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- EMDB-29001: Cryo-EM structure of Cascade-PAM complex in type I-B CAST system -

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Basic information

Entry
Database: EMDB / ID: EMD-29001
TitleCryo-EM structure of Cascade-PAM complex in type I-B CAST system
Map data
Sample
  • Complex: Ternary complex of Cascade proteins, crRNA and PAM only DNA
    • Protein or peptide: Type I-B CRISPR-associated protein Cas5
    • Protein or peptide: Type I-B CRISPR-associated protein Cas6
    • Protein or peptide: Type I-B CRISPR-associated protein Cas7
    • Protein or peptide: Type I-MYXAN CRISPR-associated Cas8a1/Cmx1
    • Protein or peptide: Type I-B CRISPR-associated protein Cas11
    • RNA: RNA
    • DNA: Target DNA strand
    • DNA: Non-target DNA strand
KeywordsCRISPR / DNA recognition / DNA BINDING PROTEIN
Function / homology
Function and homology information


defense response to virus
Similarity search - Function
CRISPR-associated protein Cas5, bacterial / CRISPR-associated protein, Cas6-related / CRISPR-associated protein Cas8a1/Csx13, Myxan subtype, N-terminal / CRISPR-associated protein Cas8a1/Csx13, Myxan subtype, C-terminal / Cas6 Crispr / CRISPR-associated protein Cas5, N-terminal
Similarity search - Domain/homology
Type I-MYXAN CRISPR-associated protein Cas5/Cmx5/DevS / CRISPR-associated protein / Type I-MYXAN CRISPR-associated Cas8a1/Cmx1 / Type I-MYXAN CRISPR-associated protein Cas6/Cmx6
Similarity search - Component
Biological speciesPeltigera membranacea (fungus) / Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsChang L / Wang S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Cell / Year: 2023
Title: Molecular mechanism for Tn7-like transposon recruitment by a type I-B CRISPR effector.
Authors: Shukun Wang / Clinton Gabel / Romana Siddique / Thomas Klose / Leifu Chang /
Abstract: Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key ...Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key feature of CASTs is their ability to recruit Tn7-like transposons to nuclease-deficient CRISPR effectors. However, how Tn7-like transposons are recruited by diverse CRISPR effectors remains poorly understood. Here, we present the cryo-EM structure of a recruitment complex comprising the Cascade complex, TniQ, TnsC, and the target DNA in the type I-B CAST from Peltigera membranacea cyanobiont 210A. Target DNA recognition by Cascade induces conformational changes in Cas6 and primes TniQ recruitment through its C-terminal domain. The N-terminal domain of TniQ is bound to the seam region of the TnsC spiral heptamer. Our findings provide insights into the diverse mechanisms for the recruitment of Tn7-like transposons to CRISPR effectors and will aid in the development of CASTs as gene knockin tools.
History
DepositionDec 1, 2022-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateJan 31, 2024-
Current statusJan 31, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29001.png

Downloads & links

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Map

FileDownload / File: emd_29001.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.054 Å
Density
Contour LevelBy AUTHOR: 0.126
Minimum - Maximum-0.6506219 - 1.3892628
Average (Standard dev.)0.0038060923 (±0.021297224)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 404.73602 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_29001_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_29001_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 Cascade proteins, crRNA and PAM only DNA

EntireName: Ternary complex of Cascade proteins, crRNA and PAM only DNA
Components
  • Complex: Ternary complex of Cascade proteins, crRNA and PAM only DNA
    • Protein or peptide: Type I-B CRISPR-associated protein Cas5
    • Protein or peptide: Type I-B CRISPR-associated protein Cas6
    • Protein or peptide: Type I-B CRISPR-associated protein Cas7
    • Protein or peptide: Type I-MYXAN CRISPR-associated Cas8a1/Cmx1
    • Protein or peptide: Type I-B CRISPR-associated protein Cas11
    • RNA: RNA
    • DNA: Target DNA strand
    • DNA: Non-target DNA strand

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Supramolecule #1: Ternary complex of Cascade proteins, crRNA and PAM only DNA

