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Yorodumi- EMDB-27671: Open state of RFC:PCNA bound to a 3' ss/dsDNA junction (DNA2) with NTD -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27671 | ||||||||||||
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Title | Open state of RFC:PCNA bound to a 3' ss/dsDNA junction (DNA2) with NTD | ||||||||||||
Map data | Main Map | ||||||||||||
Sample |
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Keywords | REPLICATION-DNA complex | ||||||||||||
Function / homology | Function and homology information DNA clamp unloading / Gap-filling DNA repair synthesis and ligation in GG-NER / Rad17 RFC-like complex / Ctf18 RFC-like complex / DNA clamp loader activity / DNA replication factor C complex / Elg1 RFC-like complex / Polymerase switching / Translesion Synthesis by POLH / Translesion synthesis by REV1 ...DNA clamp unloading / Gap-filling DNA repair synthesis and ligation in GG-NER / Rad17 RFC-like complex / Ctf18 RFC-like complex / DNA clamp loader activity / DNA replication factor C complex / Elg1 RFC-like complex / Polymerase switching / Translesion Synthesis by POLH / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / DNA replication checkpoint signaling / PCNA complex / Activation of ATR in response to replication stress / Termination of translesion DNA synthesis / sister chromatid cohesion / mitotic sister chromatid cohesion / DNA polymerase processivity factor activity / leading strand elongation / regulation of DNA replication / Gap-filling DNA repair synthesis and ligation in TC-NER / Dual incision in TC-NER / mismatch repair / translesion synthesis / DNA damage checkpoint signaling / DNA-templated DNA replication / mitotic cell cycle / cell division / DNA repair / ATP hydrolysis activity / DNA binding / ATP binding / nucleus / cytosol Similarity search - Function | ||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.76 Å | ||||||||||||
Authors | Schrecker M / Hite RK | ||||||||||||
Funding support | United States, 3 items
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Citation | Journal: Elife / Year: 2022 Title: Multistep loading of a DNA sliding clamp onto DNA by replication factor C. Authors: Marina Schrecker / Juan C Castaneda / Sujan Devbhandari / Charanya Kumar / Dirk Remus / Richard K Hite / Abstract: The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader ...The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded (ss)/double-stranded DNA (dsDNA) junctions harboring a recessed 3' end (3' ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATPγS and 3' ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and raising the question of whether PCNA ring closure is mechanistically coupled to ATP hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA. | ||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_27671.map.gz | 108.4 MB | EMDB map data format | |
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Header (meta data) | emd-27671-v30.xml emd-27671.xml | 31.2 KB 31.2 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_27671_fsc.xml | 12.7 KB | Display | FSC data file |
Images | emd_27671.png | 203.6 KB | ||
Filedesc metadata | emd-27671.cif.gz | 8.1 KB | ||
Others | emd_27671_additional_1.map.gz emd_27671_half_map_1.map.gz emd_27671_half_map_2.map.gz | 676.6 MB 200.4 MB 200.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27671 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27671 | HTTPS FTP |
-Validation report
Summary document | emd_27671_validation.pdf.gz | 1.1 MB | Display | EMDB validaton report |
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Full document | emd_27671_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | emd_27671_validation.xml.gz | 21.8 KB | Display | |
Data in CIF | emd_27671_validation.cif.gz | 28.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27671 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27671 | HTTPS FTP |
-Related structure data
Related structure data | 8dr5MC 8dqwC 8dqxC 8dqzC 8dr0C 8dr1C 8dr3C 8dr4C 8dr6C 8dr7C C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_27671.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Main Map | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.826 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: Density modified and 1.5x resampled map
File | emd_27671_additional_1.map | ||||||||||||
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Annotation | Density modified and 1.5x resampled map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half Map B
File | emd_27671_half_map_1.map | ||||||||||||
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Annotation | Half Map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half Map A
File | emd_27671_half_map_2.map | ||||||||||||
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Annotation | Half Map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : Open state of RFC:PCNA bound to a 3' ss/dsDNA junction (DNA2) with NTD
+Supramolecule #1: Open state of RFC:PCNA bound to a 3' ss/dsDNA junction (DNA2) with NTD
+Macromolecule #1: Replication factor C subunit 1
+Macromolecule #2: Replication factor C subunit 4
+Macromolecule #3: Replication factor C subunit 3
+Macromolecule #4: Replication factor C subunit 2
+Macromolecule #5: Replication factor C subunit 5
+Macromolecule #6: Proliferating cell nuclear antigen
+Macromolecule #7: DNA (5'-D(P*TP*TP*TP*CP*GP*GP*GP*GP*GP*GP*GP*CP*C)-3')
+Macromolecule #8: DNA (5'-D(P*GP*GP*CP*CP*CP*CP*CP*CP*CP*GP*GP*C)-3')
+Macromolecule #9: DNA (5'-D(P*AP*GP*GP*GP*GP*GP*GP*GP*GP*GP*G)-3')
+Macromolecule #10: DNA (5'-D(P*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*TP*TP*T)-3')
+Macromolecule #11: MAGNESIUM ION
+Macromolecule #12: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
+Macromolecule #13: GUANOSINE-5'-DIPHOSPHATE
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.0 mg/mL | ||||||||
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Buffer | pH: 7.6 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: GRAPHENE OXIDE / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS | ||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 297 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |