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- PDB-8t8e: cryoEM structure of Smc5/6 5mer -

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Basic information

Entry
Database: PDB / ID: 8t8e
TitlecryoEM structure of Smc5/6 5mer
Components
  • DNA repair protein KRE29
  • Non-structural maintenance of chromosome element 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 / regulation of telomere maintenance / protein sumoylation / double-strand break repair via homologous recombination / single-stranded DNA binding ...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 / 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 / DNA repair proteins Nse5 and Nse6 / Rad50/SbcC-type AAA domain / AAA domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA repair protein KRE29 / Non-structural maintenance of chromosome element 5 / Structural maintenance of chromosomes protein 6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae W303 (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 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
A: Structural maintenance of chromosomes protein 6
C: Non-structural maintenance of chromosome element 5
B: DNA repair protein KRE29


Theoretical massNumber of molelcules
Total (without water)246,1153
Polymers246,1153
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: 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: the visible residues from Smc6 (with E1048Q mutation)in the structure are the N terminal part of 76-271aa and C terminal part of 949-1100aa.
Source: (gene. exp.) Saccharomyces cerevisiae W303 (yeast) / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: Q12749
#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 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

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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 5mer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 234 kDa/nm / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae W303 (yeast)
Buffer solutionpH: 7.5 / Details: 25 mM Hepes, pH 7.5, 250 mM NaCl, 1 mM DTT
SpecimenConc.: 0.31 mg/ml / Embedding 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
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 53 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: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 219202 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058953
ELECTRON MICROSCOPYf_angle_d0.69812064
ELECTRON MICROSCOPYf_dihedral_angle_d5.2221164
ELECTRON MICROSCOPYf_chiral_restr0.0431361
ELECTRON MICROSCOPYf_plane_restr0.0051529

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