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- PDB-7vti: Crystal structure of the Cas13bt3-crRNA binary complex -

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Basic information

Entry
Database: PDB / ID: 7vti
TitleCrystal structure of the Cas13bt3-crRNA binary complex
Components
  • Cas13bt3
  • crRNA
KeywordsRNA BINDING PROTEIN/RNA / RNA BINDING PROTEIN-RNA COMPLEX
Function / homologyBROMIDE ION / RNA / RNA (> 10) / Uncharacterized protein
Function and homology information
Biological speciesPlanctomycetes bacterium (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.89 Å
AuthorsNakagawa, R. / Takeda, N.S. / Tomita, A. / Hirano, H. / Kusakizako, T. / Nishizawa, T. / Yamashita, K. / 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 29, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 24, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 14, 2022Group: Database references / Category: 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.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Aug 23, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Revision 1.3Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cas13bt3
B: crRNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)105,96433
Polymers104,2682
Non-polymers1,69631
Water6,107339
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12910 Å2
ΔGint-66 kcal/mol
Surface area38170 Å2
MethodPISA
Unit cell
Length a, b, c (Å)78.434, 95.452, 125.283
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein Cas13bt3


Mass: 91061.547 Da / Num. of mol.: 1 / Mutation: R840A,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: 13206.819 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 5 types, 370 molecules

#3: Chemical
ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical...
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 22 / Source method: obtained synthetically / Formula: C2H6O2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-BR / BROMIDE ION / Bromide


Mass: 79.904 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Br / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 339 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.26 Å3/Da / Density % sol: 45.6 %
Description: THE ENTRY CONTAINS FRIEDEL PAIRS IN I/F_PLUS/MINUS COLUMNS.
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / Details: 10% PEG3350, 0.2 M Sodium bromide

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL41XU / Wavelength: 0.978 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 30, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.978 Å / Relative weight: 1
ReflectionResolution: 1.89→48.99 Å / Num. obs: 146058 / % possible obs: 99.9 % / Redundancy: 13.981 % / Biso Wilson estimate: 45.565 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.128 / Rrim(I) all: 0.133 / Χ2: 1.334 / Net I/σ(I): 14.59 / Num. measured all: 2042011
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsCC1/2Rrim(I) all% possible all
1.89-213.8862.8830.9332610423652234840.522.99299.3
2-2.1414.4431.5921.9132050922192221920.7621.65100
2.14-2.3113.7520.8833.6328497720722207220.910.917100
2.31-2.5313.9250.5126.6126479219016190160.9710.532100
2.53-2.8314.4590.28512.1124897817219172190.9930.296100
2.83-3.2613.7730.1522.2220931615198151980.9980.156100
3.26-3.9913.7760.07539.2617665512823128230.9990.078100
3.99-5.6313.7830.05451.33136586991099100.9990.056100
5.63-48.9913.4890.04555.8974093550254930.9990.04799.8

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Processing

Software
NameVersionClassification
REFMAC5.8.0291refinement
XSCALEdata scaling
PDB_EXTRACT3.27data extraction
XDSdata reduction
SHELXDEphasing
RefinementMethod to determine structure: SAD / Resolution: 1.89→48.99 Å / Cor.coef. Fo:Fc: 0.963 / Cor.coef. Fo:Fc free: 0.944 / SU B: 3.688 / SU ML: 0.102 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.153 / ESU R Free: 0.147 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2429 2287 3 %RANDOM
Rwork0.1957 ---
obs0.1971 73944 99.9 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 166.37 Å2 / Biso mean: 46.065 Å2 / Biso min: 21.73 Å2
Baniso -1Baniso -2Baniso -3
1-0.73 Å2-0 Å2-0 Å2
2---2.65 Å20 Å2
3---1.92 Å2
Refinement stepCycle: final / Resolution: 1.89→48.99 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6033 856 97 339 7325
Biso mean--60.27 46.94 -
Num. residues----776
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0137290
X-RAY DIFFRACTIONr_bond_other_d0.0020.0166458
X-RAY DIFFRACTIONr_angle_refined_deg1.7971.6759957
X-RAY DIFFRACTIONr_angle_other_deg1.2861.59514871
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.485743
X-RAY DIFFRACTIONr_dihedral_angle_2_deg30.73721.21372
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.654151118
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.471554
X-RAY DIFFRACTIONr_chiral_restr0.1290.2957
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.027704
X-RAY DIFFRACTIONr_gen_planes_other0.0020.021719
LS refinement shellResolution: 1.89→1.936 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.388 165 -
Rwork0.326 5336 -
obs--98.71 %

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