EMDB-18201: E. coli plasmid-borne JetABCD(E248A) core in a cleavage-competent...

Basic information

Entry

Database: EMDB / ID: EMD-18201

• Title: E. coli plasmid-borne JetABCD(E248A) core in a cleavage-competent state

Sample

• Complex: JetABCD(E248A) loaded onto plasmid DNA
  • Protein or peptide: JetC
  • Protein or peptide: JetB
  • Protein or peptide: JetA
  • Protein or peptide: JetD(E248A)
  • DNA: Circular plasmid DNA (1840-MER)
• Ligand: ADENOSINE-5'-DIPHOSPHATE

• Keywords: SMC complexes / DNA loop extrusion / nuclease / bacterial immunity / defense system / topoisomerase / plamid restriction / MksBEFG / Wadjet / EptABCD / horizontal gene transfer / DNA binding protein / DNA kinking

Function / homology

Domain/homology:

P-loop containing region of AAA domain / Uncharacterised conserved protein UCP028408 / Wadjet protein JetD, C-terminal / Domain of unknown function DUF3322 / Wadjet protein JetD, C-terminal / Uncharacterized protein conserved in bacteria N-term (DUF3322) / Protein of unknown function DUF3375 / Protein of unknown function (DUF3375) / SbcC/RAD50-like, Walker B motif / P-loop containing nucleoside triphosphate hydrolase

Component:

DUF3322 and DUF2220 domain-containing protein / DUF3375 family protein / Chromosome partition protein Smc

• Biological species: Escherichia coli (E. coli)
• Method: single particle reconstruction / cryo EM / Resolution: 4.17 Å
• Authors: Roisne-Hamelin F / Li Y / Gruber S

Funding support

European Union, 1 items

Organization	Grant number	Country
European Research Council (ERC)	724482	European Union

• Citation: Journal: 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 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

Deposition	Aug 15, 2023	-
Header (metadata) release	Jan 31, 2024	-
Map release	Jan 31, 2024	-
Update	Mar 20, 2024	-
Current status	Mar 20, 2024	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_18201.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 1.66 Å

Density

Contour Level	By AUTHOR: 0.102
Minimum - Maximum	-0.66107005 - 1.1526163
Average (Standard dev.)	0.000082643266 (±0.019152261)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 320 320 320 Spacing 320 320 320
Cell	A=B=C: 531.2 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_18201_msk_1.map

Additional map: This is the unsharpened map.

• File: emd_18201_additional_1.map
• Annotation: This is the unsharpened map.

Half map: #2

• File: emd_18201_half_map_1.map

Half map: #1

• File: emd_18201_half_map_2.map

Sample components

Entire : JetABCD(E248A) loaded onto plasmid DNA

• Entire: Name: JetABCD(E248A) loaded onto plasmid DNA

Components

• Complex: JetABCD(E248A) loaded onto plasmid DNA
  • Protein or peptide: JetC
  • Protein or peptide: JetB
  • Protein or peptide: JetA
  • Protein or peptide: JetD(E248A)
  • DNA: Circular plasmid DNA (1840-MER)
• Ligand: ADENOSINE-5'-DIPHOSPHATE

Supramolecule #1: JetABCD(E248A) loaded onto plasmid DNA

• Supramolecule: Name: JetABCD(E248A) loaded onto plasmid DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5; Details: E. coli JetABC, JetD(E248A) and plasmid DNA were mixed and incubated with ATP prior freezing.
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: GF4-3

