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- EMDB-4410: Yeast RPA bound to ssDNA -

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Basic information

Entry
Database: EMDB / ID: EMD-4410
TitleYeast RPA bound to ssDNA
Map data4.7 Angstrom map (sharpened) corresponding to Yeast RPA Trimerisation Core (Tri-C) bound by ssDNA.
Sample
  • Complex: Replication Protein A heterotrimeric complex with ssDNADNA replication
    • Complex: Replication Protein A heterotrimeric complex with ssDNADNA replication
      • Protein or peptide: Replication factor A protein 3
      • Protein or peptide: Replication factor A protein 2
      • Protein or peptide: Replication factor A protein 1
    • Complex: Replication Protein A heterotrimeric complex with ssDNADNA replication
      • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
Function / homology
Function and homology information


Gap-filling DNA repair synthesis and ligation in GG-NER / Termination of translesion DNA synthesis / heteroduplex formation / Translesion Synthesis by POLH / Translesion synthesis by POLK / Translesion synthesis by POLI / Translesion synthesis by REV1 / sporulation / DNA replication factor A complex / telomere maintenance via telomere lengthening ...Gap-filling DNA repair synthesis and ligation in GG-NER / Termination of translesion DNA synthesis / heteroduplex formation / Translesion Synthesis by POLH / Translesion synthesis by POLK / Translesion synthesis by POLI / Translesion synthesis by REV1 / sporulation / DNA replication factor A complex / telomere maintenance via telomere lengthening / Removal of the Flap Intermediate / mitotic recombination / Activation of the pre-replicative complex / telomere maintenance via recombination / Activation of ATR in response to replication stress / reciprocal meiotic recombination / DNA unwinding involved in DNA replication / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / DNA topological change / telomere maintenance via telomerase / condensed nuclear chromosome / nucleotide-excision repair / double-strand break repair via homologous recombination / establishment of protein localization / single-stranded DNA binding / site of double-strand break / double-stranded DNA binding / sequence-specific DNA binding / DNA replication / chromosome, telomeric region / protein ubiquitination / DNA repair / mRNA binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Replication factor A protein 3, Saccharomycetes / Replication factor A protein 2 / Replication protein A, C-terminal / Replication protein A C terminal / Replication factor A protein 3 / Replication factor A protein 3 / Replication factor A protein-like / Replication factor A protein 1 / Replication factor-A protein 1, N-terminal / Replication protein A, OB domain ...Replication factor A protein 3, Saccharomycetes / Replication factor A protein 2 / Replication protein A, C-terminal / Replication protein A C terminal / Replication factor A protein 3 / Replication factor A protein 3 / Replication factor A protein-like / Replication factor A protein 1 / Replication factor-A protein 1, N-terminal / Replication protein A, OB domain / Replication protein A OB domain / : / Replication factor A, C-terminal / Replication factor-A C terminal domain / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
Replication factor A protein 1 / Replication factor A protein 2 / Replication factor A protein 3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsYates LA / Aramayo RJ / Zhang X
CitationJournal: Nat Commun / Year: 2018
Title: A structural and dynamic model for the assembly of Replication Protein A on single-stranded DNA.
Authors: Luke A Yates / Ricardo J Aramayo / Nilisha Pokhrel / Colleen C Caldwell / Joshua A Kaplan / Rajika L Perera / Maria Spies / Edwin Antony / Xiaodong Zhang /
Abstract: Replication Protein A (RPA), the major eukaryotic single stranded DNA-binding protein, binds to exposed ssDNA to protect it from nucleases, participates in a myriad of nucleic acid transactions and ...Replication Protein A (RPA), the major eukaryotic single stranded DNA-binding protein, binds to exposed ssDNA to protect it from nucleases, participates in a myriad of nucleic acid transactions and coordinates the recruitment of other important players. RPA is a heterotrimer and coats long stretches of single-stranded DNA (ssDNA). The precise molecular architecture of the RPA subunits and its DNA binding domains (DBDs) during assembly is poorly understood. Using cryo electron microscopy we obtained a 3D reconstruction of the RPA trimerisation core bound with ssDNA (∼55 kDa) at ∼4.7 Å resolution and a dimeric RPA assembly on ssDNA. FRET-based solution studies reveal dynamic rearrangements of DBDs during coordinated RPA binding and this activity is regulated by phosphorylation at S178 in RPA70. We present a structural model on how dynamic DBDs promote the cooperative assembly of multiple RPAs on long ssDNA.
History
DepositionNov 12, 2018-
Header (metadata) releaseDec 19, 2018-
Map releaseDec 19, 2018-
UpdateJan 2, 2019-
Current statusJan 2, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6i52
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4410.png

