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- PDB-8gj0: E. coli clamp loader with open clamp on primed template DNA (form 1) -

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Basic information

Entry
Database: PDB / ID: 8gj0
TitleE. coli clamp loader with open clamp on primed template DNA (form 1)
Components
  • (DNA polymerase III subunit ...DNA polymerase III holoenzyme) x 4
  • Beta sliding clamp
  • Primer
  • Template
KeywordsDNA BINDING PROTEIN/DNA / clamp loader / DNA clamp / AAA+ / ATPase / DNA BINDING PROTEIN-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 / response to radiation / 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, psi subunit / DNA polymerase III, psi subunit, subgroup / DNA polymerase III, psi subunit superfamily / DNA polymerase III psi 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 ...DNA polymerase III, psi subunit / DNA polymerase III, psi subunit, subgroup / DNA polymerase III, psi subunit superfamily / DNA polymerase III psi 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 / TETRAFLUOROALUMINATE ION / DNA / DNA (> 10) / DNA polymerase III subunit tau / Beta sliding clamp / DNA polymerase III subunit delta / DNA polymerase III subunit delta' / DNA polymerase III subunit psi
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsOakley, A.J. / Xu, Z.-Q. / Dixon, N.E.
Funding support Australia, 1items
OrganizationGrant numberCountry
Australian Research Council (ARC)DP210100365 Australia
CitationJournal: To Be Published
Title: Structural characterisation of the complete cycle of sliding clamp loading in E. coli
Authors: Xu, Z.-Q. / Oakley, A.J. / Dixon, N.E.
History
DepositionMar 14, 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: DNA polymerase III subunit psi
H: Beta sliding clamp
I: Beta sliding clamp
X: Primer
Y: Template
hetero molecules


Theoretical massNumber of molelcules
Total (without water)415,59423
Polymers413,66910
Non-polymers1,92513
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA polymerase III subunit ... , 4 types, 6 molecules ABCDEF

#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 K-12 (bacteria) / Gene: holA, b0640, JW0635 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P28630, DNA-directed DNA polymerase
#2: Protein DNA polymerase III subunit tau / DNA polymerase III holoenzyme / DNA polymerase III subunit gamma


Mass: 71228.648 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: dnaX, dnaZ, dnaZX, b0470, JW0459 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P06710, DNA-directed DNA polymerase
#3: Protein DNA polymerase III subunit delta' / DNA polymerase III holoenzyme


Mass: 36980.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: holB, b1099, JW1085 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P28631, DNA-directed DNA polymerase
#4: Protein DNA polymerase III subunit psi / DNA polymerase III holoenzyme


Mass: 15188.276 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: holD, b4372, JW4334 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P28632, DNA-directed DNA polymerase

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Protein , 1 types, 2 molecules HI

#5: Protein Beta sliding clamp / Beta clamp / Sliding clamp / Beta-clamp processivity factor / DNA polymerase III beta sliding clamp subunit


Mass: 40630.508 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: dnaN, b3701, JW3678 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P0A988

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DNA chain , 2 types, 2 molecules XY

#6: DNA chain Primer


Mass: 7097.624 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#7: DNA chain Template


Mass: 20710.217 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 4 types, 13 molecules

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


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#10: Chemical ChemComp-ALF / TETRAFLUOROALUMINATE ION


Mass: 102.975 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: AlF4
#11: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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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: E. coli clamp loader with open clamp on primed template DNA
Type: COMPLEX
Details: Clamp loader complex composed of DNA polymerase III delta tau(3) delta' chi and psi subunits. Clamp composed of DNA polymerase III beta(2) subunits.
Entity ID: #1-#7 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.419 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 7.6
Details: 30 mM Tris-HCl pH 7.6, 5 mM MgCl2, 2 mM ADP, 0.5 mM AlCl3, 5 mM NaF, 5 mM dithiothreitol, 0.25 mM EDTA, 2% glycerol.
Buffer component
IDConc.NameFormulaBuffer-ID
130 mMTris-HClTris1
23 mMMagnesium ChlorideMgCl21
32 mMdithiothreitol1
40.25 mMEDTAEthylenediaminetetraacetic acid1
52 %glycerol1
62 mMAdenosine disphosphate1
70.5 mMAluminium ChlorideAlCl31
85 mMSodium FluorideNaF1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 30 microL of 6.0 microM delta/tau3/delta' complex was mixed with chi/psi complex, beta, and p/t DNA (annealed DNA oligonucleotides) at a molar ratio of 1:1.3 and dialysed at 4 degrees C ...Details: 30 microL of 6.0 microM delta/tau3/delta' complex was mixed with chi/psi complex, beta, and p/t DNA (annealed DNA oligonucleotides) at a molar ratio of 1:1.3 and dialysed at 4 degrees C against 250 mL of 30 mM Tris-HCl pH 7.6, 5 mM MgCl2, 2 mM ADP, 0.5 mM AlCl3, 5 mM NaF, 5 mM dithiothreitol, 0.25 mM EDTA, 2% glycerol.
Specimen supportDetails: Used a Zepto Low-pressure plasma systems (PLASMA CLEANER) (Diener Electronic) at 10% power for 120 seconds.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 279 K
Details: 3 uL of sample was applied onto a Ultrafoil Au R1.2/1.3 grid. Blot for 4.5 s with no extra force before plunging into liquid ethane.

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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: 1300 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4226
EM imaging opticsEnergyfilter name: GIF Bioquantum / Details: unfiltered mode
Image scansWidth: 4096 / Height: 4096 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

Software
NameVersionClassification
phenix.real_space_refinedev_4788refinement
PHENIXdev_4788refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4RELION3.1CTF correction
7PHENIXdev-4788model fitting
9PHENIXdev-4788model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionDetails: CTF refinement per-particle was performed in RELION before final 3D reconstruction.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1433000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 75000 / Algorithm: BACK PROJECTION / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
13GLH

3glh

13GLH1PDBexperimental model
21MMI

1mmi

11MMI2PDBexperimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 136.43 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221376
ELECTRON MICROSCOPYf_angle_d0.498429239
ELECTRON MICROSCOPYf_chiral_restr0.03663369
ELECTRON MICROSCOPYf_plane_restr0.00263617
ELECTRON MICROSCOPYf_dihedral_angle_d10.42247824

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