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- PDB-8uk9: Structure of T4 Bacteriophage clamp loader mutant D110C bound to ... -

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Basic information

Entry
Database: PDB / ID: 8uk9
TitleStructure of T4 Bacteriophage clamp loader mutant D110C bound to the T4 clamp, primer-template DNA, and ATP analog
Components
  • (Sliding-clamp-loader ...) x 2
  • DNA primerPrimer (molecular biology)
  • DNA template
  • Sliding clampDNA clamp
KeywordsDNA BINDING PROTEIN/DNA / DNA Replication / AAA+ ATPase / Bacteriophage / Complex / DNA / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
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
ADENOSINE-5'-DIPHOSPHATE / ALUMINUM FLUORIDE / DNA / DNA (> 10) / Sliding clamp / Sliding-clamp-loader large subunit / Sliding-clamp-loader small subunit
Similarity search - Component
Biological speciesTequatrovirus T4
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.1 Å
AuthorsMarcus, K. / Ghaffari-Kashani, S. / Gee, C.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 12, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2024Group: Database references / Category: citation / citation_author
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.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Apr 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: DNA template
J: DNA primer
K: Sliding-clamp-loader large subunit
L: Sliding-clamp-loader large subunit
M: Sliding-clamp-loader large subunit
N: Sliding-clamp-loader large subunit
O: DNA template
P: DNA primer
Q: Sliding-clamp-loader small subunit
R: Sliding clamp
S: Sliding clamp
T: Sliding clamp
hetero molecules


Theoretical massNumber of molelcules
Total (without water)510,42942
Polymers506,31320
Non-polymers4,11622
Water0
1
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: DNA template
J: DNA primer
hetero molecules


Theoretical massNumber of molelcules
Total (without water)255,21521
Polymers253,15710
Non-polymers2,05811
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area37690 Å2
ΔGint-173 kcal/mol
Surface area90490 Å2
MethodPISA
2
K: Sliding-clamp-loader large subunit
L: Sliding-clamp-loader large subunit
M: Sliding-clamp-loader large subunit
N: Sliding-clamp-loader large subunit
O: DNA template
P: DNA primer
Q: Sliding-clamp-loader small subunit
R: Sliding clamp
S: Sliding clamp
T: Sliding clamp
hetero molecules


Theoretical massNumber of molelcules
Total (without water)255,21521
Polymers253,15710
Non-polymers2,05811
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area38350 Å2
ΔGint-184 kcal/mol
Surface area92120 Å2
MethodPISA
Unit cell
Length a, b, c (Å)95.243, 231.991, 264.511
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

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Sliding-clamp-loader ... , 2 types, 10 molecules AQBCDEKLMN

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


Mass: 21391.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tequatrovirus T4 / Gene: 62 / Production host: Escherichia coli (E. coli) / References: UniProt: P04527
#2: Protein
Sliding-clamp-loader large subunit


Mass: 35909.387 Da / Num. of mol.: 8 / Mutation: D110C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tequatrovirus T4 / Gene: 44 / Production host: Escherichia coli (E. coli) / References: UniProt: P04526

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Protein , 1 types, 6 molecules FGHRST

#3: Protein
Sliding clamp / DNA clamp / GP45


Mass: 24881.223 Da / Num. of mol.: 6
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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DNA chain , 2 types, 4 molecules IOJP

#4: DNA chain DNA template


Mass: 7347.733 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Tequatrovirus T4
#5: DNA chain DNA primer / Primer (molecular biology)


Mass: 6136.008 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Tequatrovirus T4

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Non-polymers , 3 types, 22 molecules

#6: Chemical
ChemComp-AF3 / ALUMINUM FLUORIDE / Aluminium fluoride


Mass: 83.977 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: AlF3
#7: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#8: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg

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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: 2.82 Å3/Da / Density % sol: 56.31 % / Description: Rods
Crystal growTemperature: 298 K / Method: evaporation / pH: 6.5 / Details: 0.1M MES pH 6.5, 9% PEG MME 5000, 6% 1-Propanol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 0.9794 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Oct 31, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9794 Å / Relative weight: 1
ReflectionResolution: 3.1→48.13 Å / Num. obs: 106987 / % possible obs: 71.39 % / Redundancy: 2 % / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.05745 / Rpim(I) all: 0.05745 / Rrim(I) all: 0.08124 / Net I/σ(I): 5.8
Reflection shellResolution: 3.1→3.211 Å / Redundancy: 2 % / Rmerge(I) obs: 1.571 / Num. unique obs: 470 / CC1/2: 0.169 / Rpim(I) all: 1.571 / Rrim(I) all: 2.222

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Processing

Software
NameVersionClassification
PHENIX(1.19.2_4158: ???)refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.1→48.13 Å / SU ML: 0.36 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 31.77 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.265 1999 2.62 %
Rwork0.2526 --
obs0.253 76441 71.35 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.1→48.13 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms33620 1794 0 0 35414
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00336215
X-RAY DIFFRACTIONf_angle_d0.66449329
X-RAY DIFFRACTIONf_dihedral_angle_d18.78613616
X-RAY DIFFRACTIONf_chiral_restr0.0445632
X-RAY DIFFRACTIONf_plane_restr0.0055978
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1-3.180.232780.4254277X-RAY DIFFRACTION4
3.18-3.260.4649160.4099609X-RAY DIFFRACTION8
3.26-3.360.4339300.39511120X-RAY DIFFRACTION15
3.36-3.470.3088490.3721835X-RAY DIFFRACTION25
3.47-3.590.3857970.35873597X-RAY DIFFRACTION49
3.59-3.730.39441860.39366935X-RAY DIFFRACTION94
3.73-3.90.32641980.31617411X-RAY DIFFRACTION100
3.9-4.110.29932000.29097425X-RAY DIFFRACTION100
4.11-4.370.28991990.25317406X-RAY DIFFRACTION100
4.37-4.70.23922000.22957445X-RAY DIFFRACTION100
4.7-5.180.26782010.23527471X-RAY DIFFRACTION100
5.18-5.930.29962010.26197506X-RAY DIFFRACTION100
5.93-7.460.27992040.26687577X-RAY DIFFRACTION100
7.46-48.130.19362100.19247828X-RAY DIFFRACTION100

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