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- PDB-8unf: Cryo-EM structure of T4 Bacteriophage Clamp Loader with Sliding C... -

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Basic information

Entry
Database: PDB / ID: 8unf
TitleCryo-EM structure of T4 Bacteriophage Clamp Loader with Sliding Clamp and DNA
Components
  • (Sliding-clamp-loader ...) x 2
  • Sliding clampDNA clamp
  • primer DNA
  • template DNA
KeywordsREPLICATION / active / DNA-bound
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
Sliding-clamp-loader small subunit gp62 / Sliding-clamp-loader large subunit / : / : / Bacteriophage clamp loader A subunit / Sliding-clamp-loader large subunit, AAA+ ATPase lid domain / Sliding-clamp-loader large subunit, C-terminal domain / Sliding clamp, C-terminal / Sliding clamp / gp45 sliding clamp, C terminal ...Sliding-clamp-loader small subunit gp62 / Sliding-clamp-loader large subunit / : / : / Bacteriophage clamp loader A subunit / Sliding-clamp-loader large subunit, AAA+ ATPase lid domain / Sliding-clamp-loader large subunit, C-terminal domain / Sliding clamp, C-terminal / Sliding clamp / gp45 sliding clamp, C terminal / DNA polymerase processivity factor / DNA polymerase processivity factor / : / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Chem-08T / ADENOSINE-5'-DIPHOSPHATE / DNA / DNA (> 10) / Sliding clamp / Sliding-clamp-loader large subunit / Sliding-clamp-loader small subunit
Similarity search - Component
Biological speciesTequatrovirus T4
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsHuang, 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
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: 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
DepositionOct 18, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 10, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 17, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Apr 3, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
J: primer DNA
I: template DNA
H: Sliding clamp
G: Sliding clamp
F: Sliding clamp
E: Sliding-clamp-loader large subunit
D: Sliding-clamp-loader large subunit
C: Sliding-clamp-loader large subunit
B: Sliding-clamp-loader large subunit
A: Sliding-clamp-loader small subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)254,85718
Polymers252,85610
Non-polymers2,0018
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 2 types, 2 molecules JI

#1: DNA chain primer DNA


Mass: 6136.008 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Tequatrovirus T4
#2: DNA chain template DNA


Mass: 7347.733 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Tequatrovirus T4

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Protein , 1 types, 3 molecules HGF

#3: Protein Sliding clamp / DNA clamp / GP45


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

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Sliding-clamp-loader ... , 2 types, 5 molecules EDCBA

#4: Protein
Sliding-clamp-loader large subunit


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
#5: Protein Sliding-clamp-loader small subunit / Clamp loader gp62 subunit / Gene product 62 / gp62


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

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Non-polymers , 3 types, 8 molecules

#6: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#7: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-08T / [[[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-bis(oxidanyl)oxolan-2-yl]methoxy-oxidanyl-phosphoryl]oxy-oxidanyl-phosphoryl]oxy-tris(fluoranyl)beryllium


Mass: 492.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H14BeF3N5O10P2 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1T4 Bacteriophage Clamp Loader with Sliding Clamp and DNA (gp44+gp62+gp45+DNA)COMPLEX#1-#50RECOMBINANT
2T4 Bacteriophage Sliding Clamp (gp45)COMPLEX#31RECOMBINANT
3T4 Bacteriophage Clamp Loader (gp44 + gp62)COMPLEX#4-#51RECOMBINANT
4T4 Bacteriophage Clamp Loader subunit A (gp62)COMPLEX#53RECOMBINANT
5T4 Bacteriophage Clamp Loader subunits B,C,D,E(gp44)COMPLEX#43RECOMBINANT
6primed DNA duplexCOMPLEX#1-#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.24 MDaNO
21NO
31NO
41NO
54
61NO
76
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Tequatrovirus T410665
32Tequatrovirus T410665
43Tequatrovirus T410665
54Tequatrovirus T410665
65Tequatrovirus T410665
76Tequatrovirus T410665
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
54Escherichia coli (E. coli)562
65Escherichia coli (E. coli)562
76Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3particle selectiontemplate-free Blob_picker
2SerialEMimage acquisition
4cryoSPARC3CTF correctionPatch-Based CTF Estimation
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARC3initial Euler assignmentab-initio reconstruction
11cryoSPARC3final Euler assignmentheterogenous refinement
12cryoSPARC3classificationheterogenous refinement
13cryoSPARC33D reconstructionNon-uniform refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4206415
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 204448 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00418195
ELECTRON MICROSCOPYf_angle_d0.57624794
ELECTRON MICROSCOPYf_dihedral_angle_d14.4222754
ELECTRON MICROSCOPYf_chiral_restr0.0442816
ELECTRON MICROSCOPYf_plane_restr0.0043008

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