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- PDB-8vat: Structure of the E. coli clamp loader bound to the beta clamp in ... -

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Entry
Database: PDB / ID: 8vat
TitleStructure of the E. coli clamp loader bound to the beta clamp in a Open-RNAp/t conformation
Components
  • (DNA polymerase III subunit ...DNA polymerase III holoenzyme) x 3
  • Beta sliding clamp
  • DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*CP*AP*GP*AP*CP*AP*CP*CP*AP*CP*TP*GP*C)-3')
  • RNA (5'-R(P*AP*GP*UP*GP*GP*UP*GP*UP*CP*UP*G)-3')
KeywordsREPLICATION / TRANSFERASE/DNA / Bacterial Clamp Loader Complex / TRANSFERASE-DNA complex
Function / homology
Function and homology information


DNA polymerase III, clamp loader complex / Hda-beta clamp complex / bacterial-type DNA replication / replication inhibiting complex / DNA polymerase III complex / replisome / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / DNA polymerase processivity factor activity / error-prone translesion synthesis ...DNA polymerase III, clamp loader complex / Hda-beta clamp complex / bacterial-type DNA replication / replication inhibiting complex / DNA polymerase III complex / replisome / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / DNA polymerase processivity factor activity / error-prone translesion synthesis / negative regulation of DNA-templated DNA replication initiation / 3'-5' exonuclease activity / ribonucleoside triphosphate phosphatase activity / DNA-templated DNA replication / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA damage response / ATP hydrolysis activity / protein homodimerization activity / DNA binding / ATP binding / identical protein binding / cytosol
Similarity search - Function
DNA polymerase III, delta prime subunit / DNA polymerase III, delta subunit, C-terminal / : / DNA polymerase III, delta subunit, C terminal / DNA polymerase III subunit delta', AAA+ ATPase lid domain / DNA polymerase III subunit delta, C-terminal / Processivity clamp loader gamma complex DNA pol III C-term / DNA polymerase III, delta subunit / DNA polymerase III delta, N-terminal / DNA polymerase III, delta subunit ...DNA polymerase III, delta prime subunit / DNA polymerase III, delta subunit, C-terminal / : / DNA polymerase III, delta subunit, C terminal / DNA polymerase III subunit delta', AAA+ ATPase lid domain / DNA polymerase III subunit delta, C-terminal / Processivity clamp loader gamma complex DNA pol III C-term / DNA polymerase III, delta subunit / DNA polymerase III delta, N-terminal / DNA polymerase III, delta subunit / DNA polymerase III, tau subunit, domain V / DNA polymerase III subunit tau, DnaB-binding domain IV / DNA polymerase III, tau subunit, domain V superfamily / DNA polymerase III, subunit gamma/tau, helical lid domain / DNA polymerase III subunits tau domain IV DnaB-binding / DNA polymerase III tau subunit V interacting with alpha / DNA polymerase III, subunit gamma/ tau, N-terminal / DNA polymerase III, gamma subunit, domain III / DNA polymerase III subunits gamma and tau domain III / DNA polymerase III, delta subunit / DNA polymerase III, beta sliding clamp / DNA polymerase III, beta sliding clamp, N-terminal / DNA polymerase III, beta sliding clamp, C-terminal / DNA polymerase III, beta sliding clamp, central / DNA polymerase III beta subunit, N-terminal domain / DNA polymerase III beta subunit, central domain / DNA polymerase III beta subunit, C-terminal domain / DNA polymerase III beta subunit / DNA polymerase III, clamp loader complex, gamma/delta/delta subunit, C-terminal / ClpA/B family / : / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / BERYLLIUM TRIFLUORIDE ION / DNA / DNA (> 10) / RNA / RNA (> 10) / DNA polymerase III subunit tau / Beta sliding clamp / DNA polymerase III subunit delta / DNA polymerase III subunit delta'
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLandeck, J.T. / Kelch, B.A.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM127776 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM145943 United States
CitationJournal: J Biol Chem / Year: 2024
Title: Differences between bacteria and eukaryotes in clamp loader mechanism, a conserved process underlying DNA replication.
Authors: Jacob T Landeck / Joshua Pajak / Emily K Norman / Emma L Sedivy / Brian A Kelch /
Abstract: Clamp loaders are pentameric ATPases that place circular sliding clamps onto DNA, where they function in DNA replication and genome integrity. The central activity of a clamp loader is the opening of ...Clamp loaders are pentameric ATPases that place circular sliding clamps onto DNA, where they function in DNA replication and genome integrity. The central activity of a clamp loader is the opening of the ring-shaped sliding clamp and the subsequent binding to primer-template (p/t)-junctions. The general architecture of clamp loaders is conserved across all life, suggesting that their mechanism is retained. Recent structural studies of the eukaryotic clamp loader replication factor C (RFC) revealed that it functions using a crab-claw mechanism, where clamp opening is coupled to a massive conformational change in the loader. Here we investigate the clamp loading mechanism of the Escherichia coli clamp loader at high resolution using cryo-electron microscopy. We find that the E. coli clamp loader opens the clamp using a crab-claw motion at a single pivot point, whereas the eukaryotic RFC loader uses motions distributed across the complex. Furthermore, we find clamp opening occurs in multiple steps, starting with a partly open state with a spiral conformation, and proceeding to a wide open clamp in a surprising planar geometry. Finally, our structures in the presence of p/t-junctions illustrate how the clamp closes around p/t-junctions and how the clamp loader initiates release from the loaded clamp. Our results reveal mechanistic distinctions in a macromolecular machine that is conserved across all domains of life.
History
DepositionDec 11, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 27, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase III subunit delta
B: DNA polymerase III subunit tau
C: DNA polymerase III subunit tau
D: DNA polymerase III subunit tau
E: DNA polymerase III subunit delta'
F: Beta sliding clamp
G: Beta sliding clamp
H: RNA (5'-R(P*AP*GP*UP*GP*GP*UP*GP*UP*CP*UP*G)-3')
I: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*CP*AP*GP*AP*CP*AP*CP*CP*AP*CP*TP*GP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)297,69622
Polymers295,8819
Non-polymers1,81413
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 polymerase III subunit ... , 3 types, 5 molecules ABCDE

