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- EMDB-42402: Cryo-EM structure of T4 Bacteriophage Clamp Loader with Sliding Clamp -
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Open data
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Basic information
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Title | Cryo-EM structure of T4 Bacteriophage Clamp Loader with Sliding Clamp | |||||||||
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![]() | autoinhibited / DNA-free / catalytically inactive / stable / ![]() | |||||||||
Function / homology | ![]() Rad17 RFC-like complex / Elg1 RFC-like complex / DNA replication factor C complex / DNA clamp loader activity / bidirectional double-stranded viral DNA replication / viral DNA genome replication / DNA polymerase processivity factor activity / viral transcription / ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | ![]() ![]() | |||||||||
![]() | Huang Y / Marcus K / Subramanian S / Gee LC / Gorday K / Ghaffari-Kashani S / Luo X / Zhang L / O'Donnell M / Kuriyan J | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Autoinhibition of a clamp-loader ATPase revealed by deep mutagenesis and cryo-EM. Authors: Kendra Marcus / Yongjian Huang / Subu Subramanian / Christine L Gee / Kent Gorday / Sam Ghaffari-Kashani / Xiao Ran Luo / Lisa Zheng / Michael O'Donnell / Sriram Subramaniam / John Kuriyan / ![]() ![]() Abstract: Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between aspartate residues in one ...Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between aspartate residues in one protomer, present in conserved 'DEAD-box' motifs, and arginine residues in adjacent protomers. We show that functional defects resulting from a DEAD-box mutation in the T4 bacteriophage clamp loader can be compensated by widely distributed single mutations in the ATPase domain. Using cryo-EM, we discovered an unsuspected inactive conformation of the clamp loader, in which DNA binding is blocked and the catalytic sites are disassembled. Mutations that restore function map to regions of conformational change upon activation, suggesting that these mutations may increase DNA affinity by altering the energetic balance between inactive and active states. Our results show that there are extensive opportunities for evolution to improve catalytic efficiency when an inactive intermediate is involved. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 15.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 20.8 KB 20.8 KB | Display Display | ![]() |
Images | ![]() | 159.6 KB | ||
Filedesc metadata | ![]() | 6.5 KB | ||
Others | ![]() ![]() | 28.3 MB 28.3 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8unhMC ![]() 8uh7C ![]() 8uk9C ![]() 8unfC C: citing same article ( M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 1.048 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_42402_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_42402_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
-Entire : T4 Bacteriophage Clamp Loader with Sliding Clamp
Entire | Name: T4 Bacteriophage Clamp Loader with Sliding Clamp |
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Components |
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-Supramolecule #1: T4 Bacteriophage Clamp Loader with Sliding Clamp
Supramolecule | Name: T4 Bacteriophage Clamp Loader with Sliding Clamp / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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Source (natural) | Organism: ![]() ![]() |
-Supramolecule #2: T4 Bacteriophage Sliding Clamp (gp45)
Supramolecule | Name: T4 Bacteriophage Sliding Clamp (gp45) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #3 |
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Source (natural) | Organism: ![]() ![]() |
-Supramolecule #3: T4 Bacteriophage Clamp Loader (gp44 + gp62)
Supramolecule | Name: T4 Bacteriophage Clamp Loader (gp44 + gp62) / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: ![]() ![]() |
-Macromolecule #1: Sliding-clamp-loader large subunit
Macromolecule | Name: Sliding-clamp-loader large subunit / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 35.834254 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: MITVNEKEHI LEQKYRPSTI DECILPAFDK ETFKSITSKG KIPHIILHSP SPGTGKTTVA KALCHDVNAD MMFVNGSDCK IDFVRGPLT NFASAASFDG RQKVIVIDEF DRSGLAESQR HLRSFMEAYS SNCSIIITAN NIDGIIKPLQ SRCRVITFGQ P TDEDKIEM ...String: MITVNEKEHI LEQKYRPSTI DECILPAFDK ETFKSITSKG KIPHIILHSP SPGTGKTTVA KALCHDVNAD MMFVNGSDCK IDFVRGPLT NFASAASFDG RQKVIVIDEF DRSGLAESQR HLRSFMEAYS SNCSIIITAN NIDGIIKPLQ SRCRVITFGQ P TDEDKIEM MKQMIRRLTE ICKHEGIAIA DMKVVAALVK KNFPDFRKTI GELDSYSSKG VLDAGILSLV TNDRGAIDDV LE SLKNKDV KQLRALAPKY AADYSWFVGK LAEEIYSRVT PQSIIRMYEI VGENNQYHGI AANTELHLAY LFIQLACEMQ WK UniProtKB: Sliding-clamp-loader large subunit |
-Macromolecule #2: Sliding-clamp-loader small subunit
Macromolecule | Name: Sliding-clamp-loader small subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 21.391703 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: MSLFKDDIQL NEHQVAWYSK DWTAVQSAAD SFKEKAENEF FEIIGAINNK TKCSIAQKDY SKFMVENALS QFPECMPAVY AMNLIGSGL SDEAHFNYLM AAVPRGKRYG KWAKLVEDST EVLIIKLLAK RYQVNTNDAI NYKSILTKNG KLPLVLKELK G LVTDDFLK EVTKNVKEQK QLKKLALEW UniProtKB: Sliding-clamp-loader small subunit |
-Macromolecule #3: Sliding clamp
Macromolecule | Name: Sliding clamp / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 24.881223 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: MKLSKDTTAL LKNFATINSG IMLKSGQFIM TRAVNGTTYA EANISDVIDF DVAIYDLNGF LGILSLVNDD AEISQSEDGN IKIADARST IFWPAADPST VVAPNKPIPF PVASAVTEIK AEDLQQLLRV SRGLQIDTIA ITVKEGKIVI NGFNKVEDSA L TRVKYSLT ...String: MKLSKDTTAL LKNFATINSG IMLKSGQFIM TRAVNGTTYA EANISDVIDF DVAIYDLNGF LGILSLVNDD AEISQSEDGN IKIADARST IFWPAADPST VVAPNKPIPF PVASAVTEIK AEDLQQLLRV SRGLQIDTIA ITVKEGKIVI NGFNKVEDSA L TRVKYSLT LGDYDGENTF NFIINMANMK MQPGNYKLLL WAKGKQGAAK FEGEHANYVV ALEADSTHDF UniProtKB: ![]() |
-Macromolecule #4: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 2 / Formula: AGS |
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Molecular weight | Theoretical: 523.247 Da |
Chemical component information | ![]() ChemComp-AGS: |
-Macromolecule #5: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | ![]() |
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Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 |
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Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 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![]() |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2 |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Particle selection | Number selected: 17937844 |
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Startup model | Type of model: INSILICO MODEL In silico model: 3D Ab-initio reconstruction from selected particles after 2D classification in cryoSPARC |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3) / Software - details: ab-initio reconstruction |
Final 3D classification | Number classes: 10 / Software - Name: cryoSPARC (ver. 3) / Software - details: heterogenous refinement |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3) / Software - details: heterogenous refinement |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Software - details: Non-uniform refinement / Number images used: 455041 |
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: OTHER |
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Output model | ![]() PDB-8unh: |