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Yorodumi- EMDB-20446: Cryo-EM structure of AdnA(D934A)-AdnB(D1014A) in complex with AMP... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-20446 | |||||||||
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Title | Cryo-EM structure of AdnA(D934A)-AdnB(D1014A) in complex with AMPPNP and DNA | |||||||||
Map data | AdnAB-D934A-D1014A mutant in complex with AMPPNP and DNA | |||||||||
Sample |
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Keywords | DNA / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex | |||||||||
Function / homology | Function and homology information DNA 3'-5' helicase / exonuclease activity / DNA helicase activity / isomerase activity / DNA helicase / hydrolase activity / DNA repair / DNA binding / ATP binding Similarity search - Function | |||||||||
Biological species | Mycolicibacterium smegmatis (bacteria) / Bacillus subtilis subsp. subtilis str. 168 (bacteria) / Mycobacterium smegmatis (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.5 Å | |||||||||
Authors | Jia N / Unciuleac M / Shuman S / Patel DJ | |||||||||
Citation | Journal: Proc Natl Acad Sci U S A / Year: 2019 Title: Structures and single-molecule analysis of bacterial motor nuclease AdnAB illuminate the mechanism of DNA double-strand break resection. Authors: Ning Jia / Mihaela C Unciuleac / Chaoyou Xue / Eric C Greene / Dinshaw J Patel / Stewart Shuman / Abstract: Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N- ...Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Here we report cryoelectron microscopy (cryo-EM) structures of AdnAB in three functional states: in the absence of DNA and in complex with forked duplex DNAs before and after cleavage of the 5' single-strand DNA (ssDNA) tail by the AdnA nuclease. The structures reveal the path of the 5' ssDNA through the AdnA nuclease domain and the mechanism of 5' strand cleavage; the path of the 3' tracking strand through the AdnB motor and the DNA contacts that couple ATP hydrolysis to mechanical work; the position of the AdnA iron-sulfur cluster subdomain at the Y junction and its likely role in maintaining the split trajectories of the unwound 5' and 3' strands. Single-molecule DNA curtain analysis of DSB resection reveals that AdnAB is highly processive but prone to spontaneous pausing at random sites on duplex DNA. A striking property of AdnAB is that the velocity of DSB resection slows after the enzyme experiences a spontaneous pause. Our results highlight shared as well as distinctive properties of AdnAB vis-à-vis the RecBCD and AddAB clades of bacterial DSB-resecting motor nucleases. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_20446.map.gz | 7.2 MB | EMDB map data format | |
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Header (meta data) | emd-20446-v30.xml emd-20446.xml | 16.1 KB 16.1 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_20446_fsc.xml | 10 KB | Display | FSC data file |
Images | emd_20446.png | 58.2 KB | ||
Filedesc metadata | emd-20446.cif.gz | 6.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-20446 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-20446 | HTTPS FTP |
-Validation report
Summary document | emd_20446_validation.pdf.gz | 407 KB | Display | EMDB validaton report |
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Full document | emd_20446_full_validation.pdf.gz | 406.6 KB | Display | |
Data in XML | emd_20446_validation.xml.gz | 11.1 KB | Display | |
