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- PDB-6yuf: Cohesin complex with loader gripping DNA -

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Basic information

Entry
Database: PDB / ID: 6yuf
TitleCohesin complex with loader gripping DNA
Components
  • (DNA (32-MER)) x 2
  • (Structural maintenance of chromosomes protein ...) x 2
  • Cohesin subunit rad21
  • Sister chromatid cohesion protein mis4
KeywordsDNA BINDING PROTEIN / Chromosome segregation Sister chromatid cohesion SMC complexes Cohesin ABC-ATPase Mis4-Scc2-NIPBL
Function / homology
Function and homology information


pericentric heterochromatin => GO:0005721 / meiotic cohesin complex => GO:0030893 / mitotic cohesin dsDNA (leading strand) loading / positive regulation of mitotic cohesin loading / Cohesin Loading onto Chromatin / Resolution of Sister Chromatid Cohesion / mitotic cohesin ssDNA (lagging strand) loading / maintenance of mitotic sister chromatid cohesion, centromeric / mitotic cohesin complex => GO:0030892 / : ...pericentric heterochromatin => GO:0005721 / meiotic cohesin complex => GO:0030893 / mitotic cohesin dsDNA (leading strand) loading / positive regulation of mitotic cohesin loading / Cohesin Loading onto Chromatin / Resolution of Sister Chromatid Cohesion / mitotic cohesin ssDNA (lagging strand) loading / maintenance of mitotic sister chromatid cohesion, centromeric / mitotic cohesin complex => GO:0030892 / : / SUMOylation of DNA damage response and repair proteins / nucleus leading edge / SMC loading complex / mitotic telomere tethering at nuclear periphery / Scc2-Scc4 cohesin loading complex / mitotic cohesin loading / cohesin loader activity / heterochromatin island / maintenance of mitotic sister chromatid cohesion / mitotic cohesin complex / cohesin complex / establishment of protein localization to chromatin / chromosome, subtelomeric region / rDNA heterochromatin / replication-born double-strand break repair via sister chromatid exchange / establishment of mitotic sister chromatid cohesion / condensed chromosome, centromeric region / sister chromatid cohesion / mitotic sister chromatid cohesion / nuclear chromosome / mitotic sister chromatid segregation / enzyme regulator activity / pericentric heterochromatin / double-strand break repair / single-stranded DNA binding / chromosome / double-stranded DNA binding / cell division / DNA damage response / chromatin binding / chromatin / DNA binding / ATP binding / nucleus / cytosol
Similarity search - Function
Sister chromatid cohesion C-terminal domain / HEAT repeat associated with sister chromatid cohesion protein / Scc2/Nipped-B family / Sister chromatid cohesion C-terminus / HEAT repeat associated with sister chromatid cohesion / Smc1, ATP-binding cassette domain / Rad21/Rec8-like protein, C-terminal, eukaryotic / Rad21/Rec8-like protein, N-terminal / Rad21/Rec8-like protein / Structural maintenance of chromosomes 3, ABC domain, eukaryotic ...Sister chromatid cohesion C-terminal domain / HEAT repeat associated with sister chromatid cohesion protein / Scc2/Nipped-B family / Sister chromatid cohesion C-terminus / HEAT repeat associated with sister chromatid cohesion / Smc1, ATP-binding cassette domain / Rad21/Rec8-like protein, C-terminal, eukaryotic / Rad21/Rec8-like protein, N-terminal / Rad21/Rec8-like protein / Structural maintenance of chromosomes 3, ABC domain, eukaryotic / Conserved region of Rad21 / Rec8 like protein / N terminus of Rad21 / Rec8 like protein / ScpA-like, C-terminal / Structural maintenance of chromosomes protein / SMCs flexible hinge / SMCs flexible hinge superfamily / SMC proteins Flexible Hinge Domain / SMC proteins Flexible Hinge Domain / RecF/RecN/SMC, N-terminal / RecF/RecN/SMC N terminal domain / Armadillo-type fold / Winged helix DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / BERYLLIUM TRIFLUORIDE ION / DNA / DNA (> 10) / Structural maintenance of chromosomes protein 3 / Structural maintenance of chromosomes protein 1 / Cohesin subunit rad21 / Sister chromatid cohesion protein mis4
Similarity search - Component
Biological speciesSchizosaccharomyces pombe (fission yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.94 Å
AuthorsHigashi, T.L. / Eickhoff, P. / Sousa, J.S. / Costa, A. / Uhlmann, F.
Funding support2items
OrganizationGrant numberCountry
European Research Council (ERC)
The Francis Crick Institute
CitationJournal: Mol Cell / Year: 2020
Title: A Structure-Based Mechanism for DNA Entry into the Cohesin Ring.
Authors: Torahiko L Higashi / Patrik Eickhoff / Joana S Sousa / Julia Locke / Andrea Nans / Helen R Flynn / Ambrosius P Snijders / George Papageorgiou / Nicola O'Reilly / Zhuo A Chen / Francis J ...Authors: Torahiko L Higashi / Patrik Eickhoff / Joana S Sousa / Julia Locke / Andrea Nans / Helen R Flynn / Ambrosius P Snijders / George Papageorgiou / Nicola O'Reilly / Zhuo A Chen / Francis J O'Reilly / Juri Rappsilber / Alessandro Costa / Frank Uhlmann /
Abstract: Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and ...Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.
History
DepositionApr 27, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 19, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 30, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

