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- PDB-7n6i: ATP-bound TnsC-TniQ complex from ShCAST system -

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Basic information

Entry
Database: PDB / ID: 7n6i
TitleATP-bound TnsC-TniQ complex from ShCAST system
Components
  • DNA (5'-D(P*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*T)-3')
  • TniQ (Homology model)
  • TnsC
KeywordsDNA BINDING PROTEIN/DNA / CRISPR / Transposition / AAA+ ATPase / Tn7 / DNA BINDING PROTEIN-DNA complex
Function / homologyADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10)
Function and homology information
Biological speciesScytonema hofmannii (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsPark, J. / Tsai, A.W.L. / Mehrotra, E. / Kellogg, E.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)M R00-GM124463 United States
CitationJournal: Science / Year: 2021
Title: Structural basis for target site selection in RNA-guided DNA transposition systems.
Authors: Jung-Un Park / Amy Wei-Lun Tsai / Eshan Mehrotra / Michael T Petassi / Shan-Chi Hsieh / Ailong Ke / Joseph E Peters / Elizabeth H Kellogg /
Abstract: CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron ...CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate-bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.
History
DepositionJun 8, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 28, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 25, 2021Group: Database references / Category: citation / citation_author / database_2
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 / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • EMDB-23726
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-23726
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: TniQ (Homology model)
B: TniQ (Homology model)
C: TnsC
D: TnsC
E: TnsC
F: TnsC
G: TnsC
H: TnsC
I: TnsC
J: TnsC
K: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
L: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)303,70626
Polymers299,98512
Non-polymers3,72014
Water0
1


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

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Components

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Protein , 2 types, 10 molecules ABCDEFGHIJ

#1: Protein TniQ (Homology model)


Mass: 19011.240 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli BL21 (bacteria)
#2: Protein
TnsC


Mass: 31444.617 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli BL21 (bacteria)

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DNA chain , 2 types, 2 molecules KL

#3: DNA chain DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')


Mass: 5126.320 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')


Mass: 5279.559 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 14 molecules

#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / 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: single particle reconstruction

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Sample preparation

ComponentName: ATP-bound TnsC-TniQ complex from ShCAST system / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.4 MDa / Experimental value: NO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaClSodium chloride1
225 mMHEPESC8H18N2O4S1
31 mMDTTC4H10O2S21
42 %glycerolC3H8O31
52 mMmagnesium chlorideMgCl21
62 mMATPAdenosine triphosphateC10H16N5O13P31
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Excess of TniQ was added to ATP-bound TnsC filaments
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Blot for 7 seconds before plunging

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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 magnification: 63000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: HELIUM / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4172
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
4Warp1.0.9CTF correction
5RELION3.1CTF correction
8UCSF Chimeramodel fitting
10cryoSPARC3initial Euler assignment
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 648550 / Details: Neural net based particle picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34546 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Details: Homology model was built using the crystal structure of TniQ (PDB:6V9P) by GalaxyTBM server. This model is manually docked into the cryo-EM density using UCSF Chimera.
Atomic model buildingPDB-ID: 6V9P

6v9p


Pdb chain-ID: A

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