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- EMDB-28959: DNA replication fork binding triggers structural changes in the P... -
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Open data
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Basic information
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Title | DNA replication fork binding triggers structural changes in the PriA DNA helicase that regulate the PriA-PriB replication restart pathway in E. coli | |||||||||
![]() | final sharpened map | |||||||||
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Function / homology | ![]() pre-primosome complex / DnaB-DnaC-DnaT-PriA-PriC complex / DnaB-DnaC-DnaT-PriA-PriB complex / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() ![]() | |||||||||
Method | ![]() ![]() | |||||||||
![]() | Duckworth AT / Ducos PL / McMillan SD / Satyshur KA / Blumenthal KH / Deorio HR / Larson JA / Sandler SJ / Grant T / Keck JL | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Replication fork binding triggers structural changes in the PriA helicase that govern DNA replication restart in E. coli. Authors: Alexander T Duckworth / Peter L Ducos / Sarah D McMillan / Kenneth A Satyshur / Katelien H Blumenthal / Haley R Deorio / Joseph A Larson / Steven J Sandler / Timothy Grant / James L Keck / ![]() Abstract: Bacterial replisomes often dissociate from replication forks before chromosomal replication is complete. To avoid the lethal consequences of such situations, bacteria have evolved replication restart ...Bacterial replisomes often dissociate from replication forks before chromosomal replication is complete. To avoid the lethal consequences of such situations, bacteria have evolved replication restart pathways that reload replisomes onto prematurely terminated replication forks. To understand how the primary replication restart pathway in E. coli (PriA-PriB) selectively acts on replication forks, we determined the cryogenic-electron microscopy structure of a PriA/PriB/replication fork complex. Replication fork specificity arises from extensive PriA interactions with each arm of the branched DNA. These interactions reshape the PriA protein to create a pore encircling single-stranded lagging-strand DNA while also exposing a surface of PriA onto which PriB docks. Together with supporting biochemical and genetic studies, the structure reveals a switch-like mechanism for replication restart initiation in which restructuring of PriA directly couples replication fork recognition to PriA/PriB complex formation to ensure robust and high-fidelity replication re-initiation. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 25.1 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 22.3 KB 22.3 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 6.5 KB | Display | ![]() |
Images | ![]() | 122.1 KB | ||
Others | ![]() ![]() | 25 MB 25 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8fakMC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||
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Annotation | final sharpened map | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.079 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: unfiltered / unsharpened half map 1
File | emd_28959_half_map_1.map | ||||||||||||
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Annotation | unfiltered / unsharpened half map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: unfiltered / unsharpened half map 2
File | emd_28959_half_map_2.map | ||||||||||||
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Annotation | unfiltered / unsharpened half map 2 | ||||||||||||
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Density Histograms |
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Sample components
-Entire : Complex of PriA, PriB, and replication fork
Entire | Name: Complex of PriA, PriB, and replication fork |
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Components |
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-Supramolecule #1: Complex of PriA, PriB, and replication fork
Supramolecule | Name: Complex of PriA, PriB, and replication fork / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#5 |
