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- PDB-7kfu: Cas6-RT-Cas1--Cas2 complex -

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Basic information

Entry
Database: PDB / ID: 7kfu
TitleCas6-RT-Cas1--Cas2 complex
Components
  • Cas2
  • Cas6-RT-Cas1
KeywordsHYDROLASE / Complex / CRISPR Cas Protein / Reverse Transcriptase
Biological speciesThiomicrospira sp. (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsHoel, C.M. / Wang, J.Y. / Doudna, J.A. / Brohawn, S.G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM123496 United States
CitationJournal: Nat Commun / Year: 2021
Title: Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complex.
Authors: Joy Y Wang / Christopher M Hoel / Basem Al-Shayeb / Jillian F Banfield / Stephen G Brohawn / Jennifer A Doudna /
Abstract: CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation ...CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1-Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference.
History
DepositionOct 14, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 31, 2021Provider: repository / Type: Initial release
Revision 1.1May 19, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Cas2
B: Cas2
C: Cas6-RT-Cas1
D: Cas6-RT-Cas1
E: Cas6-RT-Cas1
F: Cas6-RT-Cas1


Theoretical massNumber of molelcules
Total (without water)474,6446
Polymers474,6446
Non-polymers00
Water0
1


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

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Components

#1: Protein Cas2 /


Mass: 11583.263 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: N-terminal residues SNA are from the expression tag. Protein sequence corresponds to GenBank NCN43132.1.
Source: (gene. exp.) Thiomicrospira sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta
#2: Protein
Cas6-RT-Cas1


Mass: 112869.414 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: Protein sequence corresponds to GenBank NCN66177.1.
Source: (gene. exp.) Thiomicrospira sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta

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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: Cas6-RT-Cas1--Cas2 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.47 MDa / Experimental value: NO
Source (natural)Organism: Thiomicrospira sp. (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Rosetta
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Blot force 1, 3 second blot

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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
Image recordingElectron dose: 49.9 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategory
1RELION3particle selection
4CTFFIND4.1CTF correction
10RELION3initial Euler assignment
11RELION3.1final Euler assignment
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 129175 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 183.8 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005522332
ELECTRON MICROSCOPYf_angle_d0.821530174
ELECTRON MICROSCOPYf_chiral_restr0.053312
ELECTRON MICROSCOPYf_plane_restr0.00553870
ELECTRON MICROSCOPYf_dihedral_angle_d21.38458362

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