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- PDB-7vtn: Cryo-EM structure of the Cas13bt3-crRNA-target RNA ternary complex -

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Basic information

Entry
Database: PDB / ID: 7vtn
TitleCryo-EM structure of the Cas13bt3-crRNA-target RNA ternary complex
Components
  • Cas13bt3
  • crRNA
  • target RNA
KeywordsRNA BINDING PROTEIN/RNA / CRISPR-Cas / RNA BINDING PROTEIN-RNA COMPLEX
Function / homologyRNA / RNA (> 10) / Uncharacterized protein
Function and homology information
Biological speciesPlanctomycetes bacterium (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsNakagawa, R. / Soumya, K. / Han, A. / Takeda, N.S. / Tomita, A. / Hirano, H. / Kusakizako, T. / Tomohiro, N. / Yamashita, K. / Feng, Z. ...Nakagawa, R. / Soumya, K. / Han, A. / Takeda, N.S. / Tomita, A. / Hirano, H. / Kusakizako, T. / Tomohiro, N. / Yamashita, K. / Feng, Z. / Nishimasu, H. / Nureki, O.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED) Japan
CitationJournal: Mol Cell / Year: 2022
Title: Structure and engineering of the minimal type VI CRISPR-Cas13bt3.
Authors: Ryoya Nakagawa / Soumya Kannan / Han Altae-Tran / Satoru N Takeda / Atsuhiro Tomita / Hisato Hirano / Tsukasa Kusakizako / Tomohiro Nishizawa / Keitaro Yamashita / Feng Zhang / Hiroshi ...Authors: Ryoya Nakagawa / Soumya Kannan / Han Altae-Tran / Satoru N Takeda / Atsuhiro Tomita / Hisato Hirano / Tsukasa Kusakizako / Tomohiro Nishizawa / Keitaro Yamashita / Feng Zhang / Hiroshi Nishimasu / Osamu Nureki /
Abstract: Type VI CRISPR-Cas13 effector enzymes catalyze RNA-guided RNA cleavage and have been harnessed for various technologies, such as RNA detection, targeting, and editing. Recent studies identified ...Type VI CRISPR-Cas13 effector enzymes catalyze RNA-guided RNA cleavage and have been harnessed for various technologies, such as RNA detection, targeting, and editing. Recent studies identified Cas13bt3 (also known as Cas13X.1) as a miniature Cas13 enzyme, which can be used for knockdown and editing of target transcripts in mammalian cells. However, the action mechanism of the compact Cas13bt3 remains unknown. Here, we report the structures of the Cas13bt3-guide RNA complex and the Cas13bt3-guide RNA-target RNA complex. The structures revealed how Cas13bt3 recognizes the guide RNA and its target RNA and provided insights into the activation mechanism of Cas13bt3, which is distinct from those of the other Cas13a/d enzymes. Furthermore, we rationally engineered enhanced Cas13bt3 variants and ultracompact RNA base editors. Overall, this study improves our mechanistic understanding of the CRISPR-Cas13 enzymes and paves the way for the development of efficient Cas13-mediated transcriptome modulation technologies.
History
DepositionOct 30, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 7, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 14, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jun 26, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / refine
Item: _em_admin.last_update / _refine.ls_d_res_high / _refine.ls_d_res_low

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cas13bt3
B: crRNA
C: target RNA


Theoretical massNumber of molelcules
Total (without water)118,0873
Polymers118,0873
Non-polymers00
Water00
1


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

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Components

#1: Protein Cas13bt3


Mass: 90498.789 Da / Num. of mol.: 1 / Mutation: R84A, H89A, R739A, H744A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Planctomycetes bacterium (bacteria) / Gene: DRP66_05270 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A660UUL5
#2: RNA chain crRNA


Mass: 19603.619 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: RNA chain target RNA


Mass: 7984.773 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

Component
IDNameTypeEntity IDParent-IDSource
1Cas13bt3-crRNA-target RNACOMPLEXall0MULTIPLE SOURCES
2crRNA-target RNACOMPLEX#2-#31SYNTHETIC
3Cas13bt3COMPLEX#11RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Planctomycetes bacterium (bacteria)
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
Crystal growMethod: vapor diffusion, hanging drop

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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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 48.1 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0352 / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2380559 / Num. of class averages: 3 / Symmetry type: POINT
RefinementResolution: 3.38→3.38 Å / Cor.coef. Fo:Fc: 0.905 / SU B: 29.392 / SU ML: 0.497 / ESU R: 0.461
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.43425 --
obs0.43425 87459 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 191.023 Å2
Refinement stepCycle: 1 / Total: 7305
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.0137686
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg1.4981.70210610
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.9316.941076
ELECTRON MICROSCOPYr_dihedral_angle_2_deg2.114552
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.931101065
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.140.2071228
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.025132
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it1.98618.912763
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it3.74228.3493445
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it1.219.44923
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined16.92471392507
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3→3.078 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.619 6445 -
obs--100 %

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