Data in CIF | emd_20446_validation.cif.gz | 14.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-20446 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-20446 | HTTPS FTP |
-Related structure data
Related structure data | 6pprMC 6ppjC 6ppuC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_20446.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | AdnAB-D934A-D1014A mutant in complex with AMPPNP and DNA | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.8613 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : AdnAB-D934A-D1014A mutant in complex with AMPPNP and DNA
Entire | Name: AdnAB-D934A-D1014A mutant in complex with AMPPNP and DNA |
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Components |
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-Supramolecule #1: AdnAB-D934A-D1014A mutant in complex with AMPPNP and DNA
Supramolecule | Name: AdnAB-D934A-D1014A mutant in complex with AMPPNP and DNA type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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Molecular weight | Theoretical: 200 KDa |
-Supramolecule #2: UvrD/REP helicase
Supramolecule | Name: UvrD/REP helicase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: Mycolicibacterium smegmatis (bacteria) |
-Supramolecule #3: ATP-dependent DNA helicase (UvrD/REP)
Supramolecule | Name: ATP-dependent DNA helicase (UvrD/REP) / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
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Source (natural) | Organism: Mycolicibacterium smegmatis (bacteria) |
-Supramolecule #4: DNA
Supramolecule | Name: DNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3 |
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Source (natural) | Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria) |
-Macromolecule #1: UvrD/REP helicase
Macromolecule | Name: UvrD/REP helicase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase |
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Source (natural) | Organism: Mycobacterium smegmatis (bacteria) |
Molecular weight | Theoretical: 118.084531 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MTQVASPVVQ ARYSPVELSA ALGLFPPTDE QAAVIAAPPG PLVVIAGAGA GKTETMAARV VWLVANGFAT PSQVLGLTFT RKAAGQLLR RVRTRLARLA GAGLAPGSGA SDESATVSTY HAFAGTLLRE HGLLLPVEPD TRLLSETELW QLAYDVVCAH P GHLDTEKT ...String: MTQVASPVVQ ARYSPVELSA ALGLFPPTDE QAAVIAAPPG PLVVIAGAGA GKTETMAARV VWLVANGFAT PSQVLGLTFT RKAAGQLLR RVRTRLARLA GAGLAPGSGA SDESATVSTY HAFAGTLLRE HGLLLPVEPD TRLLSETELW QLAYDVVCAH P GHLDTEKT PAAVTAMVLR LSGALAEHLV DTDQLRDTHV ELERLVHTLP AGPYQRDRGP SQWLLRMLAT QTERTELVPL ID ALHQRMR AEKVMDFGMQ MAAAARLAAR FPQVGEQLRQ RFRVVLLDEY QDTGHAQRIA LSSLFGGGAD DGLALTAVGD PIQ SIYGWR GASATNLPRF TTDFPYSDGT PAPTLELRTS WRNPPSTLHV ANAVSEEARR RSVAVRALRP RPDAEPGTIR CALL NNVAA ERDWVADHLA RAYHGAIGRG EAAPTAAVLV RRNADAAPMA EALTARGVPV EVVGVAGLLA VPEVADLVAM LRLIA DPTA GSAVMRILTG PRWRFGARDI AALWRRAVEL DDRPKGELGT ADIVAQAAPD ADTACVADAI CDPGDAERYS PAGYER IVA LGRELTMLRA HLGHPLPELV AEVRRVLGLD AEARAARPVA AGWAGTENLD RFSDLVSDFA GHAGASVSAL LAYLDAA VE VENGLAPAEL TVSHDRVQIL TVHAAKGLEW QVVAVPHLSA RVFPSTTQAR TWLTDASDLP PLLRGDRATE SEIGVPVL D TSDIYDRKIL SDKISDHKKS LDQRRVDEER RLLYVAITRA EDTLLLSGHH WGATESKPRG PSEFLCELKT ILEEATAAG TPCGEIEHWA PDPAPGETNP LRDQVVEALW PPVASADDHV HRGAQLVAAA MAGEVSAEAD QEGWAADVDA LLAERERPPQ QEDTELPGQ LSVSTLVELS RDPKAALTRL RRRLPQRPDP HALLGTTFHE WVQRYFHAER LFDLDDLPGA VDSDSGRAVE E SLAELQDA FVKSPWAART PVEVEVPFDM VLGETVVRGR IDAVFAEPDG TTMVLAWKTG DPPETPEAKE HAAVQLAVYR LA WAAMRGC PPESVRAAFH YVRSGQTVIP ETLPGAEELV KLLAAAPTET AEEADRIT UniProtKB: DNA helicase |
