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- EMDB-25424: Mec3 focus refinement of Rad24-RFC:9-1-1:DNA in the open state -

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Open data


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Basic information

Entry
Database: EMDB / ID: EMD-25424
TitleMec3 focus refinement of Rad24-RFC:9-1-1:DNA in the open state
Map dataMec3 Focus Refinement
Sample
  • Complex: Rad24-RFC:9-1-1:DNA
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.43 Å
AuthorsCastaneda JC / Schrecker M / Hite RK / Remus D
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM107239 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM127428 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA008748 United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Mechanisms of loading and release of the 9-1-1 checkpoint clamp.
Authors: Juan C Castaneda / Marina Schrecker / Dirk Remus / Richard K Hite /
Abstract: Single-stranded or double-stranded DNA junctions with recessed 5' ends serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATR. However, ...Single-stranded or double-stranded DNA junctions with recessed 5' ends serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATR. However, the basis for 9-1-1's recruitment to 5' junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-replication factor C (RFC), in complex with 9-1-1 and a 5' junction and in a post-ATP-hydrolysis state. Unexpectedly, 9-1-1 adopts both closed and planar open states in the presence of Rad24-RFC and DNA. Moreover, Rad24-RFC associates with the DNA junction in the opposite orientation of processivity clamp loaders with Rad24 exclusively coordinating the double-stranded region. ATP hydrolysis stimulates conformational changes in Rad24-RFC, leading to disengagement of DNA-loaded 9-1-1. Together, these structures explain 9-1-1's recruitment to 5' junctions and reveal new principles of sliding clamp loading.
History
DepositionNov 12, 2021-
Header (metadata) releaseMar 23, 2022-
Map releaseMar 23, 2022-
UpdateApr 27, 2022-
Current statusApr 27, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25424.png

Downloads & links

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Map

FileDownload / File: emd_25424.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMec3 Focus Refinement
Voxel sizeX=Y=Z: 0.826 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-2.5344465 - 3.514879
Average (Standard dev.)-0.0014067745 (±0.035685044)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 317.184 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Rad24-RFC:9-1-1:DNA

EntireName: Rad24-RFC:9-1-1:DNA
Components
  • Complex: Rad24-RFC:9-1-1:DNA

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Supramolecule #1: Rad24-RFC:9-1-1:DNA

SupramoleculeName: Rad24-RFC:9-1-1:DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: 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

Concentration1.0 mg/mL
BufferpH: 7.6
Component:
ConcentrationName
25.0 mMHepes-KOH
300.0 mMKOAc
7.0 mMMg(OAc)2
GridModel: Quantifoil R1.2/1.3 / Material: GRAPHENE OXIDE / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 297 K / Instrument: FEI VITROBOT MARK IV

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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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4117022
CTF correctionSoftware - Name: CTFFIND
Initial angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.43 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 938420

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Atomic model buiding 1

Initial model(PDB ID:

1sxj

,

3a1j

)
RefinementSpace: REAL / Protocol: AB INITIO MODEL

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