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- EMDB-40079: E. coli clamp loader with closed clamp -

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Basic information

Entry
Database: EMDB / ID: EMD-40079
TitleE. coli clamp loader with closed clamp
Map dataCLC.Beta2-Closed
Sample
  • Complex: E. coli clamp loader with closed clamp
    • 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
    • Protein or peptide: Beta sliding clamp
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM 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 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 / ribosome / structural constituent of ribosome / ribonucleoprotein complex / translation / 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 / Ribosomal protein L34, conserved site / Ribosomal protein L34 signature. / Ribosomal protein L34 / Ribosomal protein L34 / 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 / Large ribosomal subunit protein bL34 / 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: 3.7 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_40079.png

Downloads & links

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Map

FileDownload / File: emd_40079.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCLC.Beta2-Closed
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 360 pix.
= 295.2 Å		0.82 Å/pix.
x 360 pix.
= 295.2 Å		0.82 Å/pix.
x 360 pix.
= 295.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0037
Minimum - Maximum-0.009739092 - 0.020211676
Average (Standard dev.)0.0000070083724 (±0.00076375547)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 295.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: CLC.Beta2-Closed half map 1

Fileemd_40079_half_map_1.map
AnnotationCLC.Beta2-Closed half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CLC.Beta2-Closed half map 2

Fileemd_40079_half_map_2.map
AnnotationCLC.Beta2-Closed 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 with closed clamp

EntireName: E. coli clamp loader with closed clamp
Components
  • Complex: E. coli clamp loader with closed clamp
    • 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
    • Protein or peptide: Beta sliding clamp
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: E. coli clamp loader with closed clamp

SupramoleculeName: E. coli clamp loader with closed clamp / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Details: Clamp loader complex composed of DNA polymerase III delta gamma(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: 331 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: 47.601766 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 TKAKKE

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: Beta sliding clamp

MacromoleculeName: Beta sliding clamp / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 40.630508 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MKFTVEREHL LKPLQQVSGP LGGRPTLPIL GNLLLQVADG TLSLTGTDLE MEMVARVALV QPHEPGATTV PARKFFDICR GLPEGAEIA VQLEGERMLV RSGRSRFSLS TLPAADFPNL DDWQSEVEFT LPQATMKRLI EATQFSMAHQ DVRYYLNGML F ETEGEELR ...String:
MKFTVEREHL LKPLQQVSGP LGGRPTLPIL GNLLLQVADG TLSLTGTDLE MEMVARVALV QPHEPGATTV PARKFFDICR GLPEGAEIA VQLEGERMLV RSGRSRFSLS TLPAADFPNL DDWQSEVEFT LPQATMKRLI EATQFSMAHQ DVRYYLNGML F ETEGEELR TVATDGHRLA VCSMPIGQSL PSHSVIVPRK GVIELMRMLD GGDNPLRVQI GSNNIRAHVG DFIFTSKLVD GR FPDYRRV LPKNPDKHLE AGCDLLKQAF ARAAILSNEK FRGVRLYVSE NQLKITANNP EQEEAEEILD VTYSGAEMEI GFN VSYVLD VLNALKCENV RMMLTDSVSS VQIEDAASQS AAYVVMPMRL

UniProtKB: Large ribosomal subunit protein bL34

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Macromolecule #6: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 6 / Number of copies: 3 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / 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: 7.5
Component:
ConcentrationNameFormula
30.0 mMHEPES
3.0 mMMagnesium ChlorideMgCl2
2.0 mMdithiothreitol
0.25 mMEDTA
2.0 %glycerol
1.0 mMATPgammaS

Details: 30 mM Na.HEPES pH 7.5, 3 mM MgCl2, 2 mM dithiothreitol, 0.25 mM EDTA, 2% glycerol, 1 mM ATPgammaS.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa
Details: Use 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
Details30 microL of 6 microM delta/gamma3/delta' mixed with chi/psi complex at a molar ratio of 1:1.2, beta2 at 1:1.3, and dialysed twice at 4 degrees C against 250 mL of 30 mM Na.HEPES pH 7.5, 3 mM MgCl2, 2 mM dithiothreitol, 0.25 mM EDTA, 2% glycerol. 1 mM ATPgammaS was added to the dialysate.

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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: 7269 / Average exposure time: 6.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.3 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 165000
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: 1376000
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: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 373000
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-8giy:
E. coli clamp loader with closed clamp

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