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- EMDB-25128: Structure of Xenopus laevis CRL2Lrr1 (State 2) -

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Basic information

Entry
Database: EMDB / ID: EMD-25128
TitleStructure of Xenopus laevis CRL2Lrr1 (State 2)
Map data
Sample
  • Complex: CRL2Lrr1
    • Protein or peptide: CULLIN_2 domain-containing protein
    • Protein or peptide: Tceb2-prov protein
    • Protein or peptide: Elongin-C
    • Protein or peptide: Lrr1
    • Protein or peptide: RING-type domain-containing protein
  • Ligand: ZINC ION
KeywordsCullin RING E3 ubiquitin ligase / DNA replication termination / LIGASE
Function / homology
Function and homology information


cullin-RING ubiquitin ligase complex / elongin complex / VCB complex / transcription elongation by RNA polymerase II / protein-macromolecule adaptor activity / ubiquitin-dependent protein catabolic process / protein ubiquitination / ubiquitin protein ligase binding / zinc ion binding
Similarity search - Function
Zinc finger, RING-H2-type / RING-H2 zinc finger domain / Cullin protein neddylation domain / Cullin, conserved site / Cullin family signature. / Elongin B / Cullin / Elongin-C / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain ...Zinc finger, RING-H2-type / RING-H2 zinc finger domain / Cullin protein neddylation domain / Cullin, conserved site / Cullin family signature. / Elongin B / Cullin / Elongin-C / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin protein neddylation domain / Cullin / Leucine-rich repeats, bacterial type / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / SKP1/BTB/POZ domain superfamily / Leucine-rich repeat / Leucine-rich repeat domain superfamily / Zinc finger RING-type profile. / Zinc finger, RING-type / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Zinc finger, RING/FYVE/PHD-type / Ubiquitin-like domain superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
CULLIN_2 domain-containing protein / RING-type domain-containing protein / Leucine rich repeat protein 1 L homeolog isoform X1 / Elongin B L homeolog / Elongin-C
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsZhou H / Brown A
Funding support United States, 3 items
OrganizationGrant numberCountry
Other private United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM141109 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM80676 United States
CitationJournal: Nucleic Acids Res / Year: 2021
Title: Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates.
Authors: Haixia Zhou / Manal S Zaher / Johannes C Walter / Alan Brown /
Abstract: When vertebrate replisomes from neighboring origins converge, the Mcm7 subunit of the replicative helicase, CMG, is ubiquitylated by the E3 ubiquitin ligase, CRL2Lrr1. Polyubiquitylated CMG is then ...When vertebrate replisomes from neighboring origins converge, the Mcm7 subunit of the replicative helicase, CMG, is ubiquitylated by the E3 ubiquitin ligase, CRL2Lrr1. Polyubiquitylated CMG is then disassembled by the p97 ATPase, leading to replication termination. To avoid premature replisome disassembly, CRL2Lrr1 is only recruited to CMGs after they converge, but the underlying mechanism is unclear. Here, we use cryogenic electron microscopy to determine structures of recombinant Xenopus laevis CRL2Lrr1 with and without neddylation. The structures reveal that CRL2Lrr1 adopts an unusually open architecture, in which the putative substrate-recognition subunit, Lrr1, is located far from the catalytic module that catalyzes ubiquitin transfer. We further demonstrate that a predicted, flexible pleckstrin homology domain at the N-terminus of Lrr1 is essential to target CRL2Lrr1 to terminated CMGs. We propose a hypothetical model that explains how CRL2Lrr1's catalytic module is positioned next to the ubiquitylation site on Mcm7, and why CRL2Lrr1 binds CMG only after replisomes converge.
History
DepositionOct 9, 2021-
Header (metadata) releaseDec 8, 2021-
Map releaseDec 8, 2021-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.004
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.004
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7shl
  • Surface level: 0.004
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25128.png

Downloads & links

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Map

FileDownload / File: emd_25128.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.825 Å
Density
Contour LevelBy AUTHOR: 0.004 / Movie #1: 0.004
Minimum - Maximum-0.017027652 - 0.03793097
Average (Standard dev.)0.00005831775 (±0.00078753376)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 264.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8250.8250.825
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z264.000264.000264.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0170.0380.000

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Supplemental data

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Mask #1

Fileemd_25128_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CRL2Lrr1

EntireName: CRL2Lrr1
Components
  • Complex: CRL2Lrr1
    • Protein or peptide: CULLIN_2 domain-containing protein
    • Protein or peptide: Tceb2-prov protein
    • Protein or peptide: Elongin-C
    • Protein or peptide: Lrr1
    • Protein or peptide: RING-type domain-containing protein
  • Ligand: ZINC ION

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Supramolecule #1: CRL2Lrr1

SupramoleculeName: CRL2Lrr1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Xenopus laevis (African clawed frog)

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Macromolecule #1: CULLIN_2 domain-containing protein

