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- EMDB-2789: Cryo-EM map of the Timeless-Tipin-RPA Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-2789
TitleCryo-EM map of the Timeless-Tipin-RPA Complex
Map dataReconstruction of the Timless-Tipin-RPA complex
Sample
  • Sample: Timeless-Tipin-RPA complex
  • Protein or peptide: Timeless
  • Protein or peptide: Timeless-interacting protein
  • Protein or peptide: Replication protein A 70
  • Protein or peptide: Replication protein A 32
  • Protein or peptide: Replication protein A 14
KeywordsTimeless / Tipin / Replication protein A / RPA / DNA replication
Function / homology
Function and homology information


Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Activation of ATR in response to replication stress / Removal of the Flap Intermediate / PCNA-Dependent Long Patch Base Excision Repair / Removal of the Flap Intermediate from the C-strand / Activation of the pre-replicative complex / Regulation of HSF1-mediated heat shock response / SUMOylation of DNA damage response and repair proteins / Gap-filling DNA repair synthesis and ligation in GG-NER ...Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Activation of ATR in response to replication stress / Removal of the Flap Intermediate / PCNA-Dependent Long Patch Base Excision Repair / Removal of the Flap Intermediate from the C-strand / Activation of the pre-replicative complex / Regulation of HSF1-mediated heat shock response / SUMOylation of DNA damage response and repair proteins / Gap-filling DNA repair synthesis and ligation in GG-NER / Fanconi Anemia Pathway / HDR through Single Strand Annealing (SSA) / Translesion synthesis by REV1 / Translesion synthesis by POLK / cellular response to bleomycin / Translesion synthesis by POLI / Recognition of DNA damage by PCNA-containing replication complex / HDR through Homologous Recombination (HRR) / G2/M DNA damage checkpoint / Translesion Synthesis by POLH / Termination of translesion DNA synthesis / Dual Incision in GG-NER / Dual incision in TC-NER / Formation of Incision Complex in GG-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / protein localization to chromosome / Processing of DNA double-strand break ends / DNA replication factor A complex / replication fork arrest / cell cycle phase transition / cellular response to cisplatin / cellular response to hydroxyurea / Regulation of TP53 Activity through Phosphorylation / lateral element / DNA replication checkpoint signaling / single-stranded telomeric DNA binding / positive regulation of circadian rhythm / regulation of DNA damage checkpoint / replication fork protection complex / mitotic intra-S DNA damage checkpoint signaling / branching involved in ureteric bud morphogenesis / regulation of double-strand break repair via homologous recombination / positive regulation of double-strand break repair / branching morphogenesis of an epithelial tube / telomeric DNA binding / replication fork processing / hemopoiesis / site of DNA damage / chromosome organization / telomere maintenance via telomerase / mismatch repair / positive regulation of double-strand break repair via homologous recombination / response to UV / condensed chromosome / homeostasis of number of cells within a tissue / telomere maintenance / regulation of mitotic cell cycle / male germ cell nucleus / DNA damage checkpoint signaling / condensed nuclear chromosome / morphogenesis of an epithelium / kidney development / meiotic cell cycle / nucleotide-excision repair / lung development / double-strand break repair via homologous recombination / regulation of circadian rhythm / base-excision repair / PML body / circadian rhythm / single-stranded DNA binding / regulation of cell population proliferation / site of double-strand break / in utero embryonic development / damaged DNA binding / chromosome, telomeric region / DNA replication / cell division / DNA repair / negative regulation of DNA-templated transcription / positive regulation of cell population proliferation / DNA damage response / chromatin binding / chromatin / negative regulation of transcription by RNA polymerase II / DNA binding / nucleoplasm / metal ion binding / identical protein binding / nucleus / cytoplasm
Similarity search - Function
Replication factor A protein 2 / 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 3 / Timeless, C-terminal / Timeless PAB domain / Replication factor-A protein 1, N-terminal domain ...Replication factor A protein 2 / 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 3 / Timeless, C-terminal / Timeless PAB domain / Replication factor-A protein 1, N-terminal domain / Replication factor A protein-like / Replication factor A protein 1 / Chromosome segregation in meiosis protein 3 / Replication factor A protein 1 / Replication factor-A protein 1, N-terminal / Chromosome segregation in meiosis protein 3 / Replication protein A, OB domain / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / Replication protein A OB domain / Timeless, N-terminal / Timeless, N-terminal / Timeless / Timeless protein / : / 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 protein A 32 kDa subunit / Replication protein A 70 kDa DNA-binding subunit / TIMELESS-interacting protein / Replication protein A 14 kDa subunit / Protein timeless homolog
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 17.0 Å
AuthorsWitosch J / Wolf E / Mizuno N
CitationJournal: Nucleic Acids Res / Year: 2014
Title: Architecture and ssDNA interaction of the Timeless-Tipin-RPA complex.
Authors: Justine Witosch / Eva Wolf / Naoko Mizuno /
Abstract: The Timeless-Tipin (Tim-Tipin) complex, also referred to as the fork protection complex, is involved in coordination of DNA replication. Tim-Tipin is suggested to be recruited to replication forks ...The Timeless-Tipin (Tim-Tipin) complex, also referred to as the fork protection complex, is involved in coordination of DNA replication. Tim-Tipin is suggested to be recruited to replication forks via Replication Protein A (RPA) but details of the interaction are unknown. Here, using cryo-EM and biochemical methods, we characterized complex formation of Tim-Tipin, RPA and single-stranded DNA (ssDNA). Tim-Tipin and RPA form a 258 kDa complex with a 1:1:1 stoichiometry. The cryo-EM 3D reconstruction revealed a globular architecture of the Tim-Tipin-RPA complex with a ring-like and a U-shaped domain covered by a RPA lid. Interestingly, RPA in the complex adopts a horse shoe-like shape resembling its conformation in the presence of long ssDNA (>30 nucleotides). Furthermore, the recruitment of the Tim-Tipin-RPA complex to ssDNA is modulated by the RPA conformation and requires RPA to be in the more compact 30 nt ssDNA binding mode. The dynamic formation and disruption of the Tim-Tipin-RPA-ssDNA complex implicates the RPA-based recruitment of Tim-Tipin to the replication fork.
History
DepositionOct 2, 2014-
Header (metadata) releaseNov 12, 2014-
Map releaseNov 12, 2014-
UpdateNov 19, 2014-
Current statusNov 19, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0733
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0733
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-12629_50...

