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- PDB-7svv: TnsBctd-TnsC complex -

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Basic information

Entry
Database: PDB / ID: 7svv
TitleTnsBctd-TnsC complex
Components
  • DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
  • DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • TnsBctd
  • TnsC filament
KeywordsDNA BINDING PROTEIN/DNA / CAST / transposase / AAA+ ATPase / AAA+ / CRISPR / Cas / DNA BINDING PROTEIN-DNA complex
Function / homologyPHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10)
Function and homology information
Biological species[Scytonema hofmanni] UTEX 2349 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.54 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 / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
2: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
A: TnsC filament
a: TnsBctd
B: TnsC filament
b: TnsBctd
C: TnsC filament
c: TnsBctd
D: TnsC filament
d: TnsBctd
E: TnsC filament
e: TnsBctd
F: TnsC filament
f: TnsBctd
G: TnsC filament
g: TnsBctd
H: TnsC filament
h: TnsBctd
I: TnsC filament
i: TnsBctd
J: TnsC filament
j: TnsBctd
hetero molecules


Theoretical massNumber of molelcules
Total (without water)352,10342
Polymers346,79822
Non-polymers5,30520
Water0
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 (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')


Mass: 5734.706 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: DNA chain DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')


Mass: 6845.593 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / Protein/peptide , 2 types, 20 molecules ABCDEFGHIJabcdefghij

#3: Protein
TnsC filament


Mass: 31444.617 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) [Scytonema hofmanni] UTEX 2349 (bacteria)
Production host: Escherichia coli (E. coli)
#4: Protein/peptide
TnsBctd


Mass: 1977.110 Da / Num. of mol.: 10
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, 20 molecules

#5: Chemical
ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Mg

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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: helical reconstruction

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Sample preparation

ComponentName: AMPPNP-bound TnsBctd-TnsC filament from ShCAST element
Type: COMPLEX / Entity ID: #1-#4 / 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 mMATPAdenosine triphosphateC10H16N5O13P31
3200 mMSodium ChlorideNaClSodium chloride1
42 mMMagnesium ChlorideMgCl21
52 %GlycerolC3H8O31
61 mMDTTC4H10O2S21
SpecimenConc.: 1 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 FIELDBright-field microscopy / 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
Helical symmertyAngular rotation/subunit: 59.3 ° / Axial rise/subunit: 7 Å / Axial symmetry: C1
3D reconstructionResolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 286988 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 7M99

7m99


Pdb chain-ID: A / Accession code: 7M99 / Source name: PDB / Type: experimental model

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