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Yorodumi- EMDB-43096: Structure of the E. coli clamp loader bound to the beta clamp in ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-43096 | ||||||||||||
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Title | Structure of the E. coli clamp loader bound to the beta clamp in an Initial-Binding conformation | ||||||||||||
Map data | |||||||||||||
Sample |
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Keywords | Bacterial Clamp Loader Complex / REPLICATION / TRANSFERASE | ||||||||||||
Function / homology | Function and homology information DNA polymerase III, clamp loader complex / DNA clamp loader activity / DNA polymerase III complex / replisome / DNA strand elongation involved in DNA replication / DNA polymerase processivity factor activity / 3'-5' exonuclease activity / ribonucleoside triphosphate phosphatase activity / DNA-templated DNA replication / DNA replication ...DNA polymerase III, clamp loader complex / DNA clamp loader activity / DNA polymerase III complex / replisome / DNA strand elongation involved in DNA replication / DNA polymerase processivity factor activity / 3'-5' exonuclease activity / ribonucleoside triphosphate phosphatase activity / 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 / cytoplasm Similarity search - Function | ||||||||||||
Biological species | Escherichia coli (E. coli) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 7.7 Å | ||||||||||||
Authors | Landeck JT / Pajak J / Kelch BA | ||||||||||||
Funding support | United States, 3 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_43096.map.gz | 91.2 MB | EMDB map data format | |
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Header (meta data) | emd-43096-v30.xml emd-43096.xml | 20.4 KB 20.4 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_43096_fsc.xml | 11.4 KB | Display | FSC data file |
Images | emd_43096.png | 32 KB | ||
Filedesc metadata | emd-43096.cif.gz | 6.9 KB | ||
Others | emd_43096_half_map_1.map.gz emd_43096_half_map_2.map.gz | 95.4 MB 95.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-43096 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-43096 | HTTPS FTP |
-Validation report
Summary document | emd_43096_validation.pdf.gz | 948.3 KB | Display | EMDB validaton report |
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Full document | emd_43096_full_validation.pdf.gz | 947.8 KB | Display | |
Data in XML | emd_43096_validation.xml.gz | 18.4 KB | Display | |
Data in CIF | emd_43096_validation.cif.gz | 23.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-43096 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-43096 | HTTPS FTP |
-Related structure data
Related structure data | 8vanMC 8valC 8vamC 8vapC 8vaqC 8varC 8vasC 8vatC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_43096.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.87 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_43096_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_43096_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Structure of the E. coli clamp loader bound to the beta clamp in ...
Entire | Name: Structure of the E. coli clamp loader bound to the beta clamp in an Initial-Binding conformation |
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Components |
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-Supramolecule #1: Structure of the E. coli clamp loader bound to the beta clamp in ...
Supramolecule | Name: Structure of the E. coli clamp loader bound to the beta clamp in an Initial-Binding conformation type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Escherichia coli (E. coli) |
-Macromolecule #1: DNA polymerase III subunit delta
Macromolecule | Name: DNA polymerase III subunit delta / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 38.745574 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: 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 |
-Macromolecule #2: DNA polymerase III subunit tau
Macromolecule | Name: DNA polymerase III subunit tau / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 41.803168 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: 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 |
-Macromolecule #3: DNA polymerase III subunit delta'
Macromolecule | Name: DNA polymerase III subunit delta' / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 37.272801 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: 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' |
-Macromolecule #4: Beta sliding clamp
Macromolecule | Name: Beta sliding clamp / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 40.922816 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: 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 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 8 |
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Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number real images: 3664 / Average electron dose: 42.8 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 1.1 µm |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |