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- PDB-7lyt: Cryo-EM structure of CasPhi-2 (Cas12j) bound to crRNA and Phospho... -

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Basic information

Entry
Database: PDB / ID: 7lyt
TitleCryo-EM structure of CasPhi-2 (Cas12j) bound to crRNA and Phosphorothioate-DNA
Components
  • CasPhi
  • NTS-DNA*
  • TS-DNA
  • crRNA
KeywordsVIRAL PROTEIN/RNA/DNA / CRISPR / CasPhi / Cas12j / Nuclease / R-loop / crRNA / PAM / RNP / Complex / VIRAL PROTEIN-RNA-DNA / VIRAL PROTEIN-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesBiggievirus Mos11
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsPausch, P. / Soczek, K. / Nogales, E. / Doudna, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Advancing Translational Sciences (NIH/NCATS)U01AI142817-02 United States
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: DNA interference states of the hypercompact CRISPR-CasΦ effector.
Authors: Patrick Pausch / Katarzyna M Soczek / Dominik A Herbst / Connor A Tsuchida / Basem Al-Shayeb / Jillian F Banfield / Eva Nogales / Jennifer A Doudna /
Abstract: CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and ...CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA, we determined cryo-EM structures of CasΦ (Cas12j) in pre- and post-DNA-binding states. The structures reveal a streamlined protein architecture that tightly encircles the CRISPR RNA and DNA target to capture, unwind and cleave DNA. Comparison of the pre- and post-DNA-binding states reveals how the protein rearranges for DNA cleavage upon target recognition. On the basis of these structures, we created and tested mutant forms of CasΦ that cut DNA up to 20-fold faster relative to wild type, showing how this system may be naturally attenuated to improve the fidelity of DNA interference. The structural and mechanistic insights into how CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing.
History
DepositionMar 8, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 4, 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 / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
A: CasPhi
B: crRNA
C: TS-DNA
D: NTS-DNA*
hetero molecules


Theoretical massNumber of molelcules
Total (without water)127,4117
Polymers127,2974
Non-polymers1143
Water00
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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DNA chain , 2 types, 2 molecules CD

#3: DNA chain TS-DNA


Mass: 13615.714 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Biggievirus Mos11
#4: DNA chain NTS-DNA*


Mass: 13072.435 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Biggievirus Mos11

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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein CasPhi


Mass: 86127.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Biggievirus Mos11 / Plasmid: pPP085 / Details (production host): Addgene #158795 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Star
#2: RNA chain crRNA


Mass: 14481.651 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Biggievirus Mos11

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Non-polymers , 2 types, 3 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

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: Cryo-EM map of CasPhi bound to crRNA and phosphorothioate-DNA in the presence of the nuclease magnesium cofactor
Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.128 MDa / Experimental value: NO
Source (natural)Organism: Biggievirus Mos11
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: NITROGEN / Humidity: 100 % / Chamber temperature: 281 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: OTHER
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.18_3845refinement
PHENIX1.18_3845refinement
EM software
IDNameVersionCategory
4cryoSPARC2.15CTF correction
10cryoSPARC2.15initial Euler assignment
11cryoSPARC2.15final Euler assignment
12cryoSPARC2.15classification
13cryoSPARC2.153D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 410553 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 67.58 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00357981
ELECTRON MICROSCOPYf_angle_d0.724311217
ELECTRON MICROSCOPYf_chiral_restr0.03931269
ELECTRON MICROSCOPYf_plane_restr0.00381113
ELECTRON MICROSCOPYf_dihedral_angle_d26.11241785

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