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- EMDB-18211: E. coli plasmid-borne JetABC in a cleavage-competent state -

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Basic information

Entry
Database: EMDB / ID: EMD-18211
TitleE. coli plasmid-borne JetABC in a cleavage-competent state
Map dataLocally filtered
Sample
  • Complex: JetABC loaded onto plasmid DNA
    • Protein or peptide: JetC
    • Protein or peptide: JetB
    • Protein or peptide: JetA
    • RNA: Circular plasmid DNA (1840-MER)
KeywordsSMC complexes / DNA loop extrusion / nuclease / bacterial immunity / defense system / topoisomerase / plamid restriction / MksBEFG / Wadjet / EptABCD / horizontal gene transfer / DNA binding protein / mechanical DNA bending
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.78 Å
AuthorsRoisne-Hamelin F / Gruber S
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)724482European Union
CitationJournal: Mol Cell / Year: 2024
Title: Structural basis for plasmid restriction by SMC JET nuclease.
Authors: Florian Roisné-Hamelin / Hon Wing Liu / Michael Taschner / Yan Li / Stephan Gruber /
Abstract: DNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of plasmids through DNA cleavage, yet the ...DNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of plasmids through DNA cleavage, yet the mechanisms for plasmid recognition are unresolved. We show that artificial DNA circularization renders linear DNA susceptible to JET nuclease cleavage. Unlike free DNA, JET cleaves immobilized plasmid DNA at a specific site, the plasmid-anchoring point, showing that the anchor hinders DNA extrusion but not DNA cleavage. Structures of plasmid-bound JetABC reveal two presumably stalled SMC motor units that are drastically rearranged from the resting state, together entrapping a U-shaped DNA segment, which is further converted to kinked V-shaped cleavage substrate by JetD nuclease binding. Our findings uncover mechanical bending of residual unextruded DNA as molecular signature for plasmid recognition and non-self DNA elimination. We moreover elucidate key elements of SMC loop extrusion, including the motor direction and the structure of a DNA-holding state.
History
DepositionAug 16, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18211.png

Downloads & links

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Map

FileDownload / File: emd_18211.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally filtered
Voxel sizeX=Y=Z: 1.66 Å
Density
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.65866953 - 1.1465867
Average (Standard dev.)0.001049293 (±0.016447073)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 531.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18211_msk_1.map
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Additional map: Unsharpened map.

Fileemd_18211_additional_1.map
AnnotationUnsharpened map.
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Additional map: Sharpened map

Fileemd_18211_additional_2.map
AnnotationSharpened map
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Half map: Half map A

Fileemd_18211_half_map_1.map
AnnotationHalf map A
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Half map: Half map B

Fileemd_18211_half_map_2.map
AnnotationHalf map B
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Sample components

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Entire : JetABC loaded onto plasmid DNA

EntireName: JetABC loaded onto plasmid DNA
Components
  • Complex: JetABC loaded onto plasmid DNA
    • Protein or peptide: JetC
    • Protein or peptide: JetB
    • Protein or peptide: JetA
    • RNA: Circular plasmid DNA (1840-MER)

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Supramolecule #1: JetABC loaded onto plasmid DNA

SupramoleculeName: JetABC loaded onto plasmid DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: E. coli JetABC and plasmid DNA were mixed and incubated with ATP prior freezing.
Source (natural)Organism: Escherichia coli (E. coli) / Strain: GF4-3

