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- EMDB-47902: Active state of wild-type EsCas13d ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-47902
TitleActive state of wild-type EsCas13d ternary complex
Map data
Sample
  • Complex: The active state of EsCas13d protein with crRNA and matched target
    • Protein or peptide: EsCas13d
    • RNA: crRNA
    • RNA: Target RNA (matched)
  • Ligand: MAGNESIUM ION
KeywordsCas13 / CRISPR / HEPN / RNA nuclease / RNA BINDING PROTEIN-RNA complex
Function / homologyUncharacterized protein
Function and homology information
Biological species[Eubacterium] siraeum DSM 15702 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.07 Å
AuthorsChou CW / Finkelstein IJ
Funding support United States, 1 items
OrganizationGrant numberCountry
Welch FoundationF-1808 United States
CitationJournal: Nat Commun / Year: 2024
Title: Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons.
Authors: Kuang Hu / Chia-Wei Chou / Claus O Wilke / Ilya J Finkelstein /
Abstract: CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can't acquire new spacers. Here, we ...CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can't acquire new spacers. Here, we report that CASTs can co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that CASTs co-occur with defense-associated CRISPR systems, with the highest prevalence for type I-B and type V CAST sub-types. Using an E. coli quantitative transposition assay and in vitro reconstitution, we show that CASTs can use CRISPR RNAs from these defense systems. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B CRISPR RNA reveals that Cas6 recognizes direct repeats via sequence-independent π - π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via an unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA or a single guide RNA reduces, but does not abrogate, off-target integration for type V CASTs. Our findings suggest that some CASTs may exploit defense-associated CRISPR arrays and that this fact must be considered when porting CASTs to heterologous bacterial hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.
History
DepositionNov 13, 2024-
Header (metadata) releaseAug 6, 2025-
Map releaseAug 6, 2025-
UpdateAug 6, 2025-
Current statusAug 6, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47902.png

Downloads & links

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Map

FileDownload / File: emd_47902.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.89 Å/pix.
x 280 pix.
= 249.96 Å		0.89 Å/pix.
x 280 pix.
= 249.96 Å		0.89 Å/pix.
x 280 pix.
= 249.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.89271 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.00086689385 - 2.2931433
Average (Standard dev.)0.0031842368 (±0.04538125)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 249.95999 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_47902_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_47902_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 : The active state of EsCas13d protein with crRNA and matched target

EntireName: The active state of EsCas13d protein with crRNA and matched target
Components
  • Complex: The active state of EsCas13d protein with crRNA and matched target
    • Protein or peptide: EsCas13d
    • RNA: crRNA
    • RNA: Target RNA (matched)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: The active state of EsCas13d protein with crRNA and matched target

SupramoleculeName: The active state of EsCas13d protein with crRNA and matched target
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: [Eubacterium] siraeum DSM 15702 (bacteria)
Molecular weightTheoretical: 135 KDa

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Macromolecule #1: EsCas13d

MacromoleculeName: EsCas13d / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: [Eubacterium] siraeum DSM 15702 (bacteria)
Molecular weightTheoretical: 110.828938 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGKKIHARDL REQRKTDRTE KFADQNKKRE AERAVPKKDA AVSVKSVSSV SSKKDNVTKS MAKAAGVKSV FAVGNTVYMT SFGRGNDAV LEQKIVDTSH EPLNIDDPAY QLNVVTMNGY SVTGHRGETV SAVTDNPLRR FNGRKKDEPE QSVPTDMLCL K PTLEKKFF ...String:
MGKKIHARDL REQRKTDRTE KFADQNKKRE AERAVPKKDA AVSVKSVSSV SSKKDNVTKS MAKAAGVKSV FAVGNTVYMT SFGRGNDAV LEQKIVDTSH EPLNIDDPAY QLNVVTMNGY SVTGHRGETV SAVTDNPLRR FNGRKKDEPE QSVPTDMLCL K PTLEKKFF GKEFDDNIHI QLIYNILDIE KILAVYSTNA IYALNNMSAD ENIENSDFFM KRTTDETFDD FEKKKESTNS RE KADFDAF EKFIGNYRLA YFADAFYVNK KNPKGKAKNV LREDKELYSV LTLIGKLRHW CVHSEEGRAE FWLYKLDELK DDF KNVLDV VYNRPVEEIN NRFIENNKVN IQILGSVYKN TDIAELVRSY YEFLITKKYK NMGFSIKKLR ESMLEGKGYA DKEY DSVRN KLYQMTDFIL YTGYINEDSD RADDLVNTLR SSLKEDDKTT VYCKEADYLW KKYRESIREV ADALDGDNIK KLSKS NIEI QEDKLRKCFI SYADSVSEFT KLIYLLTRFL SGKEINDLVT TLINKFDNIR SFLEIMDELG LDRTFTAEYS FFEGST KYL AELVELNSFV KSCSFDINAK RTMYRDALDI LGIESDKTEE DIEKMIDNIL QIDANGDKKL KKNNGLRNFI ASNVIDS NR FKYLVRYGNP KKIRETAKCK PAVRFVLNEI PDAQIERYYE ACCPKNTALC SANKRREKLA DMIAEIKFEN FSDAGNYQ K ANVTSRTSEA EIKRKNQAII RLYLTVMYIM LKNLVNVNAR YVIAFHCVER DTKLYAESGL EVGNIEKNKT NLTMAVMGV KLENGIIKTE FDKSFAENAA NRYLRNARWY KLILDNLKKS ERAVVNEFRN TVCHLNAIRN ININIKEIKE VENYFALYHY LIQKHLENR FADKKVERDT GDFISKLEEH KTYCKDFVKA YCTPFGYNLV RYKNLTIDGL FDKNYPGKDD SDEQK

UniProtKB: Uncharacterized protein

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Macromolecule #2: crRNA

MacromoleculeName: crRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: [Eubacterium] siraeum DSM 15702 (bacteria)
Molecular weightTheoretical: 16.739029 KDa
SequenceString:
CACCCGUGCA AAAAUGCAGG GGUCUAAAAC UGAGCGGAUA ACCUCUCUAU GG

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Macromolecule #3: Target RNA (matched)

MacromoleculeName: Target RNA (matched) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: [Eubacterium] siraeum DSM 15702 (bacteria)
Molecular weightTheoretical: 9.588762 KDa
SequenceString:
CGUACCAUAG AGAGGUUAUC CGCUCCACGA

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.135 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 7786 / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6e9f

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 176095
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 100 / Avg.num./class: 50000 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-9ec8:
Active state of wild-type EsCas13d ternary complex

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