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Yorodumi- EMDB-4038: Cryo-EM structure of RecBCD+DNA complex revealing activated nucle... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-4038 | |||||||||
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Title | Cryo-EM structure of RecBCD+DNA complex revealing activated nuclease domain | |||||||||
Map data | ||||||||||
Sample |
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Keywords | Helicase / Nuclease / SH3 / Homologous Recombination / hydrolase | |||||||||
Function / homology | Function and homology information exodeoxyribonuclease V / exodeoxyribonuclease V activity / exodeoxyribonuclease V complex / clearance of foreign intracellular DNA / DNA translocase activity / DNA 5'-3' helicase / single-stranded DNA helicase activity / DNA 3'-5' helicase / recombinational repair / 3'-5' DNA helicase activity ...exodeoxyribonuclease V / exodeoxyribonuclease V activity / exodeoxyribonuclease V complex / clearance of foreign intracellular DNA / DNA translocase activity / DNA 5'-3' helicase / single-stranded DNA helicase activity / DNA 3'-5' helicase / recombinational repair / 3'-5' DNA helicase activity / ATP-dependent activity, acting on DNA / DNA helicase activity / isomerase activity / DNA endonuclease activity / helicase activity / double-strand break repair via homologous recombination / response to radiation / 5'-3' DNA helicase activity / DNA recombination / DNA damage response / magnesium ion binding / ATP hydrolysis activity / DNA binding / ATP binding / cytosol Similarity search - Function | |||||||||
Biological species | Escherichia coli K12 (bacteria) / Escherichia coli (strain K12) (bacteria) / Endothia gyrosa (fungus) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.83 Å | |||||||||
Authors | Wilkinson M / Chaban Y | |||||||||
Citation | Journal: Elife / Year: 2016 Title: Mechanism for nuclease regulation in RecBCD. Authors: Martin Wilkinson / Yuriy Chaban / Dale B Wigley / Abstract: In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly ...In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly processive, duplex unwinding and degrading machines that require tight regulation. Here, we report the structure of E.coli RecBCD, determined by cryoEM at 3.8 Å resolution, with a DNA substrate that reveals how the nuclease activity of the complex is activated once unwinding progresses. Extension of the 5'-tail of the unwound duplex induces a large conformational change in the RecD subunit, that is transferred through the RecC subunit to activate the nuclease domain of the RecB subunit. The process involves a SH3 domain that binds to a region of the RecB subunit in a binding mode that is distinct from others observed previously in SH3 domains and, to our knowledge, this is the first example of peptide-binding of an SH3 domain in a bacterial system. | |||||||||
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_4038.map.gz | 20.4 MB | EMDB map data format | |
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Header (meta data) | emd-4038-v30.xml emd-4038.xml | 19.2 KB 19.2 KB | Display Display | EMDB header |
Images | emd_4038.png | 143 KB | ||
Filedesc metadata | emd-4038.cif.gz | 8.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-4038 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-4038 | HTTPS FTP |
-Validation report
Summary document | emd_4038_validation.pdf.gz | 276.1 KB | Display | EMDB validaton report |
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Full document | emd_4038_full_validation.pdf.gz | 275.2 KB | Display | |
Data in XML | emd_4038_validation.xml.gz | 5.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4038 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4038 | HTTPS FTP |
-Related structure data
Related structure data | 5ld2MC M: atomic model generated by this map C: citing same article (ref.) |
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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_4038.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 1.34 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 : Ternary complex of RecBCD with forked DNA substrate and ADPNP
Entire | Name: Ternary complex of RecBCD with forked DNA substrate and ADPNP |
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Components |
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-Supramolecule #1: Ternary complex of RecBCD with forked DNA substrate and ADPNP
Supramolecule | Name: Ternary complex of RecBCD with forked DNA substrate and ADPNP type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 |
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Source (natural) | Organism: Escherichia coli K12 (bacteria) |
Molecular weight | Theoretical: 350 KDa |
-Macromolecule #1: RecBCD enzyme subunit RecB,RecBCD enzyme subunit RecB,RecBCD enzy...
