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- EMDB-41612: Cryo-EM structure of the inner MKLN1 dimer from an autoinhibited ... -

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Basic information

Entry
Database: EMDB / ID: EMD-41612
TitleCryo-EM structure of the inner MKLN1 dimer from an autoinhibited MKLN1 tetramer
Map data
Sample
  • Complex: Cryo-EM structure of the inner MKLN1 dimer from an autoinhibited MKLN1 tetramer
    • Protein or peptide: Muskelin
KeywordsE3 ubiquitin ligase / CTLH complex / substrate adapter recruitment / PROTEIN BINDING / LIGASE
Function / homology
Function and homology information


postsynaptic specialization membrane of symmetric synapse / postsynaptic endosome membrane / neurotransmitter receptor transport postsynaptic membrane to endosome / regulation of receptor internalization / vesicle-mediated transport in synapse / ubiquitin ligase complex / ruffle / Regulation of pyruvate metabolism / cell-matrix adhesion / GABA-ergic synapse ...postsynaptic specialization membrane of symmetric synapse / postsynaptic endosome membrane / neurotransmitter receptor transport postsynaptic membrane to endosome / regulation of receptor internalization / vesicle-mediated transport in synapse / ubiquitin ligase complex / ruffle / Regulation of pyruvate metabolism / cell-matrix adhesion / GABA-ergic synapse / regulation of cell shape / actin cytoskeleton organization / cell cortex / signal transduction / protein homodimerization activity / nucleoplasm / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Muskelin, N-terminal / : / Muskelin N-terminus / CTLH, C-terminal LisH motif / C-terminal to LisH (CTLH) motif profile. / Galactose oxidase/kelch, beta-propeller / Lissencephaly type-1-like homology motif / KLHDC2/KLHL20/DRC7 Kelch-repeats domain / LIS1 homology (LisH) motif profile. / LIS1 homology motif ...Muskelin, N-terminal / : / Muskelin N-terminus / CTLH, C-terminal LisH motif / C-terminal to LisH (CTLH) motif profile. / Galactose oxidase/kelch, beta-propeller / Lissencephaly type-1-like homology motif / KLHDC2/KLHL20/DRC7 Kelch-repeats domain / LIS1 homology (LisH) motif profile. / LIS1 homology motif / Kelch motif / Kelch repeat type 1 / Kelch-type beta propeller / Galactose-binding-like domain superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsChana CK / Keszei AFA / Sicheri F
Funding support Canada, 3 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT-178026 Canada
Canadian Institutes of Health Research (CIHR)PJT-180338 Canada
Canadian Institutes of Health Research (CIHR)PJT-186218 Canada
CitationJournal: Nat Commun / Year: 2024
Title: FAM72A degrades UNG2 through the GID/CTLH complex to promote mutagenic repair during antibody maturation.
Authors: Philip Barbulescu / Chetan K Chana / Matthew K Wong / Ines Ben Makhlouf / Jeffrey P Bruce / Yuqing Feng / Alexander F A Keszei / Cassandra Wong / Rukshana Mohamad-Ramshan / Laura C McGary / ...Authors: Philip Barbulescu / Chetan K Chana / Matthew K Wong / Ines Ben Makhlouf / Jeffrey P Bruce / Yuqing Feng / Alexander F A Keszei / Cassandra Wong / Rukshana Mohamad-Ramshan / Laura C McGary / Mohammad A Kashem / Derek F Ceccarelli / Stephen Orlicky / Yifei Fang / Huihui Kuang / Mohammad Mazhab-Jafari / Rossanna C Pezo / Ashok S Bhagwat / Trevor J Pugh / Anne-Claude Gingras / Frank Sicheri / Alberto Martin /
Abstract: A diverse antibody repertoire is essential for humoral immunity. Antibody diversification requires the introduction of deoxyuridine (dU) mutations within immunoglobulin genes to initiate somatic ...A diverse antibody repertoire is essential for humoral immunity. Antibody diversification requires the introduction of deoxyuridine (dU) mutations within immunoglobulin genes to initiate somatic hypermutation (SHM) and class switch recombination (CSR). dUs are normally recognized and excised by the base excision repair (BER) protein uracil-DNA glycosylase 2 (UNG2). However, FAM72A downregulates UNG2 permitting dUs to persist and trigger SHM and CSR. How FAM72A promotes UNG2 degradation is unknown. Here, we show that FAM72A recruits a C-terminal to LisH (CTLH) E3 ligase complex to target UNG2 for proteasomal degradation. Deficiency in CTLH complex components result in elevated UNG2 and reduced SHM and CSR. Cryo-EM structural analysis reveals FAM72A directly binds to MKLN1 within the CTLH complex to recruit and ubiquitinate UNG2. Our study further suggests that FAM72A hijacks the CTLH complex to promote mutagenesis in cancer. These findings show that FAM72A is an E3 ligase substrate adaptor critical for humoral immunity and cancer development.
History
DepositionAug 14, 2023-
Header (metadata) releaseSep 11, 2024-
Map releaseSep 11, 2024-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41612.png

Downloads & links

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Map

FileDownload / File: emd_41612.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 324 pix.
= 267.3 Å		0.83 Å/pix.
x 324 pix.
= 267.3 Å		0.83 Å/pix.
x 324 pix.
= 267.3 Å

Surface

Projections

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Images are generated by Spider.