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Assembly

Deposited unit
B: Cohesin subunit rad21
D: Sister chromatid cohesion protein mis4
A: Structural maintenance of chromosomes protein 1
C: Structural maintenance of chromosomes protein 3
X: DNA (32-MER)
Y: DNA (32-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)547,28410
Polymers546,2976
Non-polymers9864
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry, prot-prot and DNA-prot XL-MS, cross-linking, fluorescence resonance energy transfer
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area20800 Å2
ΔGint-119 kcal/mol
Surface area122870 Å2
MethodPISA

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Components

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Protein , 2 types, 2 molecules BD

#1: Protein Cohesin subunit rad21 / / Double-strand-break repair protein rad21 / SCC1 homolog


Mass: 67913.711 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Gene: rad21, SPCC338.17c / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P30776
#2: Protein Sister chromatid cohesion protein mis4 / SCC2 homolog


Mass: 180924.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Gene: mis4, SPAC31A2.05c / Production host: Schizosaccharomyces pombe (fission yeast) / References: UniProt: Q09725

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Structural maintenance of chromosomes protein ... , 2 types, 2 molecules AC

#3: Protein Structural maintenance of chromosomes protein 1 / Chromosome segregation protein smc1 / Cohesin complex subunit psm1


Mass: 140728.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Gene: psm1, smc1, SPBC29A10.04 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: O94383
#4: Protein Structural maintenance of chromosomes protein 3 / Cohesin complex Psm3 subunit


Mass: 137048.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Gene: psm3, smc3, SPAC10F6.09c / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: O42649

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DNA chain , 2 types, 2 molecules XY

#5: DNA chain DNA (32-MER)


Mass: 9852.384 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (32-MER)


Mass: 9829.310 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 4 molecules

#7: Chemical ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: BeF3
#8: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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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

Component
IDNameTypeEntity IDParent-IDSource
1Cohesin complex with loader gripping DNACOMPLEX#1-#60MULTIPLE SOURCES
2Cohesin subunit rad21, Structural maintenance of chromosomes protein 1 and 3COMPLEX#1, #3-#41RECOMBINANT
3DNA (32-MER)COMPLEX#5-#61RECOMBINANT
4Sister chromatid cohesion protein mis4COMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)284812
24Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)284812
33Synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Saccharomyces cerevisiae (brewer's yeast)4932
24Schizosaccharomyces pombe (fission yeast)4896
33Synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Image recordingElectron dose: 33.8 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 883184
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 255148 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01819994
ELECTRON MICROSCOPYf_angle_d1.70427260
ELECTRON MICROSCOPYf_dihedral_angle_d32.5893079
ELECTRON MICROSCOPYf_chiral_restr0.0843159
ELECTRON MICROSCOPYf_plane_restr0.0113221

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