[English] 日本語
Yorodumi
- PDB-7svw: Strand-transfer complex of TnsB from ShCAST -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 7svw
TitleStrand-transfer complex of TnsB from ShCAST
Components
  • STC_LE_For
  • STC_LE_Rev1
  • STC_LE_Rev2
  • TnsB
KeywordsDNA BINDING PROTEIN/DNA / CAST / transposase / strand-transfer complex / CRISPR / Cas / DNA BINDING PROTEIN-DNA complex
Function / homologyDNA / DNA (> 10)
Function and homology information
Biological species[Scytonema hofmanni] UTEX 2349 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.69 Å
AuthorsPark, J. / Tsai, A.W.T. / Kellogg, E.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00-GM124463 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM.
Authors: Jung-Un Park / Amy Wei-Lun Tsai / Tiffany H Chen / Joseph E Peters / Elizabeth H Kellogg /
Abstract: CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the ...CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the transposase component, TnsB, remains uncharacterized. Using cryo-electron microscopy (cryo-EM) structure determination, we reveal the conformation of TnsB during transposon integration for the type V-K CAST system from (ShCAST). Our structure of TnsB is a tetramer, revealing strong mechanistic relationships with the overall architecture of RNaseH transposases/integrases in general, and in particular the MuA transposase from bacteriophage Mu. However, key structural differences in the C-terminal domains indicate that TnsB's tetrameric architecture is stabilized by a different set of protein-protein interactions compared with MuA. We describe the base-specific interactions along the TnsB binding site, which explain how different CAST elements can function on cognate mobile elements independent of one another. We observe that melting of the 5' nontransferred strand of the transposon end is a structural feature stabilized by TnsB and furthermore is crucial for donor-DNA integration. Although not observed in the TnsB strand-transfer complex, the C-terminal end of TnsB serves a crucial role in transposase recruitment to the target site. The C-terminal end of TnsB adopts a short, structured 15-residue "hook" that decorates TnsC filaments. Unlike full-length TnsB, C-terminal fragments do not appear to stimulate filament disassembly using two different assays, suggesting that additional interactions between TnsB and TnsC are required for redistributing TnsC to appropriate targets. The structural information presented here will help guide future work in modifying these important systems as programmable gene integration tools.
History
DepositionNov 19, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 10, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 17, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Feb 28, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
1: STC_LE_For
2: STC_LE_Rev1
3: STC_LE_Rev2
4: STC_LE_For
5: STC_LE_Rev1
6: STC_LE_Rev2
A: TnsB
B: TnsB
C: TnsB
D: TnsB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)349,69412
Polymers349,64610
Non-polymers492
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

-
Components

#1: DNA chain STC_LE_For


Mass: 21579.830 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: DNA chain STC_LE_Rev1


Mass: 13905.951 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain STC_LE_Rev2


Mass: 6062.945 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Protein
TnsB


Mass: 66637.070 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) [Scytonema hofmanni] UTEX 2349 (bacteria)
Production host: Escherichia coli (E. coli)
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

-
Experimental details

-
Experiment

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

-
Sample preparation

ComponentName: strand-transfer complex of TnsB from ShCAST element / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.35 MDa / Experimental value: NO
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
126 mMHEPESC8H18N2O4S1
220 mMPotassium ChlorideKCl1
3100 mMSodium ChlorideNaCl1
40.2 mMMagnesium ChlorideMgCl21
515 mMMagnesium AcetateMg(CH3COO)21
63 %GlycerolC3H8O31
71.5 mMDTTC4H10O2S21
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

-
Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 49 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

-
Processing

EM software
IDNameCategory
4WarpCTF correction
9cryoSPARCinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 186121
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.69 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 46568 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

+
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://pdbjlvh1.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