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- PDB-8t8f: Smc5/6 8mer -

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Basic information

Entry
Database: PDB / ID: 8t8f
TitleSmc5/6 8mer
Components
  • DNA repair protein KRE29
  • Non-structural maintenance of chromosome element 4
  • Non-structural maintenance of chromosome element 5
  • Structural maintenance of chromosomes protein 5
  • Structural maintenance of chromosomes protein 6
KeywordsDNA BINDING PROTEIN / Smc / Nse / Condensin / Cohesin / DNA binding / DNA damage / DNA repair
Function / homology
Function and homology information


Smc5-Smc6 complex / resolution of DNA recombination intermediates / DNA double-strand break attachment to nuclear envelope / chromosome separation / SUMOylation of DNA damage response and repair proteins / chromatin looping / recombinational repair / regulation of telomere maintenance / protein sumoylation / double-strand break repair via homologous recombination ...Smc5-Smc6 complex / resolution of DNA recombination intermediates / DNA double-strand break attachment to nuclear envelope / chromosome separation / SUMOylation of DNA damage response and repair proteins / chromatin looping / recombinational repair / regulation of telomere maintenance / protein sumoylation / double-strand break repair via homologous recombination / single-stranded DNA binding / site of double-strand break / damaged DNA binding / chromosome, telomeric region / DNA repair / ATP hydrolysis activity / mitochondrion / ATP binding / nucleus / cytoplasm
Similarity search - Function
DNA repair protein Nse5/Nse6 / Non-structural maintenance of chromosome element 4, C-terminal / Nse4/EID family / Nse4/EID protein, Nse3/MAGE-binding domain / DNA repair proteins Nse5 and Nse6 / Nse4 C-terminal / Binding domain of Nse4/EID3 to Nse3-MAGE / Structural maintenance of chromosomes protein 5 / Rad50/SbcC-type AAA domain / AAA domain ...DNA repair protein Nse5/Nse6 / Non-structural maintenance of chromosome element 4, C-terminal / Nse4/EID family / Nse4/EID protein, Nse3/MAGE-binding domain / DNA repair proteins Nse5 and Nse6 / Nse4 C-terminal / Binding domain of Nse4/EID3 to Nse3-MAGE / Structural maintenance of chromosomes protein 5 / Rad50/SbcC-type AAA domain / AAA domain / RecF/RecN/SMC, N-terminal / RecF/RecN/SMC N terminal domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA repair protein KRE29 / Non-structural maintenance of chromosome element 4 / Non-structural maintenance of chromosome element 5 / Structural maintenance of chromosomes protein 5 / Structural maintenance of chromosomes protein 6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae W303 (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsYu, Y. / Patel, D.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)GM145260 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Molecular basis for Nse5-6 mediated regulation of Smc5/6 functions.
Authors: Shibai Li / You Yu / Jian Zheng / Victoria Miller-Browne / Zheng Ser / Huihui Kuang / Dinshaw J Patel / Xiaolan Zhao /
Abstract: The Smc5/6 complex (Smc5/6) is important for genome replication and repair in eukaryotes. Its cellular functions are closely linked to the ATPase activity of the Smc5 and Smc6 subunits. This activity ...The Smc5/6 complex (Smc5/6) is important for genome replication and repair in eukaryotes. Its cellular functions are closely linked to the ATPase activity of the Smc5 and Smc6 subunits. This activity requires the dimerization of the motor domains of the two SMC subunits and is regulated by the six non-SMC subunits (Nse1 to Nse6). Among the NSEs, Nse5 and Nse6 form a stable subcomplex (Nse5-6) that dampens the ATPase activity of the complex. However, the underlying mechanisms and biological significance of this regulation remain unclear. Here, we address these issues using structural and functional studies. We determined cryo-EM structures of the yeast Smc5/6 derived from complexes consisting of either all eight subunits or a subset of five subunits. Both structures reveal that Nse5-6 associates with Smc6's motor domain and the adjacent coiled-coil segment, termed the neck region. Our structural analyses reveal that this binding is compatible with motor domain dimerization but results in dislodging the Nse4 subunit from the Smc6 neck. As the Nse4-Smc6 neck interaction favors motor domain engagement and thus ATPase activity, Nse6's competition with Nse4 can explain how Nse5-6 disfavors ATPase activity. Such regulation could in principle differentially affect Smc5/6-mediated processes depending on their needs of the complex's ATPase activity. Indeed, mutagenesis data in cells provide evidence that the Nse6-Smc6 neck interaction is important for the resolution of DNA repair intermediates but not for replication termination. Our results thus provide a molecular basis for how Nse5-6 modulates the ATPase activity and cellular functions of Smc5/6.
History
DepositionJun 22, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 15, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: DNA repair protein KRE29
C: Non-structural maintenance of chromosome element 5
D: Structural maintenance of chromosomes protein 5
E: Structural maintenance of chromosomes protein 6
G: Non-structural maintenance of chromosome element 4


Theoretical massNumber of molelcules
Total (without water)418,5475
Polymers418,5475
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein DNA repair protein KRE29 / / Killer toxin-resistance protein 29


Mass: 53836.758 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae W303 (yeast) / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: P40026
#2: Protein Non-structural maintenance of chromosome element 5 / Non-SMC element 5


Mass: 64079.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae W303 (yeast) / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: Q03718
#3: Protein Structural maintenance of chromosomes protein 5


Mass: 126236.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Smc5 E1015Q mutation, visible part is from 32-246aa and 883-1093aa.
Source: (gene. exp.) Saccharomyces cerevisiae W303 (yeast) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q08204
#4: Protein Structural maintenance of chromosomes protein 6 / DNA repair protein RHC18 / Rad18 homolog


Mass: 128198.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Smc6 E1048Q mutation with C terminal 2X strep tag, visible part is from 79-286aa and 923-1084aa
Source: (gene. exp.) Saccharomyces cerevisiae W303 (yeast) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q12749
#5: Protein Non-structural maintenance of chromosome element 4 / Non-SMC element 4 / Protein QRI2


Mass: 46195.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae W303 (yeast) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P43124

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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: Smc5/6 8mer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 400 kDa/nm / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: 25 mM Hepes, pH 7.5, 250 mM NaCl, 1 mM DTT
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
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 / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 53.55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 145290 / Symmetry type: POINT

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