MacromoleculeName: CULLIN_2 domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 87.244117 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSLKPRVVDF DETWNKLLTT IKAVVMLDYV ERATWNDRFS DIYALCVAYP EPLGERLYTE TKIFLENHVQ QLHTRVLDSA EQVLVMYFR YWEEYSRGAD YMDCLYRYLN TQYIKKNKLT EADLQYGYGG VDMNEPLMEI GELALDLWRK LMIEPLQDTL L IMLLREIK ...String:
MSLKPRVVDF DETWNKLLTT IKAVVMLDYV ERATWNDRFS DIYALCVAYP EPLGERLYTE TKIFLENHVQ QLHTRVLDSA EQVLVMYFR YWEEYSRGAD YMDCLYRYLN TQYIKKNKLT EADLQYGYGG VDMNEPLMEI GELALDLWRK LMIEPLQDTL L IMLLREIK RDRCGEDPNQ KVIHGVINSF VHVEQYKKKF PLKFYQEIFE SPFLAETGEY YKQEASNLLQ ESNCSQYMEK IL GRLKDEE IRCRKYLHPS SYNKVIHECQ QRMVADHLQF LHAECHNIIR QERRNDMANM YTLLRAVSSG LPHMIQELQN HIH DEGLRA ISNLSQENMP TQFVESVLEV HSKFVQLVNC VLNGDQHFMS ALDKALTCVV NYREPKSVCK APELLAKYCD NMLK KSAKG MTENEVEDKL TSFITVFKYI DDKDVFQKFY ARMLAKRLIH GLSMSMDSEE TMINKLKQAC GYEFTSKLHR MYTDM SVSA DLNNKFNNFI KSQDTVIDLG ISFQIYVLQA GAWPLTQAPS STFAIPQELE KSVQMFELFY NQHFSGRKLT WLHYLC TGE VKMNYLCKPY VAMVTTYQMA VLLAFNNSEI ITYKELQDST QMNEKELTKT IKSLLDVKMI NHDSDKEDIE GESTFSL NM NFSSKRTKFK ITTPMQKDTP QEVEQTRSAV DEDRKMYLQA AIVRIMKARK VLRHNALIQE VISQSRARFN PSISMIKK C IEVLIDKQYI ERSQASADEY SYVA

UniProtKB: CULLIN_2 domain-containing protein

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Macromolecule #2: Tceb2-prov protein

MacromoleculeName: Tceb2-prov protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 13.325927 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MDVFLMIRHH KTTIFTDAKE NTTVYELKRI VEGILKRPPE DQKLYKDDQL LDDNKTLGDC GFTSQTARPQ APATVGLAFR SSGDSFEPL RVEPFSSPPE LPDVMKPQET SGSANEQAVQ

UniProtKB: Elongin B L homeolog

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Macromolecule #3: Elongin-C

MacromoleculeName: Elongin-C / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 12.485135 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MDGEEKTYGG CEGPDAMYVK LISSDGHEFI VKREHALTSG TIKAMLSGPG QFAENETNEV NFREIPSHVL SKVCMYFTYK VRYTNSSTE IPEFPIAPEI ALELLMAANF LDC

UniProtKB: Elongin-C

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Macromolecule #4: Lrr1

MacromoleculeName: Lrr1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 46.882258 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKLQCEVEVI NRMLPTFGLK NRGKGTRAVL SVGRQEGKRG AAYLMICTLK DKSGSRYKLE NNIEQLFTRF VGEGKATLRL KEPALDICL SKAEICGLRN FISTVGLANK GTDIGTVSLP RLTPAKTSEI EKPRSKLFIT TKKDYPITKS FPYSLEHLQV S YCKLARVD ...String:
MKLQCEVEVI NRMLPTFGLK NRGKGTRAVL SVGRQEGKRG AAYLMICTLK DKSGSRYKLE NNIEQLFTRF VGEGKATLRL KEPALDICL SKAEICGLRN FISTVGLANK GTDIGTVSLP RLTPAKTSEI EKPRSKLFIT TKKDYPITKS FPYSLEHLQV S YCKLARVD MRMLCLKKLQ KLDLSNNHIK KLPKTIGDLV CLQELILNHN FLESFEVVLC STTLRDTLKS LDLSANKLKA LP VQICNFK ELVSLKLDEN ELLQLPFPIG QLSKLRFLSA TKNNLQCLPN TFKKLTLENL DLFGNPFMQA TPLVPDIQLK IPL PLLETA ARATLKYRIP YGPHLIPATL CQDLSLAKTC DCGLPCLNSF IQTIVLMNLH QVSQTVVLVD TMGGTDGPIV CYFC SLTCY SQFLDKYLQS TRV

UniProtKB: Leucine rich repeat protein 1 L homeolog isoform X1

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Macromolecule #5: RING-type domain-containing protein

MacromoleculeName: RING-type domain-containing protein / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 12.277985 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MAAAMDVDTP SGANNSAGKK RFEVKKWNAV ALWAWDIVVD NCAICRNHIM DLCIECQANQ ASATSEECTV AWGVCNHAFH FHCISRWLK TRQVCPLDNR EWEFQKYVG

UniProtKB: RING-type domain-containing protein

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / 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

Concentration0.8 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
250.0 mMKClpotassium chloride
50.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
2.0 mMTCEPtris-(2-carboxyethyl)-phosphine

Details: TCEP is freshly added.
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: NITROGEN / Chamber humidity: 100 % / Chamber temperature: 281.2 K / Instrument: FEI VITROBOT MARK IV / Details: Blot 6 seconds with the force 12.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio model
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 406755
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER / Target criteria: Correlation coefficient
Output model

PDB-7shl:
Structure of Xenopus laevis CRL2Lrr1 (State 2)

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