SupramoleculeName: Ternary complex of Cascade proteins, crRNA and PAM only DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Peltigera membranacea (fungus)

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Macromolecule #1: Type I-B CRISPR-associated protein Cas5

MacromoleculeName: Type I-B CRISPR-associated protein Cas5 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria)
Molecular weightTheoretical: 24.852906 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIAPLILYLD VPFTTFRESH AREMGKTYPV PPPATVYGML LSLVGETNVY RHCGVELAIA MLSSPKKSRI LRQMRRFKNA DFSHPENVI PCYQEILSNL KCLIWVRSDE EKIQPSLRER IQLAFDHPEL VRRFGCLFLG ESDQLIKTIK LAREDYLEGV R QWAIRDNR ...String:
MIAPLILYLD VPFTTFRESH AREMGKTYPV PPPATVYGML LSLVGETNVY RHCGVELAIA MLSSPKKSRI LRQMRRFKNA DFSHPENVI PCYQEILSNL KCLIWVRSDE EKIQPSLRER IQLAFDHPEL VRRFGCLFLG ESDQLIKTIK LAREDYLEGV R QWAIRDNR GRLTLPYWVD HVGSRNTRFL RYRIEEMDRL SPPDLAWTMV QSPI

UniProtKB: Type I-MYXAN CRISPR-associated protein Cas5/Cmx5/DevS

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Macromolecule #2: Type I-B CRISPR-associated protein Cas6

MacromoleculeName: Type I-B CRISPR-associated protein Cas6 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria)
Molecular weightTheoretical: 24.945744 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTAILIQSEE YVDLTFKLRG APIPLDNGYL TYAALSRICP PLHELKSIGI HPIAGIPTRN NLLELTAQSR LKIRIYHQQI PLIYPYLAG QAFHIGQNFY QLDIPDYKPL ISSESVYSRL VIIKGFQDST NFIEAVQRQM DNLGIQGKIE LLTRQDGTPQ R RQLTINKE ...String:
MTAILIQSEE YVDLTFKLRG APIPLDNGYL TYAALSRICP PLHELKSIGI HPIAGIPTRN NLLELTAQSR LKIRIYHQQI PLIYPYLAG QAFHIGQNFY QLDIPDYKPL ISSESVYSRL VIIKGFQDST NFIEAVQRQM DNLGIQGKIE LLTRQDGTPQ R RQLTINKE GKQFKVRGFG VKISELNPED SLTLQEQGIG GKRKMMCGIF VPATRSKEEE ET

UniProtKB: Type I-MYXAN CRISPR-associated protein Cas6/Cmx6

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Macromolecule #3: Type I-B CRISPR-associated protein Cas7

MacromoleculeName: Type I-B CRISPR-associated protein Cas7 / type: protein_or_peptide / ID: 3 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria)
Molecular weightTheoretical: 37.298996 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MMTQKKNDSN IPNYYLYGTV LTRYGLASLN HDIRRGNKTI LQKGYWNNGK IHSFVGSSAI RWALRFYLQK QGYLVNRVWD EEEHINRLT SEDFDPEKFY DDDIFGFALL ESAETEEDTS TTKRKKKQTK TSTPNQRMGA LGMNMAVSLT PYDGAVKLGA K SGREKDST ...String:
MMTQKKNDSN IPNYYLYGTV LTRYGLASLN HDIRRGNKTI LQKGYWNNGK IHSFVGSSAI RWALRFYLQK QGYLVNRVWD EEEHINRLT SEDFDPEKFY DDDIFGFALL ESAETEEDTS TTKRKKKQTK TSTPNQRMGA LGMNMAVSLT PYDGAVKLGA K SGREKDST SLHFTEYHAT RYQYYFGIDA THLKDFSRIL PMIDGIMNLP KVGGSSNIFN YPFCPDSLVF QWTNHFASYI SY CFEYCDP KSKEAKLSQE FIDEVECGQI DPSKLWIGGT IVKDLQQLDN FESSPLNKAH IYRNRNEMIE ALKTVIKRDL GLE ESK

