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- PDB-8fd3: Cryo-EM structure of Cascade-PAM complex in type I-B CAST system -

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Basic information

Entry
Database: PDB / ID: 8fd3
TitleCryo-EM structure of Cascade-PAM complex in type I-B CAST system
Components
  • (Type I-B CRISPR-associated protein ...) x 4
  • Non-target DNA strand
  • RNA
  • Target DNA strand
  • Type I-MYXAN CRISPR-associated Cas8a1/Cmx1
KeywordsDNA BINDING PROTEIN / CRISPR / DNA recognition
Function / homology
Function and homology information


defense response to virus
Similarity search - Function
CRISPR-associated protein Cas5, bacterial / CRISPR-associated protein, Cas6-related / CRISPR-associated protein Cas8a1/Csx13, Myxan subtype, N-terminal / CRISPR-associated protein Cas8a1/Csx13, Myxan subtype, C-terminal / Cas6 Crispr / CRISPR-associated protein Cas5, N-terminal
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Type I-MYXAN CRISPR-associated protein Cas5/Cmx5/DevS / CRISPR-associated protein / Type I-MYXAN CRISPR-associated Cas8a1/Cmx1 / Type I-MYXAN CRISPR-associated protein Cas6/Cmx6
Similarity search - Component
Biological speciesNostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsChang, L. / Wang, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Cell / Year: 2023
Title: Molecular mechanism for Tn7-like transposon recruitment by a type I-B CRISPR effector.
Authors: Shukun Wang / Clinton Gabel / Romana Siddique / Thomas Klose / Leifu Chang /
Abstract: Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key ...Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key feature of CASTs is their ability to recruit Tn7-like transposons to nuclease-deficient CRISPR effectors. However, how Tn7-like transposons are recruited by diverse CRISPR effectors remains poorly understood. Here, we present the cryo-EM structure of a recruitment complex comprising the Cascade complex, TniQ, TnsC, and the target DNA in the type I-B CAST from Peltigera membranacea cyanobiont 210A. Target DNA recognition by Cascade induces conformational changes in Cas6 and primes TniQ recruitment through its C-terminal domain. The N-terminal domain of TniQ is bound to the seam region of the TnsC spiral heptamer. Our findings provide insights into the diverse mechanisms for the recruitment of Tn7-like transposons to CRISPR effectors and will aid in the development of CASTs as gene knockin tools.
History
DepositionDec 1, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / 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.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Sep 27, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jan 31, 2024Group: Source and taxonomy / Structure summary / Category: entity / entity_src_gen
Item: _entity.pdbx_description / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Type I-B CRISPR-associated protein Cas5
B: Type I-B CRISPR-associated protein Cas6
C: Type I-B CRISPR-associated protein Cas7
D: Type I-B CRISPR-associated protein Cas7
E: Type I-B CRISPR-associated protein Cas7
F: Type I-B CRISPR-associated protein Cas7
G: Type I-B CRISPR-associated protein Cas7
H: Type I-B CRISPR-associated protein Cas7
I: Type I-MYXAN CRISPR-associated Cas8a1/Cmx1
J: Type I-B CRISPR-associated protein Cas11
K: Type I-B CRISPR-associated protein Cas11
L: Type I-B CRISPR-associated protein Cas11
M: RNA
N: Target DNA strand
O: Non-target DNA strand


Theoretical massNumber of molelcules
Total (without water)425,86415
Polymers425,86415
Non-polymers00
Water0
1


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

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Components

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Type I-B CRISPR-associated protein ... , 4 types, 11 molecules ABCDEFGHJKL

#1: Protein Type I-B CRISPR-associated protein Cas5


Mass: 24852.906 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria)
Gene: cas5 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IG00
#2: Protein Type I-B CRISPR-associated protein Cas6


Mass: 24945.744 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria)
Gene: cas6 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IH92
#3: Protein
Type I-B CRISPR-associated protein Cas7


Mass: 37298.996 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria)
Gene: CDG76_09080 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IG15
#5: Protein Type I-B CRISPR-associated protein Cas11


Mass: 16314.365 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria)
Gene: CDG76_09085 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IGR9

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DNA chain , 2 types, 2 molecules NO

#7: DNA chain Target DNA strand


Mass: 3003.993 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#8: DNA chain Non-target DNA strand


Mass: 13973.018 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules IM

#4: Protein Type I-MYXAN CRISPR-associated Cas8a1/Cmx1


Mass: 63474.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria)
Gene: CDG76_09085 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A235IGR9
#6: RNA chain RNA /


Mass: 22876.527 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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: Ternary complex of Cascade proteins, crRNA and PAM only DNA
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Peltigera membranacea (fungus)161997
31synthetic construct (others)32630
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 204496 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00327710
ELECTRON MICROSCOPYf_angle_d0.54337937
ELECTRON MICROSCOPYf_dihedral_angle_d16.834539
ELECTRON MICROSCOPYf_chiral_restr0.0394070
ELECTRON MICROSCOPYf_plane_restr0.0044450

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