emd_2789.tif

Downloads & links

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Map

FileDownload / File: emd_2789.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the Timless-Tipin-RPA complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.21 Å/pix.
x 128 pix.
= 282.88 Å		2.21 Å/pix.
x 128 pix.
= 282.88 Å		2.21 Å/pix.
x 128 pix.
= 282.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.21 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0733 / Movie #1: 0.0733
Minimum - Maximum-0.09360165 - 0.45139375
Average (Standard dev.)0.00368675 (±0.02693075)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 282.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.212.212.21
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z282.880282.880282.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.0940.4510.004

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Supplemental data

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Sample components

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Entire : Timeless-Tipin-RPA complex

EntireName: Timeless-Tipin-RPA complex
Components
  • Sample: Timeless-Tipin-RPA complex
  • Protein or peptide: Timeless
  • Protein or peptide: Timeless-interacting protein
  • Protein or peptide: Replication protein A 70
  • Protein or peptide: Replication protein A 32
  • Protein or peptide: Replication protein A 14

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Supramolecule #1000: Timeless-Tipin-RPA complex

SupramoleculeName: Timeless-Tipin-RPA complex / type: sample / ID: 1000 / Details: The sample was fixed using GraFix method / Oligomeric state: 1 / Number unique components: 5
Molecular weightExperimental: 270 KDa / Theoretical: 258 KDa / Method: Static light scattering

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Macromolecule #1: Timeless

MacromoleculeName: Timeless / type: protein_or_peptide / ID: 1 / Name.synonym: Tim / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: House mouse
Molecular weightExperimental: 130 KDa / Theoretical: 130 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant strain: pRARE / Recombinant plasmid: pEC
SequenceUniProtKB: Protein timeless homolog / InterPro: Timeless, N-terminal

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Macromolecule #2: Timeless-interacting protein

MacromoleculeName: Timeless-interacting protein / type: protein_or_peptide / ID: 2 / Name.synonym: Tipin / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: House mouse
Molecular weightExperimental: 31.9 KDa / Theoretical: 31.9 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant strain: pRARE / Recombinant plasmid: pEC
SequenceUniProtKB: TIMELESS-interacting protein / InterPro: Chromosome segregation in meiosis protein 3

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Macromolecule #3: Replication protein A 70

MacromoleculeName: Replication protein A 70 / type: protein_or_peptide / ID: 3 / Name.synonym: RPA70 / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: House mouse
Molecular weightExperimental: 52.3 KDa / Theoretical: 52.3 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant plasmid: pEC
SequenceUniProtKB: Replication protein A 70 kDa DNA-binding subunit / InterPro: Replication factor A protein 1

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Macromolecule #4: Replication protein A 32

MacromoleculeName: Replication protein A 32 / type: protein_or_peptide / ID: 4 / Name.synonym: RPA32 / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: House mouse
Molecular weightExperimental: 28.1 KDa / Theoretical: 28.1 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant plasmid: pEC
SequenceUniProtKB: Replication protein A 32 kDa subunit / InterPro: Replication factor A protein 2

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Macromolecule #5: Replication protein A 14

MacromoleculeName: Replication protein A 14 / type: protein_or_peptide / ID: 5 / Name.synonym: RPA14 / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: House mouse
Molecular weightExperimental: 16 KDa / Theoretical: 16 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant plasmid: pEC
SequenceUniProtKB: Replication protein A 14 kDa subunit / InterPro: Replication factor A protein 3

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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

Concentration0.3 mg/mL
BufferpH: 7 / Details: 20mM Hepes, 150mM NaCl, 5mM DTT, 1mM TCEP
GridDetails: 300 mesh Quantifoil holey carbon grids, glow-discharged
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 5 seconds with force 2, before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
DateOct 26, 2012
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 673 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 67873 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe initial model was obtained using RCT. The refinement process was carried out using SPARX.
CTF correctionDetails: Phases of individual images were flipped
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: OTHER / Software - Name: SPARX / Details: The amplitude was corrected. / Number images used: 39679
Final two d classificationNumber classes: 74

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