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- EMDB-40084: E. coli clamp loader on primed template DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-40084
TitleE. coli clamp loader on primed template DNA
Map dataCLC.DNA
Sample
  • Complex: E. coli clamp loader on primed template DNA
    • Protein or peptide: DNA polymerase III subunit delta
    • Protein or peptide: DNA polymerase III subunit tau
    • Protein or peptide: DNA polymerase III subunit delta'
    • Protein or peptide: DNA polymerase III subunit psi
    • DNA: Primer
    • DNA: Template
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: ZINC ION
Keywordsclamp 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-directed DNA polymerase ...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-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA replication / viral translational frameshifting / ATP hydrolysis activity / DNA binding / ATP binding / metal ion binding / identical protein 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
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 (E. coli) / Escherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsOakley AJ / Xu Z-Q / Dixon NE
Funding support Australia, 1 items
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, 2023-
Header (metadata) releaseMar 27, 2024-
Map releaseMar 27, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40084.png

Downloads & links

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Map

FileDownload / File: emd_40084.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCLC.DNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 280 pix.
= 235.2 Å		0.84 Å/pix.
x 280 pix.
= 235.2 Å		0.84 Å/pix.
x 280 pix.
= 235.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00484
Minimum - Maximum-0.020192524 - 0.05742476
Average (Standard dev.)-0.00001895102 (±0.0014820573)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 235.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: CLC.DNA half map 1

Fileemd_40084_half_map_1.map
AnnotationCLC.DNA half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CLC.DNA half map 2

Fileemd_40084_half_map_2.map
AnnotationCLC.DNA half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : E. coli clamp loader on primed template DNA

EntireName: E. coli clamp loader on primed template DNA
Components
  • Complex: E. coli clamp loader on primed template DNA
    • Protein or peptide: DNA polymerase III subunit delta
    • Protein or peptide: DNA polymerase III subunit tau
    • Protein or peptide: DNA polymerase III subunit delta'
    • Protein or peptide: DNA polymerase III subunit psi
    • DNA: Primer
    • DNA: Template
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: ZINC ION

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Supramolecule #1: E. coli clamp loader on primed template DNA

SupramoleculeName: E. coli clamp loader on primed template DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
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.
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12
Molecular weightTheoretical: 333 KDa

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Macromolecule #1: DNA polymerase III subunit delta

MacromoleculeName: DNA polymerase III subunit delta / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 38.745574 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MIRLYPEQLR AQLNEGLRAA YLLLGNDPLL LQESQDAVRQ VAAAQGFEEH HTFSIDPNTD WNAIFSLCQA MSLFASRQTL LLLLPENGP NAAINEQLLT LTGLLHDDLL LIVRGNKLSK AQENAAWFTA LANRSVQVTC QTPEQAQLPR WVAARAKQLN L ELDDAANQ ...String:
MIRLYPEQLR AQLNEGLRAA YLLLGNDPLL LQESQDAVRQ VAAAQGFEEH HTFSIDPNTD WNAIFSLCQA MSLFASRQTL LLLLPENGP NAAINEQLLT LTGLLHDDLL LIVRGNKLSK AQENAAWFTA LANRSVQVTC QTPEQAQLPR WVAARAKQLN L ELDDAANQ VLCYCYEGNL LALAQALERL SLLWPDGKLT LPRVEQAVND AAHFTPFHWV DALLMGKSKR ALHILQQLRL EG SEPVILL RTLQRELLLL VNLKRQSAHT PLRALFDKHR VWQNRRGMMG EALNRLSQTQ LRQAVQLLTR TELTLKQDYG QSV WAELEG LSLLLCHKPL ADVFIDG

UniProtKB: DNA polymerase III subunit delta

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Macromolecule #2: DNA polymerase III subunit tau

MacromoleculeName: DNA polymerase III subunit tau / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 71.228648 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MSYQVLARKW RPQTFADVVG QEHVLTALAN GLSLGRIHHA YLFSGTRGVG KTSIARLLAK GLNCETGITA TPCGVCDNCR EIEQGRFVD LIEIDAASRT KVEDTRDLLD NVQYAPARGR FKVYLIDEVH MLSRHSFNAL LKTLEEPPEH VKFLLATTDP Q KLPVTILS ...String:
MSYQVLARKW RPQTFADVVG QEHVLTALAN GLSLGRIHHA YLFSGTRGVG KTSIARLLAK GLNCETGITA TPCGVCDNCR EIEQGRFVD LIEIDAASRT KVEDTRDLLD NVQYAPARGR FKVYLIDEVH MLSRHSFNAL LKTLEEPPEH VKFLLATTDP Q KLPVTILS RCLQFHLKAL DVEQIRHQLE HILNEEHIAH EPRALQLLAR AAEGSLRDAL SLTDQAIASG DGQVSTQAVS AM LGTLDDD QALSLVEAMV EANGERVMAL INEAAARGIE WEALLVEMLG LLHRIAMVQL SPAALGNDMA AIELRMRELA RTI PPTDIQ LYYQTLLIGR KELPYAPDRR MGVEMTLLRA LAFHPRMPLP EPEVPRQSFA PVAPTAVMTP TQVPPQPQSA PQQA PTVPL PETTSQVLAA RQQLQRVQGA TKAKKSEPAA ATRARPVNNA ALERLASVTD RVQARPVPSA LEKAPAKKEA YRWKA TTPV MQQKEVVATP KALKKALEHE KTPELAAKLA AEAIERDPWA AQVSQLSLPK LVEQVALNAW KEESDNAVCL HLRSSQ RHL NNRGAQQKLA EALSMLKGST VELTIVEDDN PAVRTPLEWR QAIYEEKLAQ ARESIIADNN IQTLRRFFDA ELDEESI RP I

