFDLDRGVFP VVIQAVVDEG DVVEVTGHAH VLLAAFEKHM DGSFSVKPLK QK QIVDRVS YLLQEIYGIE NKNNQETKPS DDENSDNSNE CVVCLSDLRD TLILPCRHLC LCTSCADTLR YQANNCPICE LPF RALLQI RAVRKKPGAL SPVSFSPVLA QSLEHDEHSN SDSVPPGYEP ISLLEALNGL RAVSPAIPSA PLYEEITYSG ISDG LSQAS CPLAAIDHIL DSSRQKGRPQ SKAPDSTLRS PSSPIHEEDE EKLSEDVDAP PPLGGAELAL RESSSPESFI TEEVD ESSS PQQGTRAASI ENVLQDSSPE HCGRGPPADI YLPALGPDSC SVGIDEGTKH HHHHH

UniProtKB: Isoform 4 of E3 ubiquitin-protein ligase MGRN1

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Macromolecule #2: Human Modulator of Smoothened (MOSMO)

MacromoleculeName: Human Modulator of Smoothened (MOSMO) / type: protein_or_peptide / ID: 2
Details: Human MOSMO with C-terminal 3C protease site-TwinStrep tags.
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDKLTIISGC LFLAADIFAI ASIANPDWIN TGESAGALTV GLVRQCQTIH GRDRTCIPPR LPPEWVTTLF FIIMGIISLT VTCGLLVASH WRREATKYAR WIAFTGMILF CMAALIFPIG FYINEVGGQP YKLPNNTVVG SSYVLFVLSI FFTIVGLLFA GKVCLPGGTL ...String:
MDKLTIISGC LFLAADIFAI ASIANPDWIN TGESAGALTV GLVRQCQTIH GRDRTCIPPR LPPEWVTTLF FIIMGIISLT VTCGLLVASH WRREATKYAR WIAFTGMILF CMAALIFPIG FYINEVGGQP YKLPNNTVVG SSYVLFVLSI FFTIVGLLFA GKVCLPGGTL EVLFQGPGGS GSAWSHPQFE KGGGSGGGSG GSAWSHPQFE K

UniProtKB: Modulator of smoothened protein

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Macromolecule #3: Human Multiple Epidermal Growth Factor-like Domains Protein 8 (ME...

MacromoleculeName: Human Multiple Epidermal Growth Factor-like Domains Protein 8 (MEGF8) with stabilized cytoplasmic helix
type: protein_or_peptide / ID: 3
Details: Cytoplasmic helix of human MEGF8 (residues 2605-2624) were replaced with E/RK repeats (EEEEKKKREEEERRRREEEK). Human MEGF8 has a C-terminal 1D4 tag.
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: KCQCNGHADT CNEQDGTGCP CQNNTETGTC QGSSPSDRRD CYKYQCAKCR ESFHGSPLGG QQCYRLISVE QECCLDPTSQ TNCFHEPKRR ALGPGRTVLF GVQPKFTNVD IRLTLDVTFG AVDLYVSTSY DTFVVRVAPD TGVHTVHIQP PPAPPPPPPP ADGGPRGAGD ...String:
KCQCNGHADT CNEQDGTGCP CQNNTETGTC QGSSPSDRRD CYKYQCAKCR ESFHGSPLGG QQCYRLISVE QECCLDPTSQ TNCFHEPKRR ALGPGRTVLF GVQPKFTNVD IRLTLDVTFG AVDLYVSTSY DTFVVRVAPD TGVHTVHIQP PPAPPPPPPP ADGGPRGAGD PGGAGASSGP GAPAEPRVRE VWPRGLITYV TVTEPSAVLV VRGVRDRLVI TYPHEHHALK SSRFYLLLLG VGDPSGPGAN GSADSQGLLF FRQDQAHIDL FVFFSVFFSC FFLFLSLCVL LWKEEEEKKK REEEERRRRE EEKMASRPFA KVTVCFPPDP TAPASAWKPA GLPPPAFRRS EPFLAPLLLT GAGGPWGPMG GGCCPPAIPA TTAGLRAGPI TLEPTEDGMA GVATLLLQLP GGPHAPNGAC LGSALVTLRH RLHEYCGGGG GAGGSGHGTG AGRKGLLSQD NLTSMSLGTE TSQVAPA

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

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Macromolecule #4: Nanobody 270

MacromoleculeName: Nanobody 270 / type: protein_or_peptide / ID: 4 / Details: Nanobody 270 with C-terminal His6-EPEA tags. / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString:
QVQLVESGGG LVQAGGSLRL SCAASGSIFS YDDMGWYRQA PGKQRELVAT FTNVGSTNYV DSVKGRFTIS RDNAKNTVYL QMNSLKPEDT AVYYCHAYTV RRFQGMEYWG KGTQVTVSSH HHHHHEPEA

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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
SoftwareName: EPU (ver. 3.6)
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Number grids imaged: 1 / Number real images: 20432 / Average exposure time: 3.07 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: 5097418
Details: Particles initially picked from 2D templates (imported from EMD-53323 dataset).
CTF correctionSoftware - Name: cryoSPARC (ver. 4.3.1) / Software - details: Patch CTF
Details: 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:

9qru


Details: High-resolution binary helix-stabilized MEGF8-MOSMO complex (9QS3 PDB) and wild-type MMM complex (9QRU PDB) models were merged and docked in the map using fit-in-map tool in ChimeraX. ...Details: High-resolution binary helix-stabilized MEGF8-MOSMO complex (9QS3 PDB) and wild-type MMM complex (9QRU PDB) models were merged and docked in the map using fit-in-map tool in ChimeraX. Cytoplasmic helix residues in the helix-stabilized MEGF8 variant were mutated manually in Coot.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.41 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.2) / Software - details: Local refinement
Details: After particle subtraction of the detergent micelle and extracellular regions of the map (nanobody 270, MEGF8 residues 2374-2575), local refinement was performed using a mask around the ...Details: After particle subtraction of the detergent micelle and extracellular regions of the map (nanobody 270, MEGF8 residues 2374-2575), local refinement was performed using a mask around the transmembrane and cytoplasmic (MEGF8 ICD-MGRN1) domains.
Number images used: 71430
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2)
Final 3D classificationNumber classes: 3 / Avg.num./class: 70459 / Software - Name: cryoSPARC (ver. 4.5.3) / Software - details: 3D classification (without alignment)
Details: 3D classification without alignment was performed in cryoSPARC using a mask around cytoplasmic regions of the map (MEGF8 intracellular domain and MGRN1).
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

9qru

source_name: PDB, initial_model_type: experimental modelChain B (residues 2615-2749) and chains D, G, H.

9qs3
PDB Unreleased entry

source_name: PDB, initial_model_type: experimental modelChain B (residues 2374-2614), and chains A, C, E, F.
SoftwareName: UCSF ChimeraX (ver. 1.9)
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: 152.5

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