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- PDB-6e9f: EsCas13d-crRNA-target RNA ternary complex -

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Basic information

Entry
Database: PDB / ID: 6e9f
TitleEsCas13d-crRNA-target RNA ternary complex
Components
  • EsCas13d
  • RNA (27-MER)
  • crRNA (52-MER)
KeywordsRNA BINDING PROTEIN/RNA / CRISPR-Cas / RNase / Complex / RNA BINDING PROTEIN-RNA complex
Function / homologyRNA / RNA (> 10) / Uncharacterized protein
Function and homology information
Biological species[Eubacterium] siraeum DSM 15702 (bacteria)
bacterium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsZhang, C. / Lyumkis, D.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)DP5 OD021396 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)U54GM103368 United States
CitationJournal: Cell / Year: 2018
Title: Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d.
Authors: Cheng Zhang / Silvana Konermann / Nicholas J Brideau / Peter Lotfy / Xuebing Wu / Scott J Novick / Timothy Strutzenberg / Patrick R Griffin / Patrick D Hsu / Dmitry Lyumkis /
Abstract: CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems ...CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.
History
DepositionAug 1, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 3, 2018Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2019Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 13, 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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Structure viewerMolecule:
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Assembly

Deposited unit
A: EsCas13d
B: crRNA (52-MER)
C: RNA (27-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)135,8364
Polymers135,8123
Non-polymers241
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11320 Å2
ΔGint-112 kcal/mol
Surface area46280 Å2

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Components

#1: Protein EsCas13d


Mass: 110522.570 Da / Num. of mol.: 1 / Mutation: R295A, H300A, R849A, H854A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) [Eubacterium] siraeum DSM 15702 (bacteria)
Gene: EUBSIR_02687 / Production host: Escherichia coli (E. coli) / References: UniProt: B0MS50
#2: RNA chain crRNA (52-MER)


Mass: 16715.051 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) bacterium (bacteria)
#3: RNA chain RNA (27-MER)


Mass: 8574.064 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) bacterium (bacteria)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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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 EsCas13d(R295A/H300A/R849A/H854A) with crRNA, target RNA and Mg2+
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.135 MDa / Experimental value: NO
Source (natural)Organism: unidentified bacterium (bacteria)
Source (recombinant)Organism: Escherchia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HCl1
2100 mMsodium chlorideNaCl1
31 mMDTT1
45 %glycerol1
51 mMmagnesium chlorideMgCl21
60.1 %(w/v)Amphipol A8-351
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 80 %

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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 FIELD / Nominal magnification: 39000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 57.3 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2205
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

EM software
IDNameCategory
1FindEMparticle selection
2Leginonimage acquisition
4RELIONCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11cisTEMfinal Euler assignment
12RELIONclassification
13cisTEM3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51885 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient

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