Macromolecule | Name: RecBCD enzyme subunit RecB,RecBCD enzyme subunit RecB,RecBCD enzyme subunit RecB type: protein_or_peptide / ID: 1 Details: Residues 913-932 modelled as poly-Alanine in model to reflect uncertainty of the exact position of this loop due to weak density. Number of copies: 1 / Enantiomer: LEVO / EC number: exodeoxyribonuclease V |
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Source (natural) | Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12 |
Molecular weight | Theoretical: 133.717391 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: GMSDVAETLD PLRLPLQGER LIEASAGTGK TFTIAALYLR LLLGLGGSAA FPRPLTVEEL LVVTFTEAAT AELRGRIRSN IHELRIACL RETTDNPLYE RLLEEIDDKA QAAQWLLLAE RQMDEAAVFT IHGFCQRMLN LNAFESGMLF EQQLIEDESL L RYQACADF ...String: GMSDVAETLD PLRLPLQGER LIEASAGTGK TFTIAALYLR LLLGLGGSAA FPRPLTVEEL LVVTFTEAAT AELRGRIRSN IHELRIACL RETTDNPLYE RLLEEIDDKA QAAQWLLLAE RQMDEAAVFT IHGFCQRMLN LNAFESGMLF EQQLIEDESL L RYQACADF WRRHCYPLPR EIAQVVFETW KGPQALLRDI NRYLQGEAPV IKAPPPDDET LASRHAQIVA RIDTVKQQWR DA VGELDAL IESSGIDRRK FNRSNQAKWI DKISAWAEEE TNSYQLPESL EKFSQRFLED RTKAGGETPR HPLFEAIDQL LAE PLSIRD LVITRALAEI RETVAREKRR RGELGFDDML SRLDSALRSE SGEVLAAAIR TRFPVAMIDE FQDTDPQQYR IFRR IWHHQ PETALLLIGD PKQAIYAFRG ADIFTYMKAR SEVHAHYTLD TNWRSAPGMV NSVNKLFSQT DDAFMFREIP FIPVK SAGK NQALRFVFKG ETQPAMKMWL MEGESCGVGD YQSTMAQVCA AQIRDWLQAG QRGEALLMNG DDARPVRASD ISVLVR SRQ EAAQVRDALT LLEIPSVYLS NRDSVFETLE AQEMLWLLQA VMTPERENTL RSALATSMMG LNALDIETLN NDEHAWD VV VEEFDGYRQI WRKRGVMPML RALMSARNIA ENLLATAGGE RRLTDILHIS ELLQEAGTQL ESEHALVRWL SQHILEPD S NASSQQMRLE SDKHLVQIVT IHKSKGLEYP LVWLPFITNF RVQEQAFYHD RHSFEAVLDL NAAPESVDLA EAERLAEDL RLLYVALTRS VWHCSLGVAP LVRRRGDKKG DTDVHQSALG RLLQKGEPQD AAGLRTCIEA LCDDDIAWQT AQTGDNQPWQ VNDVSTAEL NAKTLQRLPG DNWRVTSYSG LQQR(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) PTLTPHQFP RGASPGTFLH SLFEDLDFTQ PVDPNWVREK LELGGFESQW EPVLTEWITA VLQAPLNETG VSLSQLSARN K QVEMEFYL PISEPLIASQ LDTLIRQFDP LSAGCPPLEF MQVRGMLKGF IDLVFRHEGR YYLLAYKSNW LGEDSSAYTQ QA MAAAMQA HRYDLQYQLY TLALHRYLRH RIADYDYEHH FGGVIYLFLR GVDKEHPQQG IYTTRPNAGL IALMDEMFAG MTL EEA UniProtKB: RecBCD enzyme subunit RecB, RecBCD enzyme subunit RecB |
-Macromolecule #2: RecBCD enzyme subunit RecC
Macromolecule | Name: RecBCD enzyme subunit RecC / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: exodeoxyribonuclease V |
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Source (natural) | Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12 |
