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- PDB-7m99: ATPgS bound TnsC filament from ShCAST system -

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Basic information

Entry
Database: PDB / ID: 7m99
TitleATPgS bound TnsC filament from ShCAST system
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')
  • TnsC
KeywordsDNA BINDING PROTEIN/DNA / CRISPR / Transposition / AAA+ ATPase / Tn7 / DNA BINDING PROTEIN-DNA complex
Function / homologyPHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10)
Function and homology information
Biological speciesScytonema hofmannii (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.2 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
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
Revision 2.0Jan 25, 2023Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Database references / Derived calculations / Polymer sequence / Source and taxonomy / Structure summary
Category: atom_site / em_software ...atom_site / em_software / entity / entity_poly / entity_poly_seq / ndb_struct_na_base_pair / ndb_struct_na_base_pair_step / pdbx_contact_author / pdbx_entity_instance_feature / pdbx_entity_src_syn / pdbx_entry_details / pdbx_poly_seq_scheme / pdbx_validate_close_contact / pdbx_validate_rmsd_angle / struct / struct_conn / struct_ref_seq
Item: _entity.formula_weight / _entity_poly.pdbx_seq_one_letter_code ..._entity.formula_weight / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _pdbx_entity_src_syn.pdbx_end_seq_num / _pdbx_entry_details.has_ligand_of_interest / _struct.title / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_ref_seq.seq_align_end
Description: Ligand geometry / Details: DNA model geometry was corrected / Provider: author / Type: Coordinate replacement

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: TnsC
B: TnsC
C: TnsC
D: TnsC
E: TnsC
F: TnsC
G: TnsC
H: TnsC
I: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
J: DNA (5'-D(P*AP*AP*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)268,26524
Polymers264,43210
Non-polymers3,83314
Water0
1


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

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Components

#1: 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)
#2: 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: 6343.091 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: 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: 6532.386 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Helical filament of TnsC monomers bound to DNA / Type: COMPLEX / Details: Reconstituted with ATPgS / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 45.4 kDa/nm / Experimental value: NO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 7.4 / Details: Sample was diluted into low salt using dialysis.
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaClSodium chloride1
225 mMHEPESC8H18N2O4S1
31 mMDTTC4H10O2S21
42 %glycerolC3H8O31
52 mMMagnesium chlorideMgCl21
6.2 mMATPgSC10H12N5O12P3S1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: This sample comprised monodisperse filaments reconstituted using dialysis.
Specimen supportDetails: Grid cleaned with solvent and examined for defects under light microscope.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Blot for 6 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: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 103.2 K / Temperature (min): 93.15 K
Image recordingAverage exposure time: 3 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3474
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
2Warp1.0.9particle selectionBoxNet2Mask_20180531
7Warp1.0.9CTF correction
8RELION3.1CTF correction
9PHENIX1.17.1model refinement
14cryoSPARC3initial Euler assignment
15RELION3.1initial Euler assignment
17RELION3.1final Euler assignment
19RELION3.1classification
21RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 60 ° / Axial rise/subunit: 6.83 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 1235415 / Details: Neural net trained particle picking.
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 99247 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingB value: 50 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient

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