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- PDB-7svu: TnsBctd-TnsC-TniQ complex -

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Basic information

Entry
Database: PDB / ID: 7svu
TitleTnsBctd-TnsC-TniQ complex
Components
  • DNA (28-MER)
  • DNA (29-MER)
  • TniQ
  • TnsB-CTD
  • TnsC
KeywordsDNA BINDING PROTEIN/DNA / CAST / transposase / AAA+ ATPase / AAA+ / CRISPR / Cas / DNA BINDING PROTEIN-DNA complex
Function / homologyADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10)
Function and homology information
Biological species[Scytonema hofmanni] UTEX 2349 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 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: Nature / Year: 2023
Title: Structures of the holo CRISPR RNA-guided transposon integration complex.
Authors: Jung-Un Park / Amy Wei-Lun Tsai / Alexandrea N Rizo / Vinh H Truong / Tristan X Wellner / Richard D Schargel / Elizabeth H Kellogg /
Abstract: CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR ...CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.
History
DepositionNov 19, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 23, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 8, 2023Group: 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.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: DNA (28-MER)
2: DNA (29-MER)
A: TnsC
a: TnsB-CTD
B: TnsC
b: TnsB-CTD
C: TnsC
c: TnsB-CTD
D: TnsC
d: TnsB-CTD
E: TnsC
e: TnsB-CTD
F: TnsC
f: TnsB-CTD
G: TnsC
H: TnsC
h: TnsB-CTD
I: TnsC
J: TnsC
j: TnsB-CTD
K: TnsC
k: TnsB-CTD
X: TniQ
Y: TniQ
hetero molecules


Theoretical massNumber of molelcules
Total (without water)425,06446
Polymers419,21824
Non-polymers5,84622
Water00
1


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

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Components

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DNA chain , 2 types, 2 molecules 12

#1: DNA chain DNA (28-MER)


Mass: 8472.441 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: DNA chain DNA (29-MER)


Mass: 9038.040 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein , 2 types, 13 molecules ABCDEFGHIJKXY

#3: Protein
TnsC


Mass: 31444.617 Da / Num. of mol.: 11
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) [Scytonema hofmanni] UTEX 2349 (bacteria)
Production host: Escherichia coli (E. coli)
#5: Protein TniQ


Mass: 19011.240 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) [Scytonema hofmanni] UTEX 2349 (bacteria)
Production host: Escherichia coli (E. coli)

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Protein/peptide , 1 types, 9 molecules abcdefhjk

#4: Protein/peptide
TnsB-CTD


Mass: 1977.110 Da / Num. of mol.: 9
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) [Scytonema hofmanni] UTEX 2349 (bacteria)
Production host: Escherichia coli (E. coli)

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Non-polymers , 2 types, 22 molecules

#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: Mg
#7: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM

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Details

Has ligand of interestN

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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: ATP-bound TnsBctd-TnsC-TniQ complex from ShCAST element
Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.4 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
125 mMHEPESC8H18N2O4S1
22 mMATPC10H16N5O13P31
3200 mMSodium ChlorideNaCl1
42 mMMagnesium ChlorideMgCl21
52 %GlycerolC3H8O31
61 mMDTTC4H10O2S21
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
4WarpCTF correction
10cryoSPARCinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61515 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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