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- EMDB-43102: Structure of the E. coli clamp loader bound to the beta clamp in ... -

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Entry
Database: EMDB / ID: EMD-43102
TitleStructure of the E. coli clamp loader bound to the beta clamp in a Open-RNAp/t conformation
Map data
Sample
  • Complex: Structure of the E. coli clamp loader bound to the beta clamp in an Open-RNAp/t conformation
    • Protein or peptide: DNA polymerase III subunit deltaDNA polymerase III holoenzyme
    • Protein or peptide: DNA polymerase III subunit tauDNA polymerase III holoenzyme
    • Protein or peptide: DNA polymerase III subunit delta'DNA polymerase III holoenzyme
    • Protein or peptide: Beta sliding clamp
    • RNA: RNA (5'-R(P*AP*GP*UP*GP*GP*UP*GP*UP*CP*UP*G)-3')
    • DNA: 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')
  • Ligand: ZINC ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: MAGNESIUM ION
KeywordsBacterial Clamp Loader Complex / REPLICATION / 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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLandeck JT / Kelch BA
Funding support United States, 2 items
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, 2023-
Header (metadata) releaseMar 27, 2024-
Map releaseMar 27, 2024-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43102.png

Downloads & links

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Map

FileDownload / File: emd_43102.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.88 Å
Density
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-1.0738153 - 1.4430507
Average (Standard dev.)0.00037868813 (±0.036763333)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 264.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43102_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43102_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of the E. coli clamp loader bound to the beta clamp in ...

EntireName: Structure of the E. coli clamp loader bound to the beta clamp in an Open-RNAp/t conformation
Components
  • Complex: Structure of the E. coli clamp loader bound to the beta clamp in an Open-RNAp/t conformation
    • Protein or peptide: DNA polymerase III subunit deltaDNA polymerase III holoenzyme
    • Protein or peptide: DNA polymerase III subunit tauDNA polymerase III holoenzyme
    • Protein or peptide: DNA polymerase III subunit delta'DNA polymerase III holoenzyme
    • Protein or peptide: Beta sliding clamp
    • RNA: RNA (5'-R(P*AP*GP*UP*GP*GP*UP*GP*UP*CP*UP*G)-3')
    • DNA: 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')
  • Ligand: ZINC ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: BERYLLIUM TRIFLUORIDE ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Structure of the E. coli clamp loader bound to the beta clamp in ...

SupramoleculeName: Structure of the E. coli clamp loader bound to the beta clamp in an Open-RNAp/t conformation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Escherichia coli (E. coli)

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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
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 38.745574 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (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 (E. coli)
Molecular weightTheoretical: 41.803168 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPHMSYQVLA RKWRPQTFAD VVGQEHVLTA LANGLSLGRI HHAYLFSGTR GVGKTSIARL LAKGLNCETG ITATPCGVCD NCREIEQGR FVDLIEIDAA SRTKVEDTRD LLDNVQYAPA RGRFKVYLID EVHMLSRHSF NALLKTLEEP PEHVKFLLAT T DPQKLPVT ...String:
GPHMSYQVLA RKWRPQTFAD VVGQEHVLTA LANGLSLGRI HHAYLFSGTR GVGKTSIARL LAKGLNCETG ITATPCGVCD NCREIEQGR FVDLIEIDAA SRTKVEDTRD LLDNVQYAPA RGRFKVYLID EVHMLSRHSF NALLKTLEEP PEHVKFLLAT T DPQKLPVT ILSRCLQFHL KALDVEQIRH QLEHILNEEH IAHEPRALQL LARAAEGSLR DALSLTDQAI ASGDGQVSTQ AV SAMLGTL DDDQALSLVE AMVEANGERV MALINEAAAR GIEWEALLVE MLGLLHRIAM VQLSPAALGN DMAAIELRMR ELA RTIPPT DIQLYYQTLL IGRKELPYAP DRRMGVEMTL LRALAFHPRM PLPEPEVPRQ