Macromolecule #1: JetC

• Macromolecule: Name: JetC / type: protein_or_peptide / ID: 1 ; Details: The last "G" in the theorical sequence is the result of a DNA cloning scar.; Number of copies: 4 / Enantiomer: LEVO
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: GF4-3
• Molecular weight: Theoretical: 124.562938 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MNQVSGLAGK ESFILTRIEL FNWGGFHGLH QAAIHQDGTA VIGPTGSGKT TLVDALMTLL CANPRYNLAS TGGHESDRDL ISYVRGVSG PGDGGEGQSH IARPGKTVTG IAATLEREGK QVRLGALLWF DSTSSSVTDM KRLWLFSDNP GQTLEHWLNV Y HEGGTRLL RQMEKEAIGL WTYPNKKQYL ARLRDFFEVG ENAFTLLNRA AGLKQLNSID EIFRELVLDD HSAFDRAAEV AN SFDGLTE IHQELETARK QQQSLQPVAL SWEKYQKQER QLADWLTLES LLPLWFAQQA SHLWREKINL LNARLAEAQT SEE QLQSQL DLQKKVVSDC MQRYLQVGGA NIDELNERIK DWQKTLGSRE ALARQYQQLT RNLGLPSDLS QPQLEANQHE AEAR CEQIA VDIKLKQEEA YQKGALSHHI TEELRERENE RAEIARRPDS NLPAHYQAFR SELAKALNVD ESELPFVAEL IQVKP EEAQ WRGAIERAVG SNRLRILVAP ESAQEALRWV NQRNNRLHVR LLEVKLPHSP ARFFDDGFTR KLLWKDHPWR EAVKAL LAE SDRHCVDSPE QLHDTPHAMT VQGLMSGKQR FYDKHDQKRL DEDWLTGFDN RDRLNFLAKE IATLQEQVKT ANAAFEF AK GEVGLLQNQA ASFQKIEQID FDSIDVPGAK SQLDALRERL ENLTRPDSDA SVAKAKLDEA QTIESELDKQ LRAANKVT N VLDTELTLAR AAERKAQQTA QQGMKEEERE LCASHFPVVT LEQLPDIRDL ERQHERGIQH EIERVKAELH RLNIELTKR MSEAKRVDTG ALVEAGADLD DIPVYLQRLQ ELTEEALPEK LNRFLDYLNR SSDDGVTQLL SHIEHEVLVI EERLNELNET MFRVDFQPD RYLRLDTKKV VHESLRTLEK AQRQLNAARF VDDNGESHYK ALQVLVAQLR DACERNRTLG AKALLDPRFR L EFAVSVMD RQSGNVIESR TGSQGGSGGE KEIIASYVLT ASLSYALCPA GSRYPLFGTI ILDEAFSRSS HAVAGRIIAA LR EFGLHAV FITPNKEMRL LRDHTRSAIV VHRRGQNSNM ASLSWEELER HYQRRGNAG

UniProtKB: Chromosome partition protein Smc

Macromolecule #2: JetB

• Macromolecule: Name: JetB / type: protein_or_peptide / ID: 2 ; Details: The last "G" in the theorical sequence is the result of a DNA cloning scar.; Number of copies: 4 / Enantiomer: LEVO
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: GF4-3
• Molecular weight: Theoretical: 28.020416 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MAGFFDKLIN RSVTANAGCE PEPSDEEVTD ESVEDSLASS ETRTLQKIRE ATQELLKYGL LEEASKPNLY RIVLSHPEEV TRILEPLDL DIGIDEIRGL LYVKVRLDET PAQDEWAHPL VRRQRLNLEQ SLLVAILRQH FVAWEQESGT GASQAQIAID D LLPQLQIY LGDPGSESKE RTRLLTLLDQ LKGHGLVTSP DAHERIVIRP IIAHLADPIN LQALLAWLRE QIAQQTSPND AP EKDSSEE DVG

Macromolecule #3: JetA

• Macromolecule: Name: 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.; Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: GF4-3
• Molecular weight: Theoretical: 57.81891 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

