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- PDB-7qcd: CryoEM structure of the Smc5/6-holocomplex (composite structure) -

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Basic information

Entry
Database: PDB / ID: 7qcd
TitleCryoEM structure of the Smc5/6-holocomplex (composite structure)
Components
  • (Non-structural maintenance of chromosome element ...) x 2
  • (Structural maintenance of chromosomes protein ...) x 2
  • E3 SUMO-protein ligase MMS21
  • Non-structural maintenance of chromosomes element 1
KeywordsRECOMBINATION / Structural Maintenance of Chromosomes / SMC / holo-complex / stalled replication fork
Function / homology
Function and homology information


Smc5-Smc6 complex / resolution of DNA recombination intermediates / DNA double-strand break attachment to nuclear envelope / SUMO ligase activity / chromosome separation / Platelet degranulation / SUMOylation of DNA damage response and repair proteins / chromatin looping / Transferases; Acyltransferases; Aminoacyltransferases / SUMO transferase activity ...Smc5-Smc6 complex / resolution of DNA recombination intermediates / DNA double-strand break attachment to nuclear envelope / SUMO ligase activity / chromosome separation / Platelet degranulation / SUMOylation of DNA damage response and repair proteins / chromatin looping / Transferases; Acyltransferases; Aminoacyltransferases / SUMO transferase activity / DNA damage tolerance / recombinational repair / regulation of telomere maintenance / protein sumoylation / protein serine/threonine kinase inhibitor activity / double-strand break repair via homologous recombination / RING-type E3 ubiquitin transferase / ubiquitin-protein transferase activity / nuclear envelope / single-stranded DNA binding / site of double-strand break / damaged DNA binding / chromosome, telomeric region / DNA repair / ATP hydrolysis activity / mitochondrion / zinc ion binding / ATP binding / nucleus / cytoplasm
Similarity search - Function
Non-structural maintenance of chromosomes element 1 / Zinc finger, RING-like / Nse1 non-SMC component of SMC5-6 complex / RING-like domain / Non-structural maintenance of chromosome element 4, C-terminal / Nse4/EID family / Nse4/EID protein, Nse3/MAGE-binding domain / Nse4 C-terminal / Binding domain of Nse4/EID3 to Nse3-MAGE / : ...Non-structural maintenance of chromosomes element 1 / Zinc finger, RING-like / Nse1 non-SMC component of SMC5-6 complex / RING-like domain / Non-structural maintenance of chromosome element 4, C-terminal / Nse4/EID family / Nse4/EID protein, Nse3/MAGE-binding domain / Nse4 C-terminal / Binding domain of Nse4/EID3 to Nse3-MAGE / : / E3 SUMO-protein ligase MMS21, N-terminal domain / E3 SUMO-protein ligase Nse2 (Mms21) / Zinc-finger of the MIZ type in Nse subunit / Zinc finger, MIZ-type / Zinc finger SP-RING-type profile. / MAGE homology domain / Melanoma-associated antigen / MAGE homology domain, winged helix WH1 motif / MAGE homology domain, winged helix WH2 motif / MAGE homology domain / Melanoma-associated antigen / Rad50/SbcC-type AAA domain / AAA domain / RecF/RecN/SMC, N-terminal / RecF/RecN/SMC N terminal domain / Zinc finger, RING/FYVE/PHD-type / Winged helix-like DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
E3 SUMO-protein ligase MMS21 / Non-structural maintenance of chromosome element 4 / Non-structural maintenance of chromosome element 3 / Non-structural maintenance of chromosomes element 1 / Structural maintenance of chromosomes protein 5 / Structural maintenance of chromosomes protein 6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8 Å
AuthorsHallett, S.T. / Oliver, A.W.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/PO18955/1 United Kingdom
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Cryo-EM structure of the Smc5/6 holo-complex.
Authors: Stephen T Hallett / Isabella Campbell Harry / Pascale Schellenberger / Lihong Zhou / Nora B Cronin / Jonathan Baxter / Thomas J Etheridge / Johanne M Murray / Antony W Oliver /
Abstract: The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also ...The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also functions as a restriction factor preventing viral replication. Here, we report the cryogenic EM (cryo-EM) structure of the six-subunit budding yeast Smc5/6 holo-complex, reconstituted from recombinant proteins expressed in insect cells - providing both an architectural overview of the entire complex and an understanding of how the Nse1/3/4 subcomplex binds to the hetero-dimeric SMC protein core. In addition, we demonstrate that a region within the head domain of Smc5, equivalent to the 'W-loop' of Smc4 or 'F-loop' of Smc1, mediates an important interaction with Nse1. Notably, mutations that alter the surface-charge profile of the region of Nse1 which accepts the Smc5-loop, lead to a slow-growth phenotype and a global reduction in the chromatin-associated fraction of the Smc5/6 complex, as judged by single molecule localisation microscopy experiments in live yeast. Moreover, when taken together, our data indicates functional equivalence between the structurally unrelated KITE and HAWK accessory subunits associated with SMC complexes.
History
DepositionNov 23, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 23, 2022Group: 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 1.2Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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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 5
B: Structural maintenance of chromosomes protein 6
C: E3 SUMO-protein ligase MMS21
D: Non-structural maintenance of chromosomes element 1
E: Non-structural maintenance of chromosome element 3
F: Non-structural maintenance of chromosome element 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)442,8399
Polymers442,6426
Non-polymers1963
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area27420 Å2
ΔGint-131 kcal/mol
Surface area155200 Å2