Molecular weight | Theoretical: 128.974102 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MLRVYHSNRL DVLEALMEFI VERERLDDPF EPEMILVQST GMAQWLQMTL SQKFGIAANI DFPLPASFIW DMFVRVLPEI PKESAFNKQ SMSWKLMTLL PQLLEREDFT LLRHYLTDDS DKRKLFQLSS KAADLFDQYL VYRPDWLAQW ETGHLVEGLG E AQAWQAPL ...String: MLRVYHSNRL DVLEALMEFI VERERLDDPF EPEMILVQST GMAQWLQMTL SQKFGIAANI DFPLPASFIW DMFVRVLPEI PKESAFNKQ SMSWKLMTLL PQLLEREDFT LLRHYLTDDS DKRKLFQLSS KAADLFDQYL VYRPDWLAQW ETGHLVEGLG E AQAWQAPL WKALVEYTHQ LGQPRWHRAN LYQRFIETLE SATTCPPGLP SRVFICGISA LPPVYLQALQ ALGKHIEIHL LF TNPCRYY WGDIKDPAYL AKLLTRQRRH SFEDRELPLF RDSENAGQLF NSDGEQDVGN PLLASWGKLG RDYIYLLSDL ESS QELDAF VDVTPDNLLH NIQSDILELE NRAVAGVNIE EFSRSDNKRP LDPLDSSITF HVCHSPQREV EVLHDRLLAM LEED PTLTP RDIIVMVADI DSYSPFIQAV FGSAPADRYL PYAISDRRAR QSHPVLEAFI SLLSLPDSRF VSEDVLALLD VPVLA ARFD ITEEGLRYLR QWVNESGIRW GIDDDNVREL ELPATGQHTW RFGLTRMLLG YAMESAQGEW QSVLPYDESS GLIAEL VGH LASLLMQLNI WRRGLAQERP LEEWLPVCRD MLNAFFLPDA ETEAAMTLIE QQWQAIIAEG LGAQYGDAVP LSLLRDE LA QRLDQERISQ RFLAGPVNIC TLMPMRSIPF KVVCLLGMND GVYPRQLAPL GFDLMSQKPK RGDRSRRDDD RYLFLEAL I SAQQKLYISY IGRSIQDNSE RFPSVLVQEL IDYIGQSHYL PGDEALNCDE SEARVKAHLT CLHTRMPFDP QNYQPGERQ SYAREWLPAA SQAGKAHSEF VQPLPFTLPE TVPLETLQRF WAHPVRAFFQ MRLQVNFRTE DSEIPDTEPF ILEGLSRYQI NQQLLNALV EQDDAERLFR RFRAAGDLPY GAFGEIFWET QCQEMQQLAD RVIACRQPGQ SMEIDLACNG VQITGWLPQV Q PDGLLRWR PSLLSVAQGM QLWLEHLVYC ASGGNGESRL FLRKDGEWRF PPLAAEQALH YLSQLIEGYR EGMSAPLLVL PE SGGAWLK TCYDAQNDAM LDDDSTLQKA RTKFLQAYEG NMMVRGEGDD IWYQRLWRQL TPETMEAIVE QSQRFLLPLF RFN QS UniProtKB: RecBCD enzyme subunit RecC |
-Macromolecule #3: RecBCD enzyme subunit RecD
Macromolecule | Name: RecBCD enzyme subunit RecD / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: exodeoxyribonuclease V |
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Source (natural) | Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12 |
Molecular weight | Theoretical: 67.047422 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MGKLQKQLLE AVEHKQLRPL DVQFALTVAG DEHPAVTLAA ALLSHDAGEG HVCLPLSRLE NNEASHPLLA TCVSEIGELQ NWEECLLAS QAVSRGDEPT PMILCGDRLY LNRMWCNERT VARFFNEVNH AIEVDEALLA QTLDKLFPVS DEINWQKVAA A VALTRRIS ...String: MGKLQKQLLE AVEHKQLRPL DVQFALTVAG DEHPAVTLAA ALLSHDAGEG HVCLPLSRLE NNEASHPLLA TCVSEIGELQ NWEECLLAS QAVSRGDEPT PMILCGDRLY LNRMWCNERT VARFFNEVNH AIEVDEALLA QTLDKLFPVS DEINWQKVAA A VALTRRIS VISGGPGTGK TTTVAKLLAA LIQMADGERC RIRLAAPTGK AAARLTESLG KALRQLPLTD EQKKRIPEDA ST LHRLLGA QPGSQRLRHH AGNPLHLDVL VVDEASMIDL PMMSRLIDAL PDHARVIFLG DRDQLASVEA GAVLGDICAY ANA GFTAER ARQLSRLTGT HVPAGTGTEA ASLRDSLCLL QKSYRFGSDS GIGQLAAAIN RGDKTAVKTV FQQDFTDIEK RLLQ SGEDY IAMLEEALAG YGRYLDLLQA RAEPDLIIQA FNEYQLLCAL REGPFGVAGL NERIEQFMQQ KRKIHRHPHS RWYEG RPVM IARNDSALGL FNGDIGIALD RGQGTRVWFA MPDGNIKSVQ PSRLPEHETT WAMTVHKSQG SEFDHAALIL PSQRTP VVT RELVYTAVTR ARRRLSLYAD ERILSAAIAT RTERRSGLAA LFSSRE UniProtKB: RecBCD enzyme subunit RecD |