-Macromolecule #2: ATP-dependent DNA helicase (UvrD/REP)
Macromolecule | Name: ATP-dependent DNA helicase (UvrD/REP) / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase |
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Source (natural) | Organism: Mycobacterium smegmatis (bacteria) |
Molecular weight | Theoretical: 110.899562 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MTTRPAESAP QTASTLLEPG SNGVVRLLGG PGTGKSSLLV DTAVQHILAG ADPESVLLLT GSARLRTAAR AAITARLLGA GTVGVVREP LVRTVHSYAF AVLRLAAQRN GDPPPRLITS AEQDGIIREL LAGDLEDGHR SPVGWPEQLW PALTTAGFAT E LRDLMARC ...String: MTTRPAESAP QTASTLLEPG SNGVVRLLGG PGTGKSSLLV DTAVQHILAG ADPESVLLLT GSARLRTAAR AAITARLLGA GTVGVVREP LVRTVHSYAF AVLRLAAQRN GDPPPRLITS AEQDGIIREL LAGDLEDGHR SPVGWPEQLW PALTTAGFAT E LRDLMARC TERGVDPIAL QRLGRTAKRP EWLAAGRFAQ AYEQIMLLRS AVGMAAPQAT VPALGAAELV GAALEALGAD DE LLDTERN RIKLLLVDDA QHLDPQAARL VRALAAGTGL TVIAGDPDQS VFGYRGADPV LLRDDTHPAI TLTQSYRCAP EIA SAITGL GQRLPGVSDT RHWTGNPQRE GTVTVRLAAS THAEGTMIAD ALRRAHLVDG IPWSQMAVIV RSVPRVGTAL ARAL TAAGV PVQDNGTDVP VGRQPAAAAL LTVLDVTATG HLDADSAVAL LTGPIGRVDP VTLRQLRRAL RRADGSQPPR DFGDL LVDA IEREPKGLSA EHARTLRRLR AVLTAARRSD ASGADPRYTL WQAWHASGLQ RRWLAASERG GSVGAQADRD LDAVTT LFD VADQYVNRTA GASLRGLVDH VTRLGAAVAR TEPETAAEAV AVLSVHGALA GEWDFVVIAG VQEGLWPNMI PRGGVLG TQ HLVDVLDGVA DMTDRTVSTR APLVAEERRL LMAAMGRART RVMITAVDSD TGDESLLPSP FCAEISAWAT EPVAEPPL V APRVLAPSAL VGRLRAVVCA PDGAVDDDAR ACAAAQLARL AAAGVPGADP SQWHAMTSLT TEEPLWSEPG HVVTLSPST LQMLTDCPLR WLLERHGGDD GRDVRSTVGS LVHALVSEPG KTESQLVNEL EKVWDDLPYD AKWYSDNELA RHRAMLETFT RWREDTRRQ LTEVATEIPV EGIVVEPGEN TPGVRVRGRL DRLERDEAGR LVVVALKTGK SPVTKDDAQN HAQLAMYQLA V AAGLLDDG DEPGGGKLVY LGKAGAAGAT EREQDPLTPD KRAEWLETVG EAAAATAGPR FVARVNNGCA NCPVRSSCPA QA NGDRP UniProtKB: DNA helicase |
-Macromolecule #3: DNA (70-MER)
Macromolecule | Name: DNA (70-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Mycolicibacterium smegmatis (bacteria) |
Molecular weight | Theoretical: 21.477703 KDa |
Sequence | String: (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DC)(DT)(DA) (DA)(DT)(DG)(DC)(DG)(DA)(DG)(DC)(DA)(DC) (DT)(DG)(DC)(DT)(DA)(DT)(DT)(DC)(DC) (DC)(DT)(DA)(DG)(DC)(DA)(DG)(DT)(DG)(DC) (DT) (DC)(DG)(DC)(DA)(DT)(DT) ...String: (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DC)(DT)(DA) (DA)(DT)(DG)(DC)(DG)(DA)(DG)(DC)(DA)(DC) (DT)(DG)(DC)(DT)(DA)(DT)(DT)(DC)(DC) (DC)(DT)(DA)(DG)(DC)(DA)(DG)(DT)(DG)(DC) (DT) (DC)(DG)(DC)(DA)(DT)(DT)(DA)(DG) (DA)(DT)(DT)(DT)(DT)(DG)(DT)(DT)(DT)(DT) (DT)(DT) (DT)(DA)(DG)(DC)(DG)(DG)(DT) (DT)(DT)(DT) |
-Macromolecule #4: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 1 / Formula: ANP |
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Molecular weight | Theoretical: 506.196 Da |
Chemical component information | ChemComp-ANP: |
-Macromolecule #5: IRON/SULFUR CLUSTER
Macromolecule | Name: IRON/SULFUR CLUSTER / type: ligand / ID: 5 / Number of copies: 1 / Formula: SF4 |
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Molecular weight | Theoretical: 351.64 Da |
Chemical component information | ChemComp-FS1: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.5 mg/mL |
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Buffer | pH: 7.5 / Component - Formula: Tris / Details: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 2.16 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |