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- PDB-8gj3: E. coli clamp loader on primed template DNA -

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Basic information

Entry
Database: PDB / ID: 8gj3
TitleE. coli clamp loader on primed template DNA
Components
  • (DNA polymerase III subunit ...) x 4
  • 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 / DNA clamp loader activity / DNA polymerase III complex / replisome / DNA polymerase processivity factor activity / 3'-5' exonuclease activity / ribonucleoside triphosphate phosphatase activity / response to radiation / DNA-templated DNA replication / DNA replication ...DNA polymerase III, clamp loader complex / DNA clamp loader activity / DNA polymerase III complex / replisome / DNA polymerase processivity factor activity / 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 / viral translational frameshifting / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding / metal ion binding
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 subunits tau domain IV DnaB-binding / DNA polymerase III tau subunit V interacting with alpha / DNA polymerase III, subunit gamma/tau, helical lid domain / DNA polymerase III clamp loader subunit, ATPase lid domain / 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, 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 / 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: 2.8 Å
AuthorsOakley, A.J. / Xu, Z.-Q. / Dixon, N.E.
Funding support Australia, 1items
OrganizationGrant numberCountry
Australian Research Council (ARC)DP210100365 Australia
CitationJournal: Nat Commun / Year: 2024
Title: Structural characterisation of the complete cycle of sliding clamp loading in Escherichia coli.
Authors: Zhi-Qiang Xu / Slobodan Jergic / Allen T Y Lo / Alok C Pradhan / Simon H J Brown / James C Bouwer / Harshad Ghodke / Peter J Lewis / Gökhan Tolun / Aaron J Oakley / Nicholas E Dixon /
Abstract: Ring-shaped DNA sliding clamps are essential for DNA replication and genome maintenance. Clamps need to be opened and chaperoned onto DNA by clamp loader complexes (CLCs). Detailed understanding of ...Ring-shaped DNA sliding clamps are essential for DNA replication and genome maintenance. Clamps need to be opened and chaperoned onto DNA by clamp loader complexes (CLCs). Detailed understanding of the mechanisms by which CLCs open and place clamps around DNA remains incomplete. Here, we present a series of six structures of the Escherichia coli CLC bound to an open or closed clamp prior to and after binding to a primer-template DNA, representing the most significant intermediates in the clamp loading process. We show that the ATP-bound CLC first binds to a clamp, then constricts to hold onto it. The CLC then expands to open the clamp with a gap large enough for double-stranded DNA to enter. Upon binding to DNA, the CLC constricts slightly, allowing clamp closing around DNA. These structures provide critical high-resolution snapshots of clamp loading by the E. coli CLC, revealing how the molecular machine works.
History
DepositionMar 14, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 27, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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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
X: Primer
Y: Template
hetero molecules


Theoretical massNumber of molelcules
Total (without water)319,35121
Polymers317,4268
Non-polymers1,92513
Water00
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


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 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'


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


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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DNA chain , 2 types, 2 molecules XY

#5: DNA chain Primer


Mass: 4856.191 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#6: DNA chain Template


Mass: 7969.154 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

#7: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


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


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


Mass: 102.975 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: AlF4
#10: 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
Has protein modificationN

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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 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-#6 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.333 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-HCl1
25 mMMagnesium ChlorideMgCl21
35 mMdithiothreitol1
40.25 mMEDTA1
52 %glycerol1
62 mMADP1
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 FIELD / Nominal defocus max: 1300 nm / Nominal defocus min: 400 nm / 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: 2909
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: 645000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 308000 / 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: 124.83 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00215232
ELECTRON MICROSCOPYf_angle_d0.493620850
ELECTRON MICROSCOPYf_chiral_restr0.03442412
ELECTRON MICROSCOPYf_plane_restr0.00252566
ELECTRON MICROSCOPYf_dihedral_angle_d10.95945555

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