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- EMDB-13894: CryoEM structure of the Smc5/6 holocomplex; map for hinge and arm... -

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Basic information

Entry
Database: EMDB / ID: EMD-13894
TitleCryoEM structure of the Smc5/6 holocomplex; map for hinge and arm region.
Map dataHinge and arm region of Smc5/6 holo-complex
Sample
  • Complex: Saccharomyces cerevisiae Smc5/6 holo-complex
KeywordsStructural Maintenance of Chromosomes / SMC / holo-complex / recombination / stalled replication fork
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.53 Å
AuthorsOLIVER AW / Hallett ST
Funding support United Kingdom, 1 items
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, 2021-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13894.png

Downloads & links

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Map

FileDownload / File: emd_13894.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHinge and arm region of Smc5/6 holo-complex
Voxel sizeX=Y=Z: 2.2 Å
Density
Contour LevelBy AUTHOR: 1.48
Minimum - Maximum-2.9363909 - 6.4493685
Average (Standard dev.)-0.0013666867 (±0.13732079)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 704.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13894_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A for hinge and arm region of Smc5/6 holo-complex

Fileemd_13894_half_map_1.map
AnnotationHalf-map A for hinge and arm region of Smc5/6 holo-complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B for hinge and arm region of Smc5/6 holo-complex

Fileemd_13894_half_map_2.map
AnnotationHalf-map B for hinge and arm region of Smc5/6 holo-complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Saccharomyces cerevisiae Smc5/6 holo-complex

EntireName: Saccharomyces cerevisiae Smc5/6 holo-complex
Components
  • Complex: Saccharomyces cerevisiae Smc5/6 holo-complex

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Supramolecule #1: Saccharomyces cerevisiae Smc5/6 holo-complex

SupramoleculeName: Saccharomyces cerevisiae Smc5/6 holo-complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Map focussed at the hinge/arm region of the holocomplex
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
50.0 mMNaClSodium chloridesodium chloride
0.5 mMC9H16ClO6PTCEP
GridModel: Quantifoil R0.6/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
Details: Grids were glow-discharged for 60 seconds at 15 mA using a PELCO easiGlow (Agar Scientific)
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 283.15 K / Instrument: LEICA EM GP
Details: The grid was held in the chamber for a period of 10 seconds, before blotting for 2.5 to 4.5 seconds using the auto-sensor. Grids were then plunged into liquid ethane before being stored ...Details: The grid was held in the chamber for a period of 10 seconds, before blotting for 2.5 to 4.5 seconds using the auto-sensor. Grids were then plunged into liquid ethane before being stored under liquid nitrogen until data collection..
DetailsFraction(s) eluting from a Superose 6 size exclusion chromatography column (Cytiva Life Sciences). Cross-linked with BS3 before grid preparation.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 4 / Average exposure time: 4.0 sec. / Average electron dose: 1.3 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 380714
Startup modelType of model: INSILICO MODEL / In silico model: ab initio using cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 3.1.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0)
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 8.53 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1.0) / Number images used: 106660
FSC plot (resolution estimation)

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