Voxel size
XYZ
EMDB info.0.41250.41250.4125
CCP4 map header0.8250.8250.825
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.23
Minimum - Maximum-0.32015845 - 0.9423437
Average (Standard dev.)0.0024185418 (±0.03280106)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 133.65 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41612_msk_1.map
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Additional map: deepEMhancer sharpened map

Fileemd_41612_additional_1.map
AnnotationdeepEMhancer sharpened map
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Half map: #2

Fileemd_41612_half_map_1.map
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Half map: #1

Fileemd_41612_half_map_2.map
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Sample components

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Entire : Cryo-EM structure of the inner MKLN1 dimer from an autoinhibited ...

EntireName: Cryo-EM structure of the inner MKLN1 dimer from an autoinhibited MKLN1 tetramer
Components
  • Complex: Cryo-EM structure of the inner MKLN1 dimer from an autoinhibited MKLN1 tetramer
    • Protein or peptide: Muskelin

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Supramolecule #1: Cryo-EM structure of the inner MKLN1 dimer from an autoinhibited ...

SupramoleculeName: Cryo-EM structure of the inner MKLN1 dimer from an autoinhibited MKLN1 tetramer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: From focused refinement of MKLN1 tetramer
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Muskelin

MacromoleculeName: Muskelin / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 87.202195 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHSAV DENLYFQGGG RAAGGAVAAA PECRLLPYAL HKWSSFSSTY LPENILVDKP NDQSSRWSSE SNYPPQYLIL KLERPAIVQ NITFGKYEKT HVCNLKKFKV FGGMNEENMT ELLSSGLKND YNKETFTLKH KIDEQMFPCR FIKIVPLLSW G PSFNFSIW ...String:
MHHHHHHSAV DENLYFQGGG RAAGGAVAAA PECRLLPYAL HKWSSFSSTY LPENILVDKP NDQSSRWSSE SNYPPQYLIL KLERPAIVQ NITFGKYEKT HVCNLKKFKV FGGMNEENMT ELLSSGLKND YNKETFTLKH KIDEQMFPCR FIKIVPLLSW G PSFNFSIW YVELSGIDDP DIVQPCLNWY SKYREQEAIR LCLKHFRQHN YTEAFESLQK KTKIALEHPM LTDIHDKLVL KG DFDACEE LIEKAVNDGL FNQYISQQEY KPRWSQIIPK STKGDGEDNR PGMRGGHQMV IDVQTETVYL FGGWDGTQDL ADF WAYSVK ENQWTCISRD TEKENGPSAR SCHKMCIDIQ RRQIYTLGRY LDSSVRNSKS LKSDFYRYDI DTNTWMLLSE DTAA DGGPK LVFDHQMCMD SEKHMIYTFG GRILTCNGSV DDSRASEPQF SGLFAFNCQC QTWKLLREDS CNAGPEDIQS RIGHC MLFH SKNRCLYVFG GQRSKTYLND FFSYDVDSDH VDIISDGTKK DSGMVPMTGF TQRATIDPEL NEIHVLSGLS KDKEKR EEN VRNSFWIYDI VRNSWSCVYK NDQAAKDNPT KSLQEEEPCP RFAHQLVYDE LHKVHYLFGG NPGKSCSPKM RLDDFWS LK LCRPSKDYLL RHCKYLIRKH RFEEKAQVDP LSALKYLQND LYITVDHSDP EETKEFQLLA SALFKSGSDF TALGFSDV D HTYAQRTQLF DTLVNFFPDS MTPPKGNLVD LITL

UniProtKB: Muskelin

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 57.08 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: AlphaFold2 model
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 159095
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

ul63

chain_id: A, source_name: AlphaFold, initial_model_type: in silico model

ul63

chain_id: B, source_name: AlphaFold, initial_model_type: in silico model
RefinementOverall B value: 168.75
Output model

PDB-8ttq:
Cryo-EM structure of the inner MKLN1 dimer from an autoinhibited MKLN1 tetramer

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