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Macromolecule #1: JetC

MacromoleculeName: JetC / type: protein_or_peptide / ID: 1
Details: The last "G" in the theorical sequence is the result of a DNA cloning scar.
Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: GF4-3
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNQVSGLAGK ESFILTRIEL FNWGGFHGLH QAAIHQDGTA VIGPTGSGKT TLVDALMTLL CANPRYNLAS TGGHESDRDL ISYVRGVSGP GDGGEGQSHI ARPGKTVTGI AATLEREGKQ VRLGALLWFD STSSSVTDMK RLWLFSDNPG QTLEHWLNVY HEGGTRLLRQ ...String:
MNQVSGLAGK ESFILTRIEL FNWGGFHGLH QAAIHQDGTA VIGPTGSGKT TLVDALMTLL CANPRYNLAS TGGHESDRDL ISYVRGVSGP GDGGEGQSHI ARPGKTVTGI AATLEREGKQ VRLGALLWFD STSSSVTDMK RLWLFSDNPG QTLEHWLNVY HEGGTRLLRQ MEKEAIGLWT YPNKKQYLAR LRDFFEVGEN AFTLLNRAAG LKQLNSIDEI FRELVLDDHS AFDRAAEVAN SFDGLTEIHQ ELETARKQQQ SLQPVALSWE KYQKQERQLA DWLTLESLLP LWFAQQASHL WREKINLLNA RLAEAQTSEE QLQSQLDLQK KVVSDCMQRY LQVGGANIDE LNERIKDWQK TLGSREALAR QYQQLTRNLG LPSDLSQPQL EANQHEAEAR CEQIAVDIKL KQEEAYQKGA LSHHITEELR ERENERAEIA RRPDSNLPAH YQAFRSELAK ALNVDESELP FVAELIQVKP EEAQWRGAIE RAVGSNRLRI LVAPESAQEA LRWVNQRNNR LHVRLLEVKL PHSPARFFDD GFTRKLLWKD HPWREAVKAL LAESDRHCVD SPEQLHDTPH AMTVQGLMSG KQRFYDKHDQ KRLDEDWLTG FDNRDRLNFL AKEIATLQEQ VKTANAAFEF AKGEVGLLQN QAASFQKIEQ IDFDSIDVPG AKSQLDALRE RLENLTRPDS DASVAKAKLD EAQTIESELD KQLRAANKVT NVLDTELTLA RAAERKAQQT AQQGMKEEER ELCASHFPVV TLEQLPDIRD LERQHERGIQ HEIERVKAEL HRLNIELTKR MSEAKRVDTG ALVEAGADLD DIPVYLQRLQ ELTEEALPEK LNRFLDYLNR SSDDGVTQLL SHIEHEVLVI EERLNELNET MFRVDFQPDR YLRLDTKKVV HESLRTLEKA QRQLNAARFV DDNGESHYKA LQVLVAQLRD ACERNRTLGA KALLDPRFRL EFAVSVMDRQ SGNVIESRTG SQGGSGGEKE IIASYVLTAS LSYALCPAGS RYPLFGTIIL DEAFSRSSHA VAGRIIAALR EFGLHAVFIT PNKEMRLLRD HTRSAIVVHR RGQNSNMASL SWEELERHYQ RRGNAG

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Macromolecule #2: JetB

MacromoleculeName: JetB / type: protein_or_peptide / ID: 2
Details: The last "G" in the theorical sequence is the result of a DNA cloning scar.
Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: GF4-3
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAGFFDKLIN RSVTANAGCE PEPSDEEVTD ESVEDSLASS ETRTLQKIRE ATQELLKYGL LEEASKPNLY RIVLSHPEEV TRILEPLDLD IGIDEIRGLL YVKVRLDETP AQDEWAHPLV RRQRLNLEQS LLVAILRQHF VAWEQESGTG ASQAQIAIDD LLPQLQIYLG ...String:
MAGFFDKLIN RSVTANAGCE PEPSDEEVTD ESVEDSLASS ETRTLQKIRE ATQELLKYGL LEEASKPNLY RIVLSHPEEV TRILEPLDLD IGIDEIRGLL YVKVRLDETP AQDEWAHPLV RRQRLNLEQS LLVAILRQHF VAWEQESGTG ASQAQIAIDD LLPQLQIYLG DPGSESKERT RLLTLLDQLK GHGLVTSPDA HERIVIRPII AHLADPINLQ ALLAWLREQI AQQTSPNDAP EKDSSEEDVG

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Macromolecule #3: JetA

MacromoleculeName: JetA / type: protein_or_peptide / ID: 3
Details: "GPAA" at the begining of the theorical sequence is the remaining of the purification tag after tag cleavage. The last "G" in the theorical sequence is the result of a DNA cloning scar.
Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: GF4-3
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GPAAMEENTR QRTENYISAK NQHPAWILLA TRRAPLVLSC LKTLFEKSHD GIPLEEAIQS LSSILIEHVS QEQYDINQDN PFLQASRELR EWIKRRLIVE RDGRIFATDA LEVAITFVES LDNRFMTSTA SRLSTVQREI ENLETRLNPN PANRVATLRR RISELERELQ ...String:
GPAAMEENTR QRTENYISAK NQHPAWILLA TRRAPLVLSC LKTLFEKSHD GIPLEEAIQS LSSILIEHVS QEQYDINQDN PFLQASRELR EWIKRRLIVE RDGRIFATDA LEVAITFVES LDNRFMTSTA SRLSTVQREI ENLETRLNPN PANRVATLRR RISELERELQ EAEAGHIEVL ETHQAVEHIR DVYNLASSLR ADFRRVEDSW READRALRQS IIGEQYHRGD IVERLLNDQD ALLNTPEGRV FDSFQQQLRQ SSELKAMSER LRVILSHPSA SDALNRLQRH DLRWLVKRLV DESQTVLQAR ARSERDVRGF MKTGLAAEHH RVGHLLNEFL NLALKLDWQR QMIRKQEVPL PAVGVAVTGI PAIERLRFKE VDDEAEQTLD LSNHAADLTQ IGDDFWDAFN GLDREVLIQQ TLQLLAKENR PVGLAELAEL LPPAHDLETF AVWIGMAREA GIEVIDSQRE FAELSDGEGR RWRFNLPTTG LESQALMDID WEG