-Macromolecule #4: Fork-Hairpin DNA (70-MER)
Macromolecule | Name: Fork-Hairpin DNA (70-MER) / type: dna / ID: 4 Details: Hairpin=38-42 Duplex=13-37&43-67 5'Tail=1-12 3'Tail=68-70 Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Endothia gyrosa (fungus) |
Molecular weight | Theoretical: 21.440707 KDa |
Sequence | String: (DT)(DT)(DT)(DT)(DT)(DT)(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)(DC)(DA)(DG)(DC)(DA)(DT)(DT)(DT) (DC) (DC)(DC)(DA)(DT)(DG)(DC) ...String: (DT)(DT)(DT)(DT)(DT)(DT)(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)(DC)(DA)(DG)(DC)(DA)(DT)(DT)(DT) (DC) (DC)(DC)(DA)(DT)(DG)(DC)(DT)(DG) (DT)(DA)(DG)(DC)(DA)(DG)(DT)(DG)(DC)(DT) (DC)(DG) (DC)(DA)(DT)(DT)(DA)(DG)(DA) (DT)(DT)(DT) |
-Macromolecule #5: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 1 / Formula: ANP |
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Molecular weight | Theoretical: 506.196 Da |
Chemical component information | ChemComp-ANP: |
-Macromolecule #6: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 5 mg/mL | |||||||||||||||
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Buffer | pH: 7.5 Component:
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Grid | Model: C-flat / Support film - Material: CARBON / Support film - topology: HOLEY Details: Glow discharge was used to thin the carbon film, but allowed to dissipate prior to use. Detergent or amphipols treatment was used to render carbon hydrophilic. See article for details. | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK III |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-30 / Number grids imaged: 1 / Number real images: 1674 / Average exposure time: 0.4 sec. / Average electron dose: 1.2 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated defocus max: 3.8000000000000003 µm / Calibrated defocus min: 0.5 µm / Calibrated magnification: 37313 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: PDB ENTRY PDB model - PDB ID: |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.83 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 74656 |
Initial angle assignment | Type: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3) |
Final angle assignment | Type: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4) |