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- EMDB-29433: Cryo-EM structure of the Cas13bt3-crRNA-target RNA ternary comple... -

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Basic information

Entry
Database: EMDB / ID: EMD-29433
TitleCryo-EM structure of the Cas13bt3-crRNA-target RNA ternary complex in activated state
Map data
Sample
  • Complex: Ternary complex of Cas13bt3 in complex with crRNA and 30 nt target RNA
    • Complex: Integrase
      • Protein or peptide: Integrase
    • Complex: crRNA-linker-target hairpin RNA
      • RNA: crRNA-linker-target hairpin RNA
KeywordsCas13bt3-crRNA-target RNA ternary complex / activated state / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homologyUncharacterized protein
Function and homology information
Biological speciesPlanctomycetota bacterium (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsGao Y / Deng X
Funding support United States, 1 items
OrganizationGrant numberCountry
Cancer Prevention and Research Institute of Texas (CPRIT)RR190046 United States
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis for the activation of a compact CRISPR-Cas13 nuclease.
Authors: Xiangyu Deng / Emmanuel Osikpa / Jie Yang / Seye J Oladeji / Jamie Smith / Xue Gao / Yang Gao /
Abstract: The CRISPR-Cas13 ribonucleases have been widely applied for RNA knockdown and transcriptional modulation owing to their high programmability and specificity. However, the large size of Cas13 ...The CRISPR-Cas13 ribonucleases have been widely applied for RNA knockdown and transcriptional modulation owing to their high programmability and specificity. However, the large size of Cas13 effectors and their non-specific RNA cleavage upon target activation limit the adeno-associated virus based delivery of Cas13 systems for therapeutic applications. Herein, we report detailed biochemical and structural characterizations of a compact Cas13 (Cas13bt3) suitable for adeno-associated virus delivery. Distinct from many other Cas13 systems, Cas13bt3 cleaves the target and other nonspecific RNA at internal "UC" sites and is activated in a target length-dependent manner. The cryo-electron microscope structure of Cas13bt3 in a fully active state illustrates the structural basis of Cas13bt3 activation. Guided by the structure, we obtain engineered Cas13bt3 variants with minimal off-target cleavage yet maintained target cleavage activities. In conclusion, our biochemical and structural data illustrate a distinct mechanism for Cas13bt3 activation and guide the engineering of Cas13bt3 applications.
History
DepositionJan 12, 2023-
Header (metadata) releaseSep 27, 2023-
Map releaseSep 27, 2023-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29433.png

Downloads & links

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Map

FileDownload / File: emd_29433.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 300 pix.
= 261. Å		0.87 Å/pix.
x 300 pix.
= 261. Å		0.87 Å/pix.
x 300 pix.
= 261. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.87 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.138
Minimum - Maximum-0.51884085 - 0.9649469
Average (Standard dev.)0.00017220096 (±0.0204329)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 261.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_29433_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29433_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of Cas13bt3 in complex with crRNA and 30 nt target RNA

EntireName: Ternary complex of Cas13bt3 in complex with crRNA and 30 nt target RNA
Components
  • Complex: Ternary complex of Cas13bt3 in complex with crRNA and 30 nt target RNA
    • Complex: Integrase
      • Protein or peptide: Integrase
    • Complex: crRNA-linker-target hairpin RNA
      • RNA: crRNA-linker-target hairpin RNA

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Supramolecule #1: Ternary complex of Cas13bt3 in complex with crRNA and 30 nt target RNA

SupramoleculeName: Ternary complex of Cas13bt3 in complex with crRNA and 30 nt target RNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Planctomycetota bacterium (bacteria)
Molecular weightTheoretical: 123.7 KDa

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Supramolecule #2: Integrase

SupramoleculeName: Integrase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #3: crRNA-linker-target hairpin RNA

SupramoleculeName: crRNA-linker-target hairpin RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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Macromolecule #1: Integrase

MacromoleculeName: Integrase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Planctomycetota bacterium (bacteria)
Molecular weightTheoretical: 90.38468 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAQVSKQTSK KRELSIDEYQ GARKWCFTIA FNKALVNRDK NDGLFVESLL RHEKYSKHDW YDEDTRALIK CSTQAANAKA EALANYFSA YRHSPGCLTF TAEDELRTIM ERAYERAIFE CRRRETEVII EFPSLFEGDR ITTAGVVFFV SFFVERRVLD R LYGAVSGL ...String:
MAQVSKQTSK KRELSIDEYQ GARKWCFTIA FNKALVNRDK NDGLFVESLL RHEKYSKHDW YDEDTRALIK CSTQAANAKA EALANYFSA YRHSPGCLTF TAEDELRTIM ERAYERAIFE CRRRETEVII EFPSLFEGDR ITTAGVVFFV SFFVERRVLD R LYGAVSGL KKNEGQYKLT RKALSMYCLK DSRFTKAWDK RVLLFRDILA QLGRIPAEAY EYYHGEQGDK KRANDNEGTN PK RHKDKFI EFALHYLEAQ HSEICFGRRH IVREEAGAGD EHKKHRTKGK VVVDFSKKDE DQSYYISKNN VIVRIDKNAG PRS YRMGLN ELKYLVLLSL QGKGDDAIAK LYRYRQHVEN ILDVVKVTDK DNHVFLPRFV LEQHGIGRKA FKQRIDGRVK HVRG VWEKK KAATNEMTLH EKARDILQYV NENCTRSFNP GEYNRLLVCL VGKDVENFQA GLKRLQLAER IDGRVYSIFA QTSTI NEMH QVVCDQILNR LCRIGDQKLY DYVGLGKKDE IDYKQKVAWF KEHISIRRGF LRKKFWYDSK KGFAKLVEEH LESGGG QRD VGLDKKYYHI DAIGRFEGAN PALYETLARD RLCLMMAQYF LGSVRKELGN KIVWSNDSIE LPVEGSVGNE KSIVFSV SD YGKLYVLDDA EFLGRICEYF MPHEKGKIRY HTVYEKGFRA YNDLQKKCVE AVLAFEEKVV KAKKMSEKEG AHYIDFRE I LAQTMCKEAE KTAVNKVARA FFAHHLKFVI DEFGLFSDVM KKYGIEKEWK FPVK

UniProtKB: Uncharacterized protein

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Macromolecule #2: crRNA-linker-target hairpin RNA

MacromoleculeName: crRNA-linker-target hairpin RNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 33.005457 KDa
SequenceString:
GGAAGAAGAG UUUAUUCAGA UAGAUUUGUC CUUCUUCGGA CAAAUCUAUC UGAAUAAACU CUUCUUCGCU GGAGCAGCCC CCGAUUUGU GGGGUGAUUA CAGC

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.8 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 49.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 37228
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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