Downloads & links

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Map

FileDownload / File: emd_4410.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation4.7 Angstrom map (sharpened) corresponding to Yeast RPA Trimerisation Core (Tri-C) bound by ssDNA.
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.123228535 - 0.16244632
Average (Standard dev.)0.0017604997 (±0.012339067)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 127.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z127.200127.200127.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-0.1230.1620.002

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Supplemental data

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Additional map: 7.5 Angstrom map (unsharpened) corresponding to yeast RPA dimer

Fileemd_4410_additional_1.map
Annotation7.5 Angstrom map (unsharpened) corresponding to yeast RPA dimer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 7.7 Angstrom map (sharpened) corresponding to Yeast RPA...

Fileemd_4410_additional_2.map
Annotation7.7 Angstrom map (sharpened) corresponding to Yeast RPA Trimerisation Core (Tri-C) bound by ssDNA with additional DNA-binding domain (DBD-B)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 8.5 Angstrom map (sharpened) corresponding to Yeast RPA...

Fileemd_4410_additional_3.map
Annotation8.5 Angstrom map (sharpened) corresponding to Yeast RPA Trimerisation Core (Tri-C) bound by ssDNA with additional DNA-binding domain (DBD-B)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 10 Angstrom map (sharpened) corresponding to Yeast RPA...

Fileemd_4410_additional_4.map
Annotation10 Angstrom map (sharpened) corresponding to Yeast RPA Trimerisation Core (Tri-C) bound by ssDNA with additional DNA-binding domain (DBD-B)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Replication Protein A heterotrimeric complex with ssDNA

EntireName: Replication Protein A heterotrimeric complex with ssDNADNA replication
Components
  • Complex: Replication Protein A heterotrimeric complex with ssDNADNA replication
    • Complex: Replication Protein A heterotrimeric complex with ssDNADNA replication
      • Protein or peptide: Replication factor A protein 3
      • Protein or peptide: Replication factor A protein 2
      • Protein or peptide: Replication factor A protein 1
    • Complex: Replication Protein A heterotrimeric complex with ssDNADNA replication
      • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')

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Supramolecule #1: Replication Protein A heterotrimeric complex with ssDNA

SupramoleculeName: Replication Protein A heterotrimeric complex with ssDNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 120 KDa

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Supramolecule #2: Replication Protein A heterotrimeric complex with ssDNA

SupramoleculeName: Replication Protein A heterotrimeric complex with ssDNA
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pRSF-duet-1

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Supramolecule #3: Replication Protein A heterotrimeric complex with ssDNA

SupramoleculeName: Replication Protein A heterotrimeric complex with ssDNA
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Replication factor A protein 3

MacromoleculeName: Replication factor A protein 3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 13.827723 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MASETPRVDP TEISNVNAPV FRIIAQIKSQ PTESQLILQS PTISSKNGSE VEMITLNNIR VSMNKTFEID SWYEFVCRNN DDGELGFLI LDAVLCKFKE NEDLSLNGVV ALQRLCKKYP EIY

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Macromolecule #2: Replication factor A protein 2

MacromoleculeName: Replication factor A protein 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 14.80987 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
TNTRVNTLTP VTIKQILESK QDIQDGPFVS HNQELHHVCF VGVVRNITDH TANIFLTIED GTGQIEVRKW SEDAAQQFEI GGYVKVFGA LKEFGGKKNI QYAVIKPIDS FNEVLTHHLE VIKCHSIASG MMK

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Macromolecule #3: Replication factor A protein 1

MacromoleculeName: Replication factor A protein 1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 20.643072 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
KFIAQRITIA RAQAENLGRS EKGDFFSVKA AISFLKVDNF AYPACSNENC NKKVLEQPDG TWRCEKCDTN NARPNWRYIL TISIIDETN QLWLTLFDDQ AKQLLGVDAN TLMSLKEEDP NEFTKITQSI QMNEYDFRIR AREDTYNDQS RIRYTVANLH S LNYRAEAD YLADELSKAL

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Macromolecule #4: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP...

MacromoleculeName: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.038899 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)

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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

Concentration0.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC4H12ClNO32-Amino-2-(hydroxymethyl)propane-1,3-diol hydrochloride
150.0 mMNaClSodium chlorideSodium Chloride
C4H10O2S2DTT
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: glow-discharged grid, wait time 30 seconds, blotting time 1 second.

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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 / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.1 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Startup modelType of model: INSILICO MODEL / In silico model: CryoSPARC generated ab initio start model
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 340864
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6i52:
Yeast RPA bound to ssDNA

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