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

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Basic information

Entry
Database: PDB / ID: 7shl
TitleStructure of Xenopus laevis CRL2Lrr1 (State 2)
Components
  • CULLIN_2 domain-containing protein
  • Elongin-C
  • Lrr1
  • RING-type domain-containing protein
  • Tceb2-prov protein
KeywordsLIGASE / Cullin RING E3 ubiquitin ligase / DNA replication termination
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 / Elongin B / Elongin-C / Cullin, conserved site / Cullin family signature. / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain ...: / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / Cullin protein neddylation domain / Elongin B / Elongin-C / Cullin, conserved site / Cullin family signature. / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. / Leucine-rich repeats, bacterial type / 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 / SKP1/BTB/POZ domain superfamily / Leucine-rich repeat profile. / Leucine-rich repeat / Zinc finger RING-type profile. / Leucine-rich repeat domain superfamily / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsZhou, H. / Brown, A.
Funding support United States, 3items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 8, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 29, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
A: CULLIN_2 domain-containing protein
B: Tceb2-prov protein
C: Elongin-C
L: Lrr1
R: RING-type domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)172,2816
Polymers172,2155
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10580 Å2
ΔGint-73 kcal/mol
Surface area54370 Å2

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Components

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Protein , 5 types, 5 molecules ABCLR

#1: Protein CULLIN_2 domain-containing protein


Mass: 87244.117 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: cul2.L, cul2, XELAEV_18030721mg / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A1L8FVB6
#2: Protein Tceb2-prov protein


Mass: 13325.927 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: elob.L, elob, tceb2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6PHL7
#3: Protein Elongin-C


Mass: 12485.135 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog)
Gene: eloc.L, eloc, tceb1, XELAEV_18031836mg, XELAEV_18033803mg
Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6PHW7
#4: Protein Lrr1


Mass: 46882.258 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: lrr1.L, lrr1, MGC82386, XELAEV_18039633mg / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A4QNS4
#5: Protein RING-type domain-containing protein


Mass: 12277.985 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: XELAEV_18023559mg / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A1L8GNG7

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Non-polymers , 1 types, 1 molecules

#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: CRL2Lrr1 / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.6 / Details: TCEP is freshly added.
Buffer component
IDConc.NameFormulaBuffer-ID
1250 mMpotassium chlorideKCl1
250 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
32 mMtris-(2-carboxyethyl)-phosphineTCEP1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: NITROGEN / Humidity: 100 % / Chamber temperature: 281.2 K / Details: Blot 6 seconds with the force 12

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 54.5 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
7Coot0.95model fitting
9PHENIX1.19.2model refinement
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 406755 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: Correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039397
ELECTRON MICROSCOPYf_angle_d0.61812704
ELECTRON MICROSCOPYf_dihedral_angle_d4.4971224
ELECTRON MICROSCOPYf_chiral_restr0.0411435
ELECTRON MICROSCOPYf_plane_restr0.0041615

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