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Macromolecule #4: Circular plasmid DNA (1840-MER)

MacromoleculeName: Circular plasmid DNA (1840-MER) / type: rna / ID: 4
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString: CTTTCTACGT GTTCCGCTTC CTTTAGCAGC CCTTGCGCCC TGAGTGCTTG CGGCAGCGTG AAGCTAATTC TGTCAGCCGT TAAGTGTTCC TGTGTCACTC AAAATTGCTT TGAGAGGCTC TAAGGGCTTC TCAGTGCGTT ACATCCCTGG CTTGTTGTCC ACAACCGTTA ...String:
CTTTCTACGT GTTCCGCTTC CTTTAGCAGC CCTTGCGCCC TGAGTGCTTG CGGCAGCGTG AAGCTAATTC TGTCAGCCGT TAAGTGTTCC TGTGTCACTC AAAATTGCTT TGAGAGGCTC TAAGGGCTTC TCAGTGCGTT ACATCCCTGG CTTGTTGTCC ACAACCGTTA AACCTTAAAA GCTTTAAAAG CCTTATATAT TCTTTTTTTT CTTATAAAAC TTAAAACCTT AGAGGCTATT TAAGTTGCTG ATTTATATTA ATTTTATTGT TCAAACATGA GAGCTTAGTA CGTGAAACAT GAGAGCTTAG TACGTTAGCC ATGAGAGCTT AGTACGTTAG CCATGAGGGT TTAGTTCGTT AAACATGAGA GCTTAGTACG TTAAACATGA GAGCTTAGTA CGTGAAACAT GAGAGCTTAG TACGTACTAT CAACAGGTTG AACTGCTGAT CAACAGATCC TCTACGCGGC CGCGGTACCA TAACTTCGTA TAGCATACAT TATACGAAGT TATCTGCCAG GCACATGGGT TTTACTAGTA TCGATTCGCG ACCTACTCCG GAATATTAGG TCTCAgctct tcGGAGACCC ACTGCTTGAG CCTAGAAGAT CCGGCTGCTA ACAAAGCCCG AAAGGAAGCT GAGTTGGCTG CTGCCACCGC TGAGCAATAA CTATCATAAC CCCTAGGTGC CATTTCATTA CCTCTTTCTC CGCACCCGAC ATAGATCTGG GCCAACTTTT GGCGAAAATG AGACGTTGAT CGGCACGTAA GAGGGTCCAA CTTTCACCAT AATGAAATAA GATCACTACC GGGCGTATTT TTTGAGTTAT CGAGATTTTC AGGAGCTAAG GAAGCTAAAA TGGAGAAAAA AATCACTGGA TATACCACCG TTGATATATC CCAATGGCAT CGTAAAGAAC ATTTTGAGGC ATTTCAGTCA GTTGCTCAAT GTACCTATAA CCAGACCGTT CAGCTGGATA TTACGGCCTT TTTAAAGACC GTAAAGAAAA ATAAGCACAA GTTTTATCCG GCCTTTATTC ACATTCTTGC CCGCCTGATG AATGCTCATC CGGAATTCCG TATGGCAATG AAAGACGGTG AGCTGGTGAT ATGGGATAGT GTTCACCCTT GTTACACCGT TTTCCATGAG CAAACTGAAA CGTTTTCATC GCTCTGGAGT GAATACCACG ACGATTTCCG GCAGTTTCTA CACATATATT CGCAAGATGT GGCGTGTTAC GGTGAAAACC TGGCCTATTT CCCTAAAGGG TTTATTGAGA ATATGTTTTT CGTCTCAGCC AATCCCTGGG TGAGTTTCAC CAGTTTTGAT TTAAACGTGG CCAATATGGA CAACTTCTTC GCCCCCGTTT TCACCATGGG CAAATATTAT ACGCAAGGCG ACAAGGTGCT GATGCCGCTG GCGATTCAGG TTCATCATGC CGTTTGTGAT GGCTTCCATG TCGGCAGAAT GCTTAATGAA TTACAACAGT ACTGCGATGA GTGGCAGGGC GGGGCGTAAT TTTTTTAAGG CAGTTATTGG TGCCCTTAAA CGCCTGGTTG CTACGCCTGA ATAAGTGATA ATAAGCGGAT GAATGGCAGA AATTCGAAAG CAAATTCGAC CCGGTCGTCG GTTCAGGGCA GGGTCGTTAA ATAGCCGCTT ATGTCTATTG CTGGTTTACC GGTTTATTGA CTACCGGAAG CAGTGTGACC GTGTGCTTCT CAAATGCCTG AGGCCAGTTT GCTCAGGCTC TCCCCGTGGA GGTAATAATT GACGATATGA TCATTTATTC TGCCTCCCAG CTGACATTCA TCCGGGGTCA GCACCGTTTC TGCGGACTGG

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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
GridModel: Au-flat 1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 13292 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.78 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 187999

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