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- PDB-7rzy: CryoEM structure of Vibrio cholerae transposon Tn6677 AAA+ ATPase TnsC -

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Basic information

Entry
Database: PDB / ID: 7rzy
TitleCryoEM structure of Vibrio cholerae transposon Tn6677 AAA+ ATPase TnsC
ComponentsTn6677 Vibrio cholerae transposon TnsC (VchTnsC)
KeywordsDNA BINDING PROTEIN / CRISPR / transposon / ATPase / AAA+ / TnsC
Function / homologyADENOSINE-5'-TRIPHOSPHATE
Function and homology information
Biological speciesVibrio cholerae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsHoffmann, F.T. / Kim, M. / Beh, L.Y. / Wang, J. / Vo, P.L.H. / Gelsinger, D.R. / Acree, C. / Mohabir, J.T. / Fernandez, I.S. / Sternberg, S.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2HG011650-01 United States
CitationJournal: Nature / Year: 2022
Title: Selective TnsC recruitment enhances the fidelity of RNA-guided transposition.
Authors: Florian T Hoffmann / Minjoo Kim / Leslie Y Beh / Jing Wang / Phuc Leo H Vo / Diego R Gelsinger / Jerrin Thomas George / Christopher Acree / Jason T Mohabir / Israel S Fernández / Samuel H Sternberg /
Abstract: Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site ...Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins. How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous-hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.
History
DepositionAug 28, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 8, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 7, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Sep 21, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: Tn6677 Vibrio cholerae transposon TnsC (VchTnsC)
2: Tn6677 Vibrio cholerae transposon TnsC (VchTnsC)
3: Tn6677 Vibrio cholerae transposon TnsC (VchTnsC)
4: Tn6677 Vibrio cholerae transposon TnsC (VchTnsC)
5: Tn6677 Vibrio cholerae transposon TnsC (VchTnsC)
6: Tn6677 Vibrio cholerae transposon TnsC (VchTnsC)
7: Tn6677 Vibrio cholerae transposon TnsC (VchTnsC)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)266,72314
Polymers263,1727
Non-polymers3,5507
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Tn6677 Vibrio cholerae transposon TnsC (VchTnsC)


Mass: 37596.043 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Production host: Escherichia coli (E. coli)
#2: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: VchTnsC / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.238 MDa / Experimental value: YES
Source (natural)Organism: Vibrio cholerae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Homemade
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 65000 X / Calibrated magnification: 65000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 550 nm / Calibrated defocus max: 1900 nm / Cs: 0.001 mm / C2 aperture diameter: 51 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 70 K
Image recordingElectron dose: 25 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement
EM software
IDNameVersionCategory
2Leginonimage acquisition
4RELION3.1CTF correction
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
12RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 109000 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE
RefinementResolution: 3.6→150.52 Å / Cor.coef. Fo:Fc: 0.787 / SU B: 58.051 / SU ML: 0.814
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.44314 --
obs0.44314 66202 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 78.326 Å2
Baniso -1Baniso -2Baniso -3
1--1.07 Å20.12 Å20.09 Å2
2---1 Å2-0.05 Å2
3---2.08 Å2
Refinement stepCycle: 1 / Total: 17507
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0030.01317884
ELECTRON MICROSCOPYr_bond_other_d0.0010.01717248
ELECTRON MICROSCOPYr_angle_refined_deg1.3021.64824220
ELECTRON MICROSCOPYr_angle_other_deg0.9971.5839767
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.58852184
ELECTRON MICROSCOPYr_dihedral_angle_2_deg28.83621.97924
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.651153206
ELECTRON MICROSCOPYr_dihedral_angle_4_deg11.18115133
ELECTRON MICROSCOPYr_chiral_restr
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.0219894
ELECTRON MICROSCOPYr_gen_planes_other0.0020.023997
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it0.4898.4168736
ELECTRON MICROSCOPYr_mcbond_other0.4898.4168735
ELECTRON MICROSCOPYr_mcangle_it0.93912.61910920
ELECTRON MICROSCOPYr_mcangle_other0.93912.61910921
ELECTRON MICROSCOPYr_scbond_it0.1438.3739148
ELECTRON MICROSCOPYr_scbond_other0.1438.3739148
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other0.36812.57513301
ELECTRON MICROSCOPYr_long_range_B_refined2.54620249
ELECTRON MICROSCOPYr_long_range_B_other2.54620250
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.6→3.693 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.096 4825 -
obs--100 %

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