#1: Protein DNA polymerase III subunit delta / DNA polymerase III holoenzyme


Mass: 38745.574 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: holA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P28630
#2: Protein DNA polymerase III subunit tau / DNA polymerase III holoenzyme / DNA polymerase III subunit gamma


Mass: 41803.168 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: dnaX, dnaZ, dnaZX, b0470, JW0459 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P06710, DNA-directed DNA polymerase
#3: Protein DNA polymerase III subunit delta' / DNA polymerase III holoenzyme


Mass: 37272.801 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: holB / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P28631

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Protein / RNA chain / DNA chain , 3 types, 4 molecules FGHI

#4: Protein Beta sliding clamp


Mass: 40922.816 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: dnaN / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A988
#5: RNA chain RNA (5'-R(P*AP*GP*UP*GP*GP*UP*GP*UP*CP*UP*G)-3')


Mass: 3540.122 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*CP*AP*GP*AP*CP*AP*CP*CP*AP*CP*TP*GP*C)-3')


Mass: 9067.865 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 13 molecules

#7: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#9: Chemical ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: BeF3 / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / 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

ComponentName: Structure of the E. coli clamp loader bound to the beta clamp in an Open-RNAp/t conformation
Type: COMPLEX / Entity ID: #1-#6 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2300 nm / Nominal defocus min: 1100 nm
Image recordingElectron dose: 45.2 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 3132
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
8Cootmodel fitting
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3383843
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173962 / Symmetry type: POINT
Atomic model buildingB value: 92.3 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
13GLF

3glf

13GLF1PDBexperimental model
22POL

2pol

12POL2PDBexperimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 66.98 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003919820
ELECTRON MICROSCOPYf_angle_d0.69127038
ELECTRON MICROSCOPYf_chiral_restr0.04023125
ELECTRON MICROSCOPYf_plane_restr0.00483405
ELECTRON MICROSCOPYf_dihedral_angle_d13.09992966

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