GPAAMEENTR QRTENYISAK NQHPAWILLA TRRAPLVLSC LKTLFEKSHD GIPLEEAIQS LSSILIEHVS QEQYDINQDN PFLQASREL REWIKRRLIV ERDGRIFATD ALEVAITFVE SLDNRFMTST ASRLSTVQRE IENLETRLNP NPANRVATLR R RISELERE LQEAEAGHIE VLETHQAVEH IRDVYNLASS LRADFRRVED SWREADRALR QSIIGEQYHR GDIVERLLND QD ALLNTPE GRVFDSFQQQ LRQSSELKAM SERLRVILSH PSASDALNRL QRHDLRWLVK RLVDESQTVL QARARSERDV RGF MKTGLA AEHHRVGHLL NEFLNLALKL DWQRQMIRKQ EVPLPAVGVA VTGIPAIERL RFKEVDDEAE QTLDLSNHAA DLTQ IGDDF WDAFNGLDRE VLIQQTLQLL AKENRPVGLA ELAELLPPAH DLETFAVWIG MAREAGIEVI DSQREFAELS DGEGR RWRF NLPTTGLESQ ALMDIDWEG

UniProtKB: DUF3375 family protein

Macromolecule #4: JetD(E248A)

• Macromolecule: Name: JetD(E248A) / type: protein_or_peptide / ID: 4 ; Details: "GSLEVLFQ" at the C-terminus in the theorical sequence is the remaining of the tag after tag cleavage. The sequence carries the "E248A" mutation (nuclease dead).; Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Escherichia coli (E. coli)
• Molecular weight: Theoretical: 43.938492 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MYSPDELREK LARQWDSAKL RAERLLSPGN WPLCLPIGKP STKIFAEQTQ RVLQHVQRWR QVAVGHVEWE AVSFRASDTP VLMPLRWIL NSPSEWINAA ADPVVSREFR LLEGIIEQVN PIFHPLLVKH RSLWRHKDPQ GVISAATLAC RLEPGCAKGL P LRLLSGQG VDTKFIENNI SLLTRLLDVR FSGEASEQGL TTFLDAFDES SHWVLVVPLS PGLLPFKKCR VTTAELAETT LP ASQVLVV ANEQCLHHLP ALSDTIAILG CGLDVQWLSS SVLDEKRVAY WGDMDSWGLL MLARARRCRP TLDALLMNRE LFE QYASHS AVPEPVIAKE AVPDGLLNEE ADFYRYLTRL PRGRLEQEFL PVGIAEEALL RWGKESGSLE VLFQ

UniProtKB: DUF3322 and DUF2220 domain-containing protein

Macromolecule #5: Circular plasmid DNA (1840-MER)

• Macromolecule: Name: Circular plasmid DNA (1840-MER) / type: dna / ID: 5 ; Details: Only a portion of the plasmid (pSG6085, 1840 bp) has been modelled as polyAT track, because the complex is expected to bind in random position to the DNA and the resolution of DNA do not allow any sequence assignement.; Number of copies: 4 / Classification: DNA
• Source (natural): Organism: Escherichia coli (E. coli)
• Molecular weight: Theoretical: 12.303033 KDa

Sequence

String:

(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT) (DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT) (DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA) (DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DA) (DT)

Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE

• Macromolecule: Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 4 / Formula: ADP
• Molecular weight: Theoretical: 427.201 Da

Chemical component information

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

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Buffer: pH: 7.5
• Grid: Model: Au-flat 1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

Electron microscopy

• Microscope: TFS KRIOS
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.8 µm
• Image recording: Film or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 37902 / Average electron dose: 40.0 e/Ų
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Startup model: Type of model: INSILICO MODEL
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD
• Final angle assignment: Type: MAXIMUM LIKELIHOOD
• Final reconstruction: Applied symmetry - Point group: C₂ (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.17 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 235956

Atomic model buiding 1

Initial model

PDB ID	Chain	Details
8as8	source_name: PDB, initial_model_type: experimental model
	source_name: AlphaFold, initial_model_type: in silico model
	source_name: Other, initial_model_type: in silico model	Idealized B-form DNA was flexibly fitted

• Refinement: Protocol: FLEXIBLE FIT

Output model

PDB-8q72:
E. coli plasmid-borne JetABCD(E248A) core in a cleavage-competent state