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
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 37.272801 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPHMRWYPWL RPDFEKLVAS YQAGRGHHAL LIQALPGMGD DALIYALSRY LLCQQPQGHK SCGHCRGCQL MQAGTHPDYY TLAPEKGKN TLGVDAVREV TEKLNEHARL GGAKVVWVTD AALLTDAAAN ALLKTLEEPP AETWFFLATR EPERLLATLR S RCRLHYLA ...String:
GPHMRWYPWL RPDFEKLVAS YQAGRGHHAL LIQALPGMGD DALIYALSRY LLCQQPQGHK SCGHCRGCQL MQAGTHPDYY TLAPEKGKN TLGVDAVREV TEKLNEHARL GGAKVVWVTD AALLTDAAAN ALLKTLEEPP AETWFFLATR EPERLLATLR S RCRLHYLA PPPEQYAVTW LSREVTMSQD ALLAALRLSA GSPGAALALF QGDNWQARET LCQALAYSVP SGDWYSLLAA LN HEQAPAR LHWLATLLMD ALKRHHGAAQ VTNVDVPGLV AELANHLSPS RLQAILGDVC HIREQLMSVT GINRELLITD LLL RIEHYL QPGVVLPVPH L

UniProtKB: DNA polymerase III subunit delta'

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Macromolecule #4: Beta sliding clamp

MacromoleculeName: Beta sliding clamp / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 40.922816 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPHMKFTVER EHLLKPLQQV SGPLGGRPTL PILGNLLLQV ADGTLSLTGT DLEMEMVARV ALVQPHEPGA TTVPARKFFD ICRGLPEGA EIAVQLEGER MLVRSGRSRF SLSTLPAADF PNLDDWQSEV EFTLPQATMK RLIEATQFSM AHQDVRYYLN G MLFETEGE ...String:
GPHMKFTVER EHLLKPLQQV SGPLGGRPTL PILGNLLLQV ADGTLSLTGT DLEMEMVARV ALVQPHEPGA TTVPARKFFD ICRGLPEGA EIAVQLEGER MLVRSGRSRF SLSTLPAADF PNLDDWQSEV EFTLPQATMK RLIEATQFSM AHQDVRYYLN G MLFETEGE ELRTVATDGH RLAVCSMPIG QSLPSHSVIV PRKGVIELMR MLDGGDNPLR VQIGSNNIRA HVGDFIFTSK LV DGRFPDY RRVLPKNPDK HLEAGCDLLK QAFARAAILS NEKFRGVRLY VSENQLKITA NNPEQEEAEE ILDVTYSGAE MEI GFNVSY VLDVLNALKC ENVRMMLTDS VSSVQIEDAA SQSAAYVVMP MRL

UniProtKB: Beta sliding clamp

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Macromolecule #5: RNA (5'-R(P*AP*GP*UP*GP*GP*UP*GP*UP*CP*UP*G)-3')

MacromoleculeName: RNA (5'-R(P*AP*GP*UP*GP*GP*UP*GP*UP*CP*UP*G)-3') / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.540122 KDa
SequenceString:
AGUGGUGUCU G

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Macromolecule #6: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*CP*AP*GP*AP*CP*AP*CP*CP*AP*CP*TP...

MacromoleculeName: 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')
type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.067865 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DC)(DA)(DG)(DA)(DC)(DA)(DC)(DC)(DA)(DC) (DT)(DG)(DC)(DA)(DC)(DA)(DC)(DA)(DC) (DA)

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #8: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 3 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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Macromolecule #9: BERYLLIUM TRIFLUORIDE ION

MacromoleculeName: BERYLLIUM TRIFLUORIDE ION / type: ligand / ID: 9 / Number of copies: 3 / Formula: BEF
Molecular weightTheoretical: 66.007 Da
Chemical component information

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

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Macromolecule #10: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 10 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 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: 8
GridModel: UltrAuFoil R2/2 / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 1.1 µm
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 3132 / Average electron dose: 45.2 e/Å2

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Image processing

Particle selectionNumber selected: 3383843
Startup modelType of model: INSILICO MODEL / In silico model: generate an ab-initio in cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 173962
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

3glf

source_name: PDB, initial_model_type: experimental model

2pol

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 92.3
Output model

PDB-8vat:
Structure of the E. coli clamp loader bound to the beta clamp in a Open-RNAp/t conformation

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