[English] 日本語
Yorodumi
- PDB-7ufi: VchTnsC AAA+ ATPase with DNA, single heptamer -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 7ufi
TitleVchTnsC AAA+ ATPase with DNA, single heptamer
Components
  • DNA (5'-D(P*CP*TP*CP*CP*AP*GP*TP*AP*CP*AP*GP*CP*GP*CP*GP*GP*CP*TP*GP*AP*A)-3')
  • DNA (5'-D(P*TP*TP*CP*AP*GP*CP*CP*GP*CP*GP*CP*TP*GP*TP*AP*CP*TP*GP*GP*AP*G)-3')
  • VchTnsC
KeywordsDNA BINDING PROTEIN/DNA / transposon / AAA+ / DNA BINDING PROTEIN-DNA complex
Function / homologyADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10)
Function and homology information
Biological speciesVibrio cholerae (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsFernandez, 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
DepositionMar 22, 2022Deposition 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

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: DNA (5'-D(P*CP*TP*CP*CP*AP*GP*TP*AP*CP*AP*GP*CP*GP*CP*GP*GP*CP*TP*GP*AP*A)-3')
B: DNA (5'-D(P*TP*TP*CP*AP*GP*CP*CP*GP*CP*GP*CP*TP*GP*TP*AP*CP*TP*GP*GP*AP*G)-3')
1: VchTnsC
2: VchTnsC
3: VchTnsC
4: VchTnsC
5: VchTnsC
6: VchTnsC
7: VchTnsC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)261,89616
Polymers258,3459
Non-polymers3,5507
Water0
1


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

-
Components

#1: DNA chain DNA (5'-D(P*CP*TP*CP*CP*AP*GP*TP*AP*CP*AP*GP*CP*GP*CP*GP*GP*CP*TP*GP*AP*A)-3')


Mass: 6433.163 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: DNA chain DNA (5'-D(P*TP*TP*CP*AP*GP*CP*CP*GP*CP*GP*CP*TP*GP*TP*AP*CP*TP*GP*GP*AP*G)-3')


Mass: 6455.159 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Protein
VchTnsC


Mass: 35065.273 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)
#4: 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

-
Experimental details

-
Experiment

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

-
Sample preparation

ComponentName: VchTNsC AAA+ ATPase with ATP and DNA (single heptamer form)
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
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: NITROGEN

-
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 defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 65 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

-
Processing

SoftwareName: REFMAC / Version: 5.8.0238 / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105000 / Algorithm: FOURIER SPACE / Symmetry type: POINT
RefinementResolution: 3.4→147.9 Å / Cor.coef. Fo:Fc: 0.898 / SU B: 29.851 / SU ML: 0.449 / ESU R: 0.868
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.3519 --
obs0.3519 108072 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: 139.732 Å2
Baniso -1Baniso -2Baniso -3
1--0.4 Å2-1.78 Å20.29 Å2
2--3.22 Å2-1.29 Å2
3----2.81 Å2
Refinement stepCycle: 1 / Total: 18319
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0030.01318773
ELECTRON MICROSCOPYr_bond_other_d0.0020.01717307
ELECTRON MICROSCOPYr_angle_refined_deg1.3671.62125612
ELECTRON MICROSCOPYr_angle_other_deg1.1311.61540214
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.23252170
ELECTRON MICROSCOPYr_dihedral_angle_2_deg35.59621.97924
ELECTRON MICROSCOPYr_dihedral_angle_3_deg19.955153199
ELECTRON MICROSCOPYr_dihedral_angle_4_deg21.30915133
ELECTRON MICROSCOPYr_chiral_restr0.0620.22457
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.0220090
ELECTRON MICROSCOPYr_gen_planes_other0.0020.023855
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_it4.9714.1628701
ELECTRON MICROSCOPYr_mcbond_other4.96614.1628700
ELECTRON MICROSCOPYr_mcangle_it8.31421.26510864
ELECTRON MICROSCOPYr_mcangle_other8.31521.26510865
ELECTRON MICROSCOPYr_scbond_it4.66915.94310072
ELECTRON MICROSCOPYr_scbond_other4.66815.94310072
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other8.60723.70614749
ELECTRON MICROSCOPYr_long_range_B_refined13.99921011
ELECTRON MICROSCOPYr_long_range_B_other13.99921012
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.4→3.488 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.457 8003 -
obs--100 %

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more