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- EMDB-26280: RFC bound to PCNA and two primer/template DNA molecules -

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Basic information

Entry
Database: EMDB / ID: EMD-26280
TitleRFC bound to PCNA and two primer/template DNA molecules
Map data
Sample
  • Complex: RFC bound to PCNA and two primer/template DNA molecules
Keywordssliding clamp / DNA replication&repair / AAA+ / clamp loader / BRCT domain / REPLICATION
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsGaubitz C / Liu X / Pajak J / Kelch B
Funding support United States, Switzerland, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM127776 United States
Swiss National Science Foundation168972 Switzerland
Swiss National Science Foundation177859 Switzerland
CitationJournal: Elife / Year: 2022
Title: A second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNA.
Authors: Xingchen Liu / Christl Gaubitz / Joshua Pajak / Brian A Kelch /
Abstract: Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are ...Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are common clamp-loading targets in DNA repair, yet these substrates would be sterically blocked given the known mechanism for binding of primer-template DNA. Here, we report the discovery of a second DNA binding site in the yeast clamp loader replication factor C (RFC) that aids in binding to nicked or gapped DNA. This DNA binding site is on the external surface and is only accessible in the open conformation of RFC. Initial DNA binding at this site thus provides access to the primary DNA binding site in the central chamber. Furthermore, we identify that this site can partially unwind DNA to create an extended single-stranded gap for DNA binding in RFC's central chamber and subsequent ATPase activation. Finally, we show that deletion of the BRCT domain, a major component of the external DNA binding site, results in defective yeast growth in the presence of DNA damage where nicked or gapped DNA intermediates occur. We propose that RFC's external DNA binding site acts to enhance DNA binding and clamp loading, particularly at DNA architectures typically found in DNA repair.
History
DepositionFeb 20, 2022-
Header (metadata) releaseJul 6, 2022-
Map releaseJul 6, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26280.png

Downloads & links

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Map

FileDownload / File: emd_26280.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.009
Minimum - Maximum-0.01590293 - 0.051565606
Average (Standard dev.)0.00028060557 (±0.0025586823)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 254.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_26280_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_26280_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_26280_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RFC bound to PCNA and two primer/template DNA molecules

EntireName: RFC bound to PCNA and two primer/template DNA molecules
Components
  • Complex: RFC bound to PCNA and two primer/template DNA molecules

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Supramolecule #1: RFC bound to PCNA and two primer/template DNA molecules

SupramoleculeName: RFC bound to PCNA and two primer/template DNA molecules
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 367 KDa

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 10331440
Startup modelType of model: EMDB MAP
EMDB ID:

25615

Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 43129
FSC plot (resolution estimation)

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