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- EMDB-52316: USP1-UAF1 bound to Lys63-linked diubiquitin -

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Basic information

Entry
Database: EMDB / ID: EMD-52316
TitleUSP1-UAF1 bound to Lys63-linked diubiquitin
Map data
Sample
  • Complex: USP1-UAF1 bound to Lys63-linked diubiquitin
    • Protein or peptide: Ubiquitin carboxyl-terminal hydrolase 1
    • Protein or peptide: WD repeat-containing protein 48
    • Protein or peptide: Polyubiquitin-B
  • Protein or peptide: Polyubiquitin-B
  • Ligand: ZINC ION
KeywordsUSP1 / deubiquitinating enzyme / cysteine protease / complex / HYDROLASE
Function / homology
Function and homology information


regulation of protein monoubiquitination / positive regulation of error-prone translesion synthesis / Signaling by cytosolic PDGFRA and PDGFRB fusion proteins / monoubiquitinated protein deubiquitination / seminiferous tubule development / deubiquitinase activator activity / symbiont entry into host cell via disruption of host cell glycocalyx / skeletal system morphogenesis / skin development / symbiont entry into host cell via disruption of host cell envelope ...regulation of protein monoubiquitination / positive regulation of error-prone translesion synthesis / Signaling by cytosolic PDGFRA and PDGFRB fusion proteins / monoubiquitinated protein deubiquitination / seminiferous tubule development / deubiquitinase activator activity / symbiont entry into host cell via disruption of host cell glycocalyx / skeletal system morphogenesis / skin development / symbiont entry into host cell via disruption of host cell envelope / virus tail / homeostasis of number of cells / protein deubiquitination / embryonic organ development / single fertilization / positive regulation of double-strand break repair via homologous recombination / regulation of DNA repair / response to UV / Fanconi Anemia Pathway / ubiquitin binding / Recognition of DNA damage by PCNA-containing replication complex / positive regulation of epithelial cell proliferation / skeletal system development / double-strand break repair via homologous recombination / positive regulation of receptor signaling pathway via JAK-STAT / regulation of protein stability / multicellular organism growth / late endosome / peptidase activity / single-stranded DNA binding / double-stranded DNA binding / spermatogenesis / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / lysosome / Ub-specific processing proteases / cysteine-type endopeptidase activity / DNA repair / intracellular membrane-bounded organelle / DNA damage response / proteolysis / DNA binding / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
WDR48/Bun107 / Ubiquitin specific peptidase 1 / : / Domain of unknown function (DUF3337) / : / Ubiquitin specific protease (USP) domain signature 2. / Ubiquitin specific protease (USP) domain signature 1. / Ubiquitin specific protease, conserved site / Peptidase C19, ubiquitin carboxyl-terminal hydrolase / Ubiquitin carboxyl-terminal hydrolase ...WDR48/Bun107 / Ubiquitin specific peptidase 1 / : / Domain of unknown function (DUF3337) / : / Ubiquitin specific protease (USP) domain signature 2. / Ubiquitin specific protease (USP) domain signature 1. / Ubiquitin specific protease, conserved site / Peptidase C19, ubiquitin carboxyl-terminal hydrolase / Ubiquitin carboxyl-terminal hydrolase / Ubiquitin specific protease domain / Ubiquitin specific protease (USP) domain profile. / Pectate lyase superfamily protein / Rhamnogalacturonase A/epimerase, pectate lyase-like / Pectin lyase fold / Pectin lyase fold/virulence factor / Papain-like cysteine peptidase superfamily / WD domain, G-beta repeat / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Ubiquitin carboxyl-terminal hydrolase 1 / Tail fiber / WD repeat-containing protein 48
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsKeijzer N / Sakoltchik J / Sixma TK
Funding support Netherlands, 2 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)TOP714.016.002 Netherlands
Health-HollandLSHM21048-H045 Netherlands
CitationJournal: Nat Commun / Year: 2025
Title: USP1/UAF1 targets polyubiquitinated PCNA with an exo-cleavage mechanism that can temporarily enrich for monoubiquitinated PCNA.
Authors: Niels Keijzer / Jan Sakoltchik / Kaustav Majumder / Nina van Lil / Farid El Oualid / Alexander Fish / Titia K Sixma /
Abstract: DNA damage tolerance (DDT) is an important pathway that allows cells to bypass DNA lesions during replication. DDT is orchestrated by ubiquitination of PCNA, where monoubiquitinated PCNA (PCNA-Ub) ...DNA damage tolerance (DDT) is an important pathway that allows cells to bypass DNA lesions during replication. DDT is orchestrated by ubiquitination of PCNA, where monoubiquitinated PCNA (PCNA-Ub) initiates recruitment of TLS polymerases but also serves as a substrate for further ubiquitination, forming K63-polyubiquitinated PCNA that leads to HR-mediated bypass mechanisms. Recent work on USP1/UAF1 inhibition revealed that formation of K48-linked chains also occurs on PCNA, resulting in its proteasomal degradation. USP1/UAF1 is established as deubiquitinating enzyme (DUB) for PCNA-Ub, but little is known about removal of ubiquitin chains on PCNA. Here we show that USP1/UAF1 cleaves both K48 and K63-linked ubiquitin chains on PCNA efficiently, using an exo-cleavage mechanism. Kinetic analysis reveals that USP1/UAF1 prefers cleaving the ubiquitin-ubiquitin bond over cleavage of the ubiquitin-PCNA bond and therefore treats poly- and monoubiquitinated PCNA as different substrates. A cryo-EM structure of USP1/UAF1 with a K63-diubiquitin and structure-based mutagenesis suggests that this mechanistic preference is maintained in evolution. This unusual mechanism can cause temporal enrichment of monoubiquitinated PCNA during polyubiquitination. It will be interesting to see how this affects DDT pathway balance.
History
DepositionDec 11, 2024-
Header (metadata) releaseFeb 12, 2025-
Map releaseFeb 12, 2025-
UpdateAug 20, 2025-
Current statusAug 20, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52316.png

