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Yorodumi- PDB-8unh: Cryo-EM structure of T4 Bacteriophage Clamp Loader with Sliding Clamp -
+Open data
-Basic information
Entry | Database: PDB / ID: 8unh | ||||||
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Title | Cryo-EM structure of T4 Bacteriophage Clamp Loader with Sliding Clamp | ||||||
Components |
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Keywords | REPLICATION / autoinhibited / DNA-free / catalytically inactive / stable | ||||||
Function / homology | Function and homology information DNA clamp loader activity / bidirectional double-stranded viral DNA replication / viral DNA genome replication / DNA polymerase processivity factor activity / viral transcription / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / DNA replication / ATP hydrolysis activity / DNA binding / ATP binding Similarity search - Function | ||||||
Biological species | Tequatrovirus T4 | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.21 Å | ||||||
Authors | Huang, Y. / Marcus, K. / Subramanian, S. / Gee, L.C. / Gorday, K. / Ghaffari-Kashani, S. / Luo, X. / Zhang, L. / O'Donnell, M. / Subramanian, S. / Kuriyan, J. | ||||||
Funding support | United States, 1items
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Citation | Journal: Nat Struct Mol Biol / Year: 2024 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
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8unh.cif.gz | 410 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8unh.ent.gz | 309.5 KB | Display | PDB format |
PDBx/mmJSON format | 8unh.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/un/8unh ftp://data.pdbj.org/pub/pdb/validation_reports/un/8unh | HTTPS FTP |
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-Related structure data
Related structure data | 42402MC 8uh7C 8uk9C 8unfC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 35834.254 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Tequatrovirus T4 / Gene: 44 / Production host: Escherichia coli (E. coli) / References: UniProt: P04526 #2: Protein | | Mass: 21391.703 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Tequatrovirus T4 / Gene: 62 / Production host: Escherichia coli (E. coli) / References: UniProt: P04527 #3: Protein | Mass: 24881.223 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Tequatrovirus T4 / Gene: 45 / Production host: Escherichia coli (E. coli) / References: UniProt: P04525 #4: Chemical | #5: Chemical | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Molecular weight |
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Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 7.5 | ||||||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 17937844 | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 455041 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: REAL | ||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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