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- EMDB-53338: Focused refinement of the MGRN1 ubiquitin ligase in complex with ... -

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Basic information

Entry
Database: EMDB / ID: EMD-53338
TitleFocused refinement of the MGRN1 ubiquitin ligase in complex with MEGF8, MOSMO and nanobody 992
Map data
Sample
  • Complex: Human MEGF8-MOSMO-MGRN1 (MMM) ternary complex bound to nanobody 992
    • Complex: MOSMO
      • Protein or peptide: Human Modulator of Smoothened (MOSMO)
    • Complex: MEGF8
      • Protein or peptide: Human Multiple Epidermal Growth Factor-like Domains Protein 8 (MEGF8)
    • 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.66 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 / Rohatgi R / Lartey D / Gao L / Pardon E / Milenkovic L / Chalk R / Steyaert J / Bazan F ...Authors: Williams C / Nocka LM / Hedger G / Carrique L / Pusapati GV / Parashara P / Latorraca NR / Sarkar P / Rohatgi R / Lartey D / Gao L / Pardon E / Milenkovic L / Chalk R / Steyaert J / Bazan F / Rouse SL / Marqusee S / Kong JH / Siebold C
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_53338.png

Downloads & links

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Map

FileDownload / File: emd_53338.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: 0.1
Minimum - Maximum-0.40522817 - 0.7317654
Average (Standard dev.)-0.0004315014 (±0.005947573)
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_53338_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_53338_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_53338_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Human MEGF8-MOSMO-MGRN1 (MMM) ternary complex bound to nanobody 992

EntireName: Human MEGF8-MOSMO-MGRN1 (MMM) ternary complex bound to nanobody 992
Components
  • Complex: Human MEGF8-MOSMO-MGRN1 (MMM) ternary complex bound to nanobody 992
    • Complex: MOSMO
      • Protein or peptide: Human Modulator of Smoothened (MOSMO)
    • Complex: MEGF8
      • Protein or peptide: Human Multiple Epidermal Growth Factor-like Domains Protein 8 (MEGF8)
    • Complex: MGRN1
      • Protein or peptide: Human Mahogunin Ring Finger 1 (MGRN1) E3 ligase

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Supramolecule #1: Human MEGF8-MOSMO-MGRN1 (MMM) ternary complex bound to nanobody 992

SupramoleculeName: Human MEGF8-MOSMO-MGRN1 (MMM) ternary complex bound to nanobody 992
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 992, from E. coli WK6 cells, was added to MMM complex and the MMM-nanobody 992 complex formed on size-exclusion chromatography. Focused refinement of cytoplasmic regions of MEGF8 (residues 2614-2749) and MGRN1 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 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: #3
Details: MEGF8 was co-expressed with MOSMO and MGRN1, then the complex was purified on size-exclusion chromatography with nanobody 992.
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: MGRN1

SupramoleculeName: MGRN1 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #1
Details: MGRN1 was co-expressed with MEGF8 and MOSMO to form the MMM complex.

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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 R317E 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 cleavage site-TwinStrep tags. Cys164 is palmitoylated (PLM).
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
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 #3: Human Multiple Epidermal Growth Factor-like Domains Protein 8 (MEGF8)

MacromoleculeName: Human Multiple Epidermal Growth Factor-like Domains Protein 8 (MEGF8)
type: protein_or_peptide / ID: 3
Details: Human MEGF8 (residues 2312-2778) with C-terminal 1D4 epitope tag.
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
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: Isoform 2 of Multiple epidermal growth factor-like domains protein 8

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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: 11391 / 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: 1959769
Details: Blob picker initially was used to pick particles containing a detergent micelle.
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: MMM model (9QRU PDB) and an AlphaFold2 model of nanobody 992 were merged and 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: 4.66 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.2) / Software - details: Local refinement
Details: First, particle subtraction was performed using a mask encompassing the detergent micelle. Secondly, local refinement of cytoplasmic regions of the map (MEGF8 residues 2614-2749, MGRN1) was carried out.
Number images used: 83974
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: 62596 / Software - Name: cryoSPARC (ver. 4.5.3) / Software - details: 3D classification
Details: 3D classification without alignment was performed using a mask around cytoplasmic regions of MEGF8 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 modelThe initial model consisted of chains A, B, D, F, G, H for PDB entry 9QRU.
source_name: AlphaFold, initial_model_type: in silico modelNanobody 992
SoftwareName: Coot (ver. 0.9.8.95)
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: 205.3

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