UniProtKB: CRISPR-associated protein

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Macromolecule #4: Type I-MYXAN CRISPR-associated Cas8a1/Cmx1

MacromoleculeName: Type I-MYXAN CRISPR-associated Cas8a1/Cmx1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria)
Molecular weightTheoretical: 63.474328 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MHHHHHHHHI VSTQPKISLS LHAADTTIMH RVGMTGLYMT LKRLEKQYPL SRQRGGHISW FLTADTIELF WEGSDFIALS WLINESFQL DDTGLIHLVG LDNDRIDLRQ KIHIHEGICG VFLRLNKFYQ AGEIINTELR FEEKQVEYQY KSLTWYAHQT F AEKLCEAD ...String:
MHHHHHHHHI VSTQPKISLS LHAADTTIMH RVGMTGLYMT LKRLEKQYPL SRQRGGHISW FLTADTIELF WEGSDFIALS WLINESFQL DDTGLIHLVG LDNDRIDLRQ KIHIHEGICG VFLRLNKFYQ AGEIINTELR FEEKQVEYQY KSLTWYAHQT F AEKLCEAD TQQLRHDYIQ ITSWLYLGGI VRHARTQNTT KLEEKPEYAL ALLFVPVVCH YCLLHIPSED LKERKPHRYL VV IPEIKDF EDASQRRWRL QQLETKQFHV SSLGEAGLLY YSLDDIQPEV AYYQACQVWL YEKTNKASRQ RTLMSIEEIK IDK NILITY QQVQKYFKTN YQIIKYKQIF IKVNPIRSLI ADNLVKGIHW WSNFWEKLVI EDSKEYLFNQ LFSNREGFII MAEN SEEDK QYLIFIKVFQ QAMKGNFAKI YAKTEEGKDP PIKKKVERLR AELNYCYDEL SFKEYLSDFL VRGGLNKYFN EHQEE IALL IKKSPWQEIR IWSLLAIASY KPKDKLTNRD DSSLSNNQKL EEVNDDSEEE

UniProtKB: Type I-MYXAN CRISPR-associated Cas8a1/Cmx1

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Macromolecule #5: Type I-B CRISPR-associated protein Cas11

MacromoleculeName: Type I-B CRISPR-associated protein Cas11 / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Nostoc sp. 'Peltigera membranacea cyanobiont' 210A (bacteria)
Molecular weightTheoretical: 16.314365 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAENSEEDKQ YLIFIKVFQQ AMKGNFAKIY AKTEEGKDPP IKKKVERLRA ELNYCYDELS FKEYLSDFLV RGGLNKYFNE HQEEIALLI KKSPWQEIRI WSLLAIASYK PKDKLTNRDD SSLSNNQKLE EVNDDSEEE

UniProtKB: Type I-MYXAN CRISPR-associated Cas8a1/Cmx1

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Macromolecule #6: RNA

MacromoleculeName: RNA / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 22.876527 KDa
SequenceString:
UUGCUCAAGA GAAGUCAUUU AAUAAGGCCA CUGUUAAACG UAGGUGAGUC GUGGCUUUAU GCCGUUAGGC G

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Macromolecule #7: Target DNA strand

MacromoleculeName: Target DNA strand / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.003993 KDa
SequenceString:
(DC)(DA)(DT)(DG)(DA)(DT)(DC)(DT)(DA)(DC)

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Macromolecule #8: Non-target DNA strand

MacromoleculeName: Non-target DNA strand / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.973018 KDa
SequenceString:
(DG)(DT)(DA)(DG)(DA)(DT)(DC)(DA)(DT)(DG) (DG)(DA)(DG)(DA)(DA)(DG)(DT)(DC)(DA)(DT) (DT)(DT)(DA)(DA)(DT)(DA)(DA)(DG)(DG) (DC)(DC)(DA)(DC)(DT)(DG)(DT)(DT)(DA)(DA) (DA) (DC)(DG)(DT)(DA)(DG)

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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: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8fcj

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 204496

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