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Source (natural) | Organism: ![]() ![]() ![]() |
Molecular weight | Theoretical: 134 KDa |
-Macromolecule #1: Primosomal replication protein N
Macromolecule | Name: Primosomal replication protein N / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() ![]() |
Molecular weight | Theoretical: 11.459194 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: MTNRLVLSGT VCRAPLRKVS PSGIPHCQFV LEHRSVQEEA GFHRQAWCQM PVIVSGHENQ AITHSITVGS RITVQGFISC HKAKNGLSK MVLHAEQIEL IDSGD |
-Macromolecule #3: Primosomal protein N'
Macromolecule | Name: Primosomal protein N' / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO EC number: ![]() |
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Source (natural) | Organism: ![]() ![]() ![]() |
Molecular weight | Theoretical: 81.76582 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: MPVAHVALPV PLPRTFDYLL PEGMTVKAGC RVRVPFGKQQ ERIGIVVSVS DASELPLNEL KAVVEVLDSE PVFTHSVWRL LLWAADYYH HPIGDVLFHA LPILLRQGRP AANAPMWYWF ATEQGQAVDL NSLKRSPKQQ QALAALRQGK IWRDQVATLE F NDAALQAL ...String: MPVAHVALPV PLPRTFDYLL PEGMTVKAGC RVRVPFGKQQ ERIGIVVSVS DASELPLNEL KAVVEVLDSE PVFTHSVWRL LLWAADYYH HPIGDVLFHA LPILLRQGRP AANAPMWYWF ATEQGQAVDL NSLKRSPKQQ QALAALRQGK IWRDQVATLE F NDAALQAL RKKGLCDLAS ETPEFSDWRT NYAVSGERLR LNTEQATAVG AIHSAADTFS AWLLAGVTGS GKTEVYLSVL EN VLAQGKQ ALVMVPEIGL TPQTIARFRE RFNAPVEVLH SGLNDSERLS AWLKAKNGEA AIVIGTRSAL FTPFKNLGVI VID EEHDSS YKQQEGWRYH ARDLAVYRAH SEQIPIILGS ATPALETLCN VQQKKYRLLR LTRRAGNARP AIQHVLDLKG QKVQ AGLAP ALITRMRQHL QADNQVILFL NRRGFAPALL CHDCGWIAEC PRCDHYYTLH QAQHHLRCHH CDSQRPVPRQ CPSCG STHL VPVGLGTEQL EQTLAPLFPG VPISRIDRDT TSRKGALEQQ LAEVHRGGAR ILIGTQMLAK GHHFPDVTLV ALLDVD GAL FSADFRSAER FAQLYTQVAG RAGRAGKQGE VVLQTHHPEH PLLQTLLYKG YDAFAEQALA ERRMMQLPPW TSHVIVR AE DHNNQHAPLF LQQLRNLILS SPLADEKLWV LGPVPALAPK RGGRWRWQIL LQHPSRVRLQ HIINGTLALI NTIPDSRK V KWVLDVDPIE G |
-Macromolecule #2: DNA (5'-D(P*CP*AP*GP*AP*CP*TP*CP*AP*TP*TP*TP*AP*GP*CP*CP*CP*TP*TP...
Macromolecule | Name: DNA (5'-D(P*CP*AP*GP*AP*CP*TP*CP*AP*TP*TP*TP*AP*GP*CP*CP*CP*TP*TP*AP*TP*CP*CP*G)-3') type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 12.206812 KDa |
Sequence | String: (DG)(DC)(DC)(DG)(DC)(DA)(DG)(DA)(DC)(DT) (DC)(DA)(DT)(DT)(DT)(DA)(DG)(DC)(DC)(DC) (DT)(DT)(DA)(DT)(DC)(DC)(DG)(DT)(DA) (DT)(DT)(DG)(DC)(DG)(DG)(DT)(DC)(DT)(DC) (DG) |
-Macromolecule #4: DNA (5'-D(P*CP*GP*GP*AP*TP*AP*AP*GP*GP*GP*CP*TP*GP*AP*GP*CP*AP*CP...
Macromolecule | Name: DNA (5'-D(P*CP*GP*GP*AP*TP*AP*AP*GP*GP*GP*CP*TP*GP*AP*GP*CP*AP*CP*GP*CP*CP*GP*A)-3') type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 12.399983 KDa |
Sequence | String: (DC)(DG)(DA)(DG)(DA)(DC)(DC)(DG)(DC)(DA) (DA)(DT)(DA)(DC)(DG)(DG)(DA)(DT)(DA)(DA) (DG)(DG)(DG)(DC)(DT)(DG)(DA)(DG)(DC) (DA)(DC)(DG)(DC)(DC)(DG)(DA)(DC)(DG)(DA) (DA) |
-Macromolecule #5: DNA (5'-D(P*TP*CP*GP*GP*CP*GP*TP*GP*CP*TP*C)-3')
Macromolecule | Name: DNA (5'-D(P*TP*CP*GP*GP*CP*GP*TP*GP*CP*TP*C)-3') / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 4.567944 KDa |
Sequence | String: (DT)(DT)(DC)(DG)(DT)(DC)(DG)(DG)(DC)(DG) (DT)(DG)(DC)(DT)(DC) |
-Macromolecule #6: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Experimental details
-Structure determination
Method | ![]() |
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Aggregation state | particle |
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Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 8 |
Grid | Model: Quantifoil / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD![]() |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | #0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 (6k x 4k) / #0 - Number grids imaged: 1 / #0 - Number real images: 1600 / #0 - Average electron dose: 100.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: FEI FALCON III (4k x 4k) / #1 - Detector mode: INTEGRATING / #1 - Number grids imaged: 1 / #1 - Number real images: 1200 / #1 - Average electron dose: 60.0 e/Å2 |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
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Output model | ![]() PDB-8fak: |