UniProtKB: DNA polymerase III subunit tau

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Macromolecule #3: DNA polymerase III subunit delta'

MacromoleculeName: DNA polymerase III subunit delta' / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 36.980484 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MRWYPWLRPD FEKLVASYQA GRGHHALLIQ ALPGMGDDAL IYALSRYLLC QQPQGHKSCG HCRGCQLMQA GTHPDYYTLA PEKGKNTLG VDAVREVTEK LNEHARLGGA KVVWVTDAAL LTDAAANALL KTLEEPPAET WFFLATREPE RLLATLRSRC R LHYLAPPP ...String:
MRWYPWLRPD FEKLVASYQA GRGHHALLIQ ALPGMGDDAL IYALSRYLLC QQPQGHKSCG HCRGCQLMQA GTHPDYYTLA PEKGKNTLG VDAVREVTEK LNEHARLGGA KVVWVTDAAL LTDAAANALL KTLEEPPAET WFFLATREPE RLLATLRSRC R LHYLAPPP EQYAVTWLSR EVTMSQDALL AALRLSAGSP GAALALFQGD NWQARETLCQ ALAYSVPSGD WYSLLAALNH EQ APARLHW LATLLMDALK RHHGAAQVTN VDVPGLVAEL ANHLSPSRLQ AILGDVCHIR EQLMSVTGIN RELLITDLLL RIE HYLQPG VVLPVPHL

UniProtKB: DNA polymerase III subunit delta'

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Macromolecule #4: DNA polymerase III subunit psi

MacromoleculeName: DNA polymerase III subunit psi / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 15.188276 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
MTSRRDWQLQ QLGITQWSLR RPGALQGEIA IAIPAHVRLV MVANDLPALT DPLVSDVLRA LTVSPDQVLQ LTPEKIAMLP QGSHCNSWR LGTDEPLSLE GAQVASPALT DLRANPTARA ALWQQICTYE HDFFPRND

UniProtKB: DNA polymerase III subunit psi

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Macromolecule #5: Primer

MacromoleculeName: Primer / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 4.856191 KDa
SequenceString:
(DT)(DT)(DA)(DG)(DT)(DT)(DA)(DC)(DA)(DA) (DC)(DA)(DT)(DA)(DC)(DT)

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Macromolecule #6: Template

MacromoleculeName: Template / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 7.969154 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DA)(DG)(DT)(DA)(DT)(DG)(DT)(DT)(DG) (DT)(DA)(DA)(DC)(DT)(DA)

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Macromolecule #7: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 3 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #9: TETRAFLUOROALUMINATE ION

MacromoleculeName: TETRAFLUOROALUMINATE ION / type: ligand / ID: 9 / Number of copies: 3 / Formula: ALF
Molecular weightTheoretical: 102.975 Da
Chemical component information

ChemComp-ALF:
TETRAFLUOROALUMINATE ION

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Macromolecule #10: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 10 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
Component:
ConcentrationNameFormula
30.0 mMTris-HCl
5.0 mMMagnesium ChlorideMgCl2
5.0 mMdithiothreitol
0.25 mMEDTA
2.0 %glycerol
2.0 mMADP
0.5 mMAluminium ChlorideAlCl3
5.0 mMSodium FluorideNaF

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.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa
Details: Used a Zepto Low-pressure plasma systems (PLASMA CLEANER) (Diener Electronic) at 10% power for 120 seconds.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV
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..
Details30 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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Details: unfiltered mode
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 1 / Number real images: 2909 / Average exposure time: 6.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.3 µm / Nominal defocus min: 0.4 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 645000
Startup modelType of model: OTHER / Details: Ab-initio model from RELION
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 308000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 4 / Avg.num./class: 120000 / Software - Name: RELION (ver. 3.1)
Details: Most particles were classified to two classes that were used for final 3D reconstruction.

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Atomic model buiding 1

Initial model
PDB IDChain

3glh

source_name: PDB, initial_model_type: experimental model

1mmi

source_name: PDB, initial_model_type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

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

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