Downloads & links

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Map

FileDownload / File: emd_52316.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 360 pix.
= 300.96 Å		0.84 Å/pix.
x 360 pix.
= 300.96 Å		0.84 Å/pix.
x 360 pix.
= 300.96 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.836 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0192
Minimum - Maximum-0.34302434 - 0.5805047
Average (Standard dev.)0.000013237443 (±0.0099755805)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 300.96002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_52316_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_52316_half_map_2.map
Projections & Slices
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Sample components

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Entire : USP1-UAF1 bound to Lys63-linked diubiquitin

EntireName: USP1-UAF1 bound to Lys63-linked diubiquitin
Components
  • Complex: USP1-UAF1 bound to Lys63-linked diubiquitin
    • Protein or peptide: Ubiquitin carboxyl-terminal hydrolase 1
    • Protein or peptide: WD repeat-containing protein 48
    • Protein or peptide: Polyubiquitin-B
  • Protein or peptide: Polyubiquitin-B
  • Ligand: ZINC ION

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Supramolecule #1: USP1-UAF1 bound to Lys63-linked diubiquitin

SupramoleculeName: USP1-UAF1 bound to Lys63-linked diubiquitin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2, #4
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Ubiquitin carboxyl-terminal hydrolase 1

MacromoleculeName: Ubiquitin carboxyl-terminal hydrolase 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: ubiquitinyl hydrolase 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 91.785992 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: HHHHHHDYDI PTTENLYFQG AMGNRLSLKF FQKKETKRAL DFTDSQENEE KASEYRASEI DQVVPAAQSS PINCEKRENL LPFVGLNNL GNTCYLNSIL QVLYFCPGFK SGVKHLFNII SRKKEALKDE ANQKDKGNCK EDSLASYELI CSLQSLIISV E QLQASFLL ...String:
HHHHHHDYDI PTTENLYFQG AMGNRLSLKF FQKKETKRAL DFTDSQENEE KASEYRASEI DQVVPAAQSS PINCEKRENL LPFVGLNNL GNTCYLNSIL QVLYFCPGFK SGVKHLFNII SRKKEALKDE ANQKDKGNCK EDSLASYELI CSLQSLIISV E QLQASFLL NPEKYTDELA TQPRRLLNTL RELNPMYEGY LQHDAQEVLQ CILGNIQETC QLLKKEEVKN VAELPTKVEE IP HPKEEMN GINSIEMDSM RHSEDFKEKL PKGNGKRKSD TEFGNMKKKV KLSKEHQSLE ENQRQTRSKR KATSDTLESP PKI IPKYIS ENESPRPSQK KSRVKINWLK SATKQPSILS KFCSLGKITT NQGVKGQSKE NECDPEEDLG KCESDNTTNG CGLE SPGNT VTPVNVNEVK PINKGEEQIG FELVEKLFQG QLVLRTRCLE CESLTERRED FQDISVPVQE DELSKVEESS EISPE PKTE MKTLRWAISQ FASVERIVGE DKYFCENCHH YTEAERSLLF DKMPEVITIH LKCFAASGLE FDCYGGGLSK INTPLL TPL KLSLEEWSTK PTNDSYGLFA VVMHSGITIS SGHYTASVKV TDLNSLELDK GNFVVDQMCE IGKPEPLNEE EARGVVE NY NDEEVSIRVG GNTQPSKVLN KKNVEAIGLL AAQKSKADYE LYNKASNPDK VASTAFAENR NSETSDTTGT HESDRNKE S SDQTGINISG FENKISYVVQ SLKEYEGKWL LFDDSEVKVT EEKDFLNSLS PSTSPTSTPY LLFYKKLERP LSNLEPAVS RHAVPSLSRS TRG