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Components

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

#1: Protein Structural maintenance of chromosomes protein 5


Mass: 126236.180 Da / Num. of mol.: 1 / Mutation: E1015Q
Source method: isolated from a genetically manipulated source
Details: Saccharomyces cerevisiase Smc5 fused to C-terminal FLAG-tag.
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: SMC5, YOL034W / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q08204
#2: Protein Structural maintenance of chromosomes protein 6 / DNA repair protein RHC18 / Rad18 homolog


Mass: 128198.742 Da / Num. of mol.: 1 / Mutation: E1048Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: SMC6, RHC18, YLR383W, L3502.2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q12749

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Protein , 2 types, 2 molecules CD

#3: Protein E3 SUMO-protein ligase MMS21 / E3 SUMO-protein transferase MMS21 / Methyl methanesulfonate-sensitivity protein 21 / Non-structural ...E3 SUMO-protein transferase MMS21 / Methyl methanesulfonate-sensitivity protein 21 / Non-structural maintenance of chromosome element 2 / Non-SMC element 2


Mass: 31817.775 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Saccharomyces cerevisiae Nse2 fused to N-terminal HA-tag.
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MMS21, NSE2, YEL019C / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P38632, Transferases; Acyltransferases; Aminoacyltransferases
#4: Protein Non-structural maintenance of chromosomes element 1 / Non-SMC element 1


Mass: 40943.113 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Saccharomyces cerevisiae Nse1 fused to N-terminal TEV-cleavable His6-tag.
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: NSE1, YLR007W / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q07913, RING-type E3 ubiquitin transferase

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Non-structural maintenance of chromosome element ... , 2 types, 2 molecules EF

#5: Protein Non-structural maintenance of chromosome element 3 / Non-SMC element 3


Mass: 34005.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Saccharomyces cerevisiae Nse3, untagged.
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: NSE3, YDR288W / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q05541
#6: Protein Non-structural maintenance of chromosome element 4 / Non-SMC element 4 / Protein QRI2


Mass: 81441.133 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Saccharomyces cerevisiae Nse4 fused to a C-terminal HALO-myc tag.
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: NSE4, QRI2, YDL105W, D2354 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P43124

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Non-polymers , 1 types, 3 molecules

#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / 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: Smc5/6 holo-complex / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
250 mMsodium chlorideNaCl1
30.5 mMTCEPC9H16ClO6P1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Glow-discharged for a period of 60 seconds at 15mA, PELCO easiGlow, Leica Microsystems, Germany.
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 293 K
Details: Held in chamber for a period of 10 seconds, before blotting for 2.5 to 4.5 seconds (auto-sensor) then plunged into liquid ethane before being stored under liquid nitrogen.

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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 holderCryogen: NITROGEN
Image recordingElectron dose: 1.3 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 4

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2Topaz0.2.4particle selectionTopaz, integrated into CryoSPARC was used to select particles after training
3EPUimage acquisition
5CTFFIND4.1.4CTF correction
8PyMOLmodel fitting
9UCSF ChimeraXmodel fitting
10Cootmodel fitting
12PHENIXmodel refinement
13cryoSPARC3.1.0initial Euler assignment
14cryoSPARC3.1.0final Euler assignment
15cryoSPARC3.1.0classification
16cryoSPARC3.1.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 8 Å / Resolution method: OTHER / Num. of particles: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00425515
ELECTRON MICROSCOPYf_angle_d0.69534289
ELECTRON MICROSCOPYf_dihedral_angle_d14.0129892
ELECTRON MICROSCOPYf_chiral_restr0.0423897
ELECTRON MICROSCOPYf_plane_restr0.0044434

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