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- PDB-8uh7: Structure of T4 Bacteriophage clamp loader bound to the T4 clamp,... -

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Basic information

Entry
Database: PDB / ID: 8uh7
TitleStructure of T4 Bacteriophage clamp loader bound to the T4 clamp, primer-template DNA, and ATP analog
Components
  • (Sliding-clamp-loader ...) x 2
  • Primer DNA strand
  • Sliding clamp
  • Template DNA strand
KeywordsREPLICATION/DNA / Clamp loader / T4 / AAA+ATPase / REPLICATION / REPLICATION-DNA complex
Function / homology
Function and homology information


Rad17 RFC-like complex / DNA clamp loader activity / DNA replication factor C complex / Elg1 RFC-like complex / 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-templated DNA replication ...Rad17 RFC-like complex / DNA clamp loader activity / DNA replication factor C complex / Elg1 RFC-like complex / 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-templated DNA replication / DNA replication / DNA repair / 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
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 2.628 Å
AuthorsGee, C.L. / Marcus, K. / Kelch, B.A. / Makino, D.L.
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 7, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 10, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
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
A: Sliding-clamp-loader small subunit
B: Sliding-clamp-loader large subunit
C: Sliding-clamp-loader large subunit
D: Sliding-clamp-loader large subunit
E: Sliding-clamp-loader large subunit
F: Sliding clamp
G: Sliding clamp
H: Sliding clamp
I: Template DNA strand
J: Primer DNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)258,94818
Polymers256,94710
Non-polymers2,0018
Water61334
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area35000 Å2
ΔGint-159 kcal/mol
Surface area91840 Å2
MethodPISA
Unit cell
Length a, b, c (Å)93.351, 118.325, 132.882
Angle α, β, γ (deg.)90.00, 102.05, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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

#1: Protein Sliding-clamp-loader small subunit


Mass: 21391.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tequatrovirus / Gene: 62 / Production host: Escherichia coli (E. coli) / References: UniProt: P04527
#2: Protein
Sliding-clamp-loader large subunit


Mass: 36189.602 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tequatrovirus / Gene: 44 / Production host: Escherichia coli (E. coli) / References: UniProt: P04526

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Protein , 1 types, 3 molecules FGH

#3: Protein Sliding clamp


Mass: 25162.592 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tequatrovirus / Gene: 45 / Production host: Escherichia coli (E. coli) / References: UniProt: P04525

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DNA chain , 2 types, 2 molecules IJ

#4: DNA chain Template DNA strand


Mass: 9172.891 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Tequatrovirus
#5: DNA chain Primer DNA strand


Mass: 6136.008 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Tequatrovirus

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Non-polymers , 4 types, 42 molecules

#6: 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
#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-ADP / ADENOSINE-5'-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
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 34 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.8 Å3/Da / Density % sol: 56.11 %
Crystal growTemperature: 295 K / Method: vapor diffusion / pH: 7.5 / Details: 20% PEG4k, 0.1M Tris pH7.5, 10mM MgCl2, 20mM NaCl

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 0.9739 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Nov 25, 2008
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9739 Å / Relative weight: 1
ReflectionResolution: 2.628→49.649 Å / Num. obs: 55509 / % possible obs: 92.9 % / Redundancy: 6.3 % / CC1/2: 0.978 / Rmerge(I) obs: 0.325 / Rpim(I) all: 0.141 / Rrim(I) all: 0.355 / Net I/σ(I): 6.9
Reflection shellResolution: 2.628→2.922 Å / Num. unique obs: 2775 / CC1/2: 0.306 / Rpim(I) all: 0.865

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Processing

Software
NameVersionClassification
PHENIX1.14_3260refinement
XDSdata reduction
Aimlessdata scaling
PHENIXphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS / Resolution: 2.628→49.649 Å / SU ML: 0.34 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 25.79 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2445 1998 3.6 %
Rwork0.1934 --
obs0.1952 55478 65.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.628→49.649 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms16745 897 124 34 17800
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00318166
X-RAY DIFFRACTIONf_angle_d0.52724753
X-RAY DIFFRACTIONf_dihedral_angle_d12.3610867
X-RAY DIFFRACTIONf_chiral_restr0.0422812
X-RAY DIFFRACTIONf_plane_restr0.0043001
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.628-2.6940.280530.364373X-RAY DIFFRACTION1
2.6936-2.76640.3858170.3351466X-RAY DIFFRACTION8
2.7664-2.84780.2963350.3403913X-RAY DIFFRACTION16
2.8478-2.93970.4168570.31391538X-RAY DIFFRACTION27
2.9397-3.04470.3842820.29322201X-RAY DIFFRACTION38
3.0447-3.16660.33861220.27823253X-RAY DIFFRACTION56
3.1666-3.31070.33251680.26394498X-RAY DIFFRACTION78
3.3107-3.48520.31252100.24665629X-RAY DIFFRACTION97
3.4852-3.70350.27562170.20845775X-RAY DIFFRACTION100
3.7035-3.98940.24392150.1845770X-RAY DIFFRACTION100
3.9894-4.39060.2122170.15995806X-RAY DIFFRACTION100
4.3906-5.02540.17322180.15015818X-RAY DIFFRACTION100
5.0254-6.32940.2432180.1895851X-RAY DIFFRACTION100
6.3294-49.6490.1982190.15545889X-RAY DIFFRACTION99

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