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- PDB-7m9b: ADP-AlF3 bound TnsC structure in closed form -

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Basic information

Entry
Database: PDB / ID: 7m9b
TitleADP-AlF3 bound TnsC structure in closed form
Components
  • (DNA (27-MER)) x 2
  • TnsC
KeywordsDNA BINDING PROTEIN/DNA / CRISPR / Transposition / AAA+ ATPase / Tn7 / DNA BINDING PROTEIN-DNA complex
Function / homologyADENOSINE-5'-DIPHOSPHATE / DNA / DNA (> 10)
Function and homology information
Biological speciesScytonema hofmannii (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 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)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
DepositionMar 30, 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
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  • EMDB-23722
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: TnsC
B: TnsC
C: TnsC
D: TnsC
E: TnsC
F: TnsC
G: TnsC
H: TnsC
I: TnsC
J: TnsC
K: TnsC
L: TnsC
M: DNA (27-MER)
N: DNA (27-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)398,18724
Polymers393,91514
Non-polymers4,27210
Water0
1


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

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Components

#1: Protein
TnsC


Mass: 31444.617 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli BL21 (bacteria)
#2: DNA chain DNA (27-MER)


Mass: 8411.627 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (27-MER)


Mass: 8168.248 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY

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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: Head to head dimer of TnsC hexamers bound to DNA / Type: COMPLEX / Details: Reconstituted with ADP-AlF3, in closed state / Entity ID: #1-#3 / Source: RECOMBINANT
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
125 mMHEPESC8H18N2O4S1
21 mMDTTC4H10O2S21
32 %glycerolC3H8O31
46 mMMagnesium ChlorideMgCl21
52 mMADPAdenosine diphosphateC10H15N5O10P21
6200 mMSodium ChlorideNaClSodium chloride1
76 mMAluminum ChlorideAlCl31
860 mMSodium FluorideNaF1
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid 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 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: NITROGEN
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
14cryoSPARCinitial Euler assignment
17RELION3.1final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 754099
Symmetry
IDImage processing-IDEntry-IDPoint symmetry
117M9B
217M9B
317M9BC1 (asymmetric)
417M9B
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 121512 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6AZ0

6az0

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