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- EMDB-32118: Cryo-EM structure of the Cas13bt3-crRNA-target RNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-32118
TitleCryo-EM structure of the Cas13bt3-crRNA-target RNA ternary complex
Map data
Sample
  • Complex: Cas13bt3-crRNA-target RNA
    • Complex: crRNA-target RNA
      • RNA: crRNA
      • RNA: target RNA
    • Complex: Cas13bt3
      • Protein or peptide: Cas13bt3
Function / homologyUncharacterized protein
Function and homology information
Biological speciesPlanctomycetes bacterium (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsNakagawa R / Soumya K / Han A / Takeda NS / Tomita A / Hirano H / Kusakizako T / Tomohiro N / Yamashita K / Feng Z ...Nakagawa R / Soumya K / Han A / Takeda NS / Tomita A / Hirano H / Kusakizako T / Tomohiro N / Yamashita K / Feng Z / Nishimasu H / Nureki O
Funding support Japan, 1 items
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, 2021-
Header (metadata) releaseSep 7, 2022-
Map releaseSep 7, 2022-
UpdateSep 14, 2022-
Current statusSep 14, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32118.png

Downloads & links

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Map

FileDownload / File: emd_32118.map.gz / Format: CCP4 / Size: 9.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.21733 Å
Density
Contour LevelBy AUTHOR: 0.0348
Minimum - Maximum-0.20375037 - 0.29954737
Average (Standard dev.)0.0005145754 (±0.007485715)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions136136136
Spacing136136136
CellA=B=C: 165.55688 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_32118_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_32118_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_32118_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Cas13bt3-crRNA-target RNA

EntireName: Cas13bt3-crRNA-target RNA
Components
  • Complex: Cas13bt3-crRNA-target RNA
    • Complex: crRNA-target RNA
      • RNA: crRNA
      • RNA: target RNA
    • Complex: Cas13bt3
      • Protein or peptide: Cas13bt3

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Supramolecule #1: Cas13bt3-crRNA-target RNA

SupramoleculeName: Cas13bt3-crRNA-target RNA / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: crRNA-target RNA

SupramoleculeName: crRNA-target RNA / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Planctomycetes bacterium (bacteria)

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Supramolecule #3: Cas13bt3

SupramoleculeName: Cas13bt3 / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #1

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Macromolecule #1: Cas13bt3

MacromoleculeName: Cas13bt3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Planctomycetes bacterium (bacteria)
Molecular weightTheoretical: 90.498789 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GGMAQVSKQT SKKRELSIDE YQGARKWCFT IAFNKALVNR DKNDGLFVES LLRHEKYSKH DWYDEDTRAL IKCSTQAANA KAEALANYF SAYRHSPGCL TFTAEDELRT IMERAYERAI FECRRRETEV IIEFPSLFEG DRITTAGVVF FVSFFVERRV L DRLYGAVS ...String:
GGMAQVSKQT SKKRELSIDE YQGARKWCFT IAFNKALVNR DKNDGLFVES LLRHEKYSKH DWYDEDTRAL IKCSTQAANA KAEALANYF SAYRHSPGCL TFTAEDELRT IMERAYERAI FECRRRETEV IIEFPSLFEG DRITTAGVVF FVSFFVERRV L DRLYGAVS GLKKNEGQYK LTRKALSMYC LKDSRFTKAW DKRVLLFRDI LAQLGRIPAE AYEYYHGEQG DKKRANDNEG TN PKRHKDK FIEFALHYLE AQHSEICFGR RHIVREEAGA GDEHKKHRTK GKVVVDFSKK DEDQSYYISK NNVIVRIDKN AGP RSYRMG LNELKYLVLL SLQGKGDDAI AKLYRYRQHV ENILDVVKVT DKDNHVFLPR FVLEQHGIGR KAFKQRIDGR VKHV RGVWE KKKAATNEMT LHEKARDILQ YVNENCTRSF NPGEYNRLLV CLVGKDVENF QAGLKRLQLA ERIDGRVYSI FAQTS TINE MHQVVCDQIL NRLCRIGDQK LYDYVGLGKK DEIDYKQKVA WFKEHISIRR GFLRKKFWYD SKKGFAKLVE EHLESG GGQ RDVGLDKKYY HIDAIGRFEG ANPALYETLA RDRLCLMMAQ YFLGSVRKEL GNKIVWSNDS IELPVEGSVG NEKSIVF SV SDYGKLYVLD DAEFLGRICE YFMPHEKGKI RYHTVYEKGF RAYNDLQKKC VEAVLAFEEK VVKAKKMSEK EGAHYIDF R EILAQTMCKE AEKTAVNKVA RAFFAHHLKF VIDEFGLFSD VMKKYGIEKE WKFPVK

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Macromolecule #2: crRNA

MacromoleculeName: crRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 19.603619 KDa
SequenceString:
GCUUGGCAAC CAUUCAAAUA UGUAUGCUGG AGCAGCCCCC GAUUUGUGGG GUGAUUACAG C

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Macromolecule #3: target RNA

MacromoleculeName: target RNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.984773 KDa
SequenceString:
AUACAUAUUU GAAUGGUUGC CAAGC

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 48.1 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 3 / Resolution.type: BY AUTHOR / Resolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 2380559
FSC plot (resolution estimation)

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