UniProtKB: Ubiquitin carboxyl-terminal hydrolase 1

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Macromolecule #2: WD repeat-containing protein 48

MacromoleculeName: WD repeat-containing protein 48 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 80.130719 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: WSHPQFEKGA LEVLFQGPGM AAHHRQNTAG RRKVQVSYVI RDEVEKYNRN GVNALQLDPA LNRLFTAGRD SIIRIWSVNQ HKQDPYIAS MEHHTDWVND IVLCCNGKTL ISASSDTTVK VWNAHKGFCM STLRTHKDYV KALAYAKDKE LVASAGLDRQ I FLWDVNTL ...String:
WSHPQFEKGA LEVLFQGPGM AAHHRQNTAG RRKVQVSYVI RDEVEKYNRN GVNALQLDPA LNRLFTAGRD SIIRIWSVNQ HKQDPYIAS MEHHTDWVND IVLCCNGKTL ISASSDTTVK VWNAHKGFCM STLRTHKDYV KALAYAKDKE LVASAGLDRQ I FLWDVNTL TALTASNNTV TTSSLSGNKD SIYSLAMNQL GTIIVSGSTE KVLRVWDPRT CAKLMKLKGH TDNVKALLLN RD GTQCLSG SSDGTIRLWS LGQQRCIATY RVHDEGVWAL QVNDAFTHVY SGGRDRKIYC TDLRNPDIRV LICEEKAPVL KME LDRSAD PPPAIWVATT KSTVNKWTLK GIHNFRASGD YDNDCTNPIT PLCTQPDQVI KGGASIIQCH ILNDKRHILT KDTN NNVAY WDVLKACKVE DLGKVDFEDE IKKRFKMVYV PNWFSVDLKT GMLTITLDES DCFAAWVSAK DAGFSSPDGS DPKLN LGGL LLQALLEYWP RTHVNPMDEE ENEVNHVNGE QENRVQKGNG YFQVPPHTPV IFGEAGGRTL FRLLCRDSGG ETESML LNE TVPQWVIDIT VDKNMPKFNK IPFYLQPHAS SGAKTLKKDR LSASDMLQVR KVMEHVYEKI INLDNESQTT SSSNNEK PG EQEKEEDIAV LAEEKIELLC QDQVLDPNMD LRTVKHFIWK SGGDLTLHYL RSPRNSRHAV PSLSRSTRGS

UniProtKB: WD repeat-containing protein 48

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Macromolecule #3: Polyubiquitin-B

MacromoleculeName: Polyubiquitin-B / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.604884 KDa
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRG(ABU)

UniProtKB: Tail fiber

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Macromolecule #4: Polyubiquitin-B

MacromoleculeName: Polyubiquitin-B / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.54777 KDa
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQ(DAB)ESTLHLV LRLRGG

UniProtKB: Tail fiber

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration0.75 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMHepesHepes
150.0 mMNaClsodium chloride
2.0 mMTCEPTris(2-carboxyethyl)phosphine hydrochloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
DetailsCollected on Krios 1 at Netherlands Center for Electron Nanoscopy (NeCEN)
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 5833 / Average exposure time: 2.71 sec. / Average electron dose: 50.0 e/Å2
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: 2.4 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 10500
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: 3110350
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 299304
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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