+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27666 | ||||||||||||
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Title | Intermediate state of RFC:PCNA bound to a 3' ss/dsDNA junction | ||||||||||||
Map data | RFC-PCNA intermediate state | ||||||||||||
Sample |
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Keywords | REPLICATION-DNA complex | ||||||||||||
Function / homology | Function and homology information DNA clamp unloading / Rad17 RFC-like complex / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / meiotic mismatch repair / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / DNA clamp loader activity ...DNA clamp unloading / Rad17 RFC-like complex / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / meiotic mismatch repair / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / DNA clamp loader activity / Polymerase switching / SUMOylation of DNA replication proteins / positive regulation of DNA metabolic process / maintenance of DNA trinucleotide repeats / PCNA complex / establishment of mitotic sister chromatid cohesion / DNA replication checkpoint signaling / Activation of ATR in response to replication stress / lagging strand elongation / postreplication repair / sister chromatid cohesion / silent mating-type cassette heterochromatin formation / mitotic sister chromatid cohesion / leading strand elongation / DNA polymerase processivity factor activity / error-free translesion synthesis / Gap-filling DNA repair synthesis and ligation in TC-NER / subtelomeric heterochromatin formation / mismatch repair / translesion synthesis / positive regulation of DNA repair / replication fork / positive regulation of DNA replication / DNA damage checkpoint signaling / nucleotide-excision repair / DNA-templated DNA replication / mitotic cell cycle / chromosome, telomeric region / cell division / DNA repair / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding / nucleus / cytosol Similarity search - Function | ||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.92 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_27666.map.gz | 108.2 MB | EMDB map data format | |
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Header (meta data) | emd-27666-v30.xml emd-27666.xml | 29.8 KB 29.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_27666_fsc.xml | 12.7 KB | Display | FSC data file |
Images | emd_27666.png | 183.1 KB | ||
Filedesc metadata | emd-27666.cif.gz | 8.1 KB | ||
Others | emd_27666_additional_1.map.gz emd_27666_half_map_1.map.gz emd_27666_half_map_2.map.gz | 676.6 MB 200.3 MB 200.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27666 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27666 | HTTPS FTP |
-Related structure data
Related structure data | 8dqzMC 8dqwC 8dqxC 8dr0C 8dr1C 8dr3C 8dr4C 8dr5C 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_27666.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | RFC-PCNA intermediate state | ||||||||||||||||||||
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 sampled map
File | emd_27666_additional_1.map | ||||||||||||
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Annotation | Density modified and 1.5x sampled map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: A
File | emd_27666_half_map_1.map | ||||||||||||
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Annotation | A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: B
File | emd_27666_half_map_2.map | ||||||||||||
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Annotation | B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : Intermediate state of RFC:PCNA bound to a 3' ss/dsDNA junction
+Supramolecule #1: Intermediate state of RFC:PCNA bound to a 3' ss/dsDNA junction
+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*TP*TP*TP*CP*GP*GP*GP*GP*GP*GP*GP*CP*CP*CP*CP...
+Macromolecule #8: DNA (5'-D(P*CP*CP*CP*CP*GP*GP*GP*GP*CP*CP*CP*CP*CP*CP*CP*GP*GP*C)-3')
+Macromolecule #9: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
+Macromolecule #10: MAGNESIUM ION
+Macromolecule #11: 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 / 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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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |