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- EMDB-34495: type I-B Cascade bound to a PAM-containing dsDNA target at 3.8 an... -

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Basic information

Entry
Database: EMDB / ID: EMD-34495
Titletype I-B Cascade bound to a PAM-containing dsDNA target at 3.8 angstrom resolution.
Map data
Sample
  • Complex: Cryo-EM structure of Synechocystis sp. PCC6714 Cascade bound to a PAM-containing dsDNA target at 3.8 angstrom resolution.
    • RNA: CRISPR RNA
    • DNA: Target DNA
    • DNA: Non target DNA
    • Protein or peptide: CRISPR associated protein Cas5
    • Protein or peptide: CRISPR associated protein Cas7
    • Protein or peptide: CRISPR associated protein Cas8
    • Protein or peptide: CRISPR associated protein Cas11b
KeywordsType I-B / CRISPR-Cas / Cascade / RNA BINDING PROTEIN
Function / homologyCRISPR type MYXAN-associated protein Cmx8 / CRISPR-associated protein Cas5, N-terminal / defense response to virus / Uncharacterized protein / Fruiting body developmental protein R-like protein / Type I-MYXAN CRISPR-associated protein Cmx8
Function and homology information
Biological speciesSynechocystis sp. PCC 6714 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsXiao Y / Lu M / Yu C / Zhang Y
Funding support China, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31970547 China
Ministry of Science and Technology (MoST, China)2018YFA0902000 China
CitationJournal: Nat Commun / Year: 2024
Title: Structure and genome editing of type I-B CRISPR-Cas.
Authors: Meiling Lu / Chenlin Yu / Yuwen Zhang / Wenjun Ju / Zhi Ye / Chenyang Hua / Jinze Mao / Chunyi Hu / Zhenhuang Yang / Yibei Xiao /
Abstract: Type I CRISPR-Cas systems employ multi-subunit effector Cascade and helicase-nuclease Cas3 to target and degrade foreign nucleic acids, representing the most abundant RNA-guided adaptive immune ...Type I CRISPR-Cas systems employ multi-subunit effector Cascade and helicase-nuclease Cas3 to target and degrade foreign nucleic acids, representing the most abundant RNA-guided adaptive immune systems in prokaryotes. Their ability to cause long fragment deletions have led to increasing interests in eukaryotic genome editing. While the Cascade structures of all other six type I systems have been determined, the structure of the most evolutionarily conserved type I-B Cascade is still missing. Here, we present two cryo-EM structures of the Synechocystis sp. PCC 6714 (Syn) type I-B Cascade, revealing the molecular mechanisms that underlie RNA-directed Cascade assembly, target DNA recognition, and local conformational changes of the effector complex upon R-loop formation. Remarkably, a loop of Cas5 directly intercalated into the major groove of the PAM and facilitated PAM recognition. We further characterized the genome editing profiles of this I-B Cascade-Cas3 in human CD3 T cells using mRNA-mediated delivery, which led to unidirectional 4.5 kb deletion in TRAC locus and achieved an editing efficiency up to 41.2%. Our study provides the structural basis for understanding target DNA recognition by type I-B Cascade and lays foundation for harnessing this system for long range genome editing in human T cells.
History
DepositionOct 15, 2022-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_34495.png

Downloads & links

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Map

FileDownload / File: emd_34495.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.)
1.32 Å/pix.
x 300 pix.
= 396. Å		1.32 Å/pix.
x 300 pix.
= 396. Å		1.32 Å/pix.
x 300 pix.
= 396. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.32 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-0.38769025 - 1.4840822
Average (Standard dev.)0.0013211941 (±0.053808544)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 396.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_34495_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_34495_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 : Cryo-EM structure of Synechocystis sp. PCC6714 Cascade bound to a...

EntireName: Cryo-EM structure of Synechocystis sp. PCC6714 Cascade bound to a PAM-containing dsDNA target at 3.8 angstrom resolution.
Components
  • Complex: Cryo-EM structure of Synechocystis sp. PCC6714 Cascade bound to a PAM-containing dsDNA target at 3.8 angstrom resolution.
    • RNA: CRISPR RNA
    • DNA: Target DNA
    • DNA: Non target DNA
    • Protein or peptide: CRISPR associated protein Cas5
    • Protein or peptide: CRISPR associated protein Cas7
    • Protein or peptide: CRISPR associated protein Cas8
    • Protein or peptide: CRISPR associated protein Cas11b

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Supramolecule #1: Cryo-EM structure of Synechocystis sp. PCC6714 Cascade bound to a...

SupramoleculeName: Cryo-EM structure of Synechocystis sp. PCC6714 Cascade bound to a PAM-containing dsDNA target at 3.8 angstrom resolution.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Synechocystis sp. PCC 6714 (bacteria)

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Macromolecule #1: CRISPR RNA

MacromoleculeName: CRISPR RNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 22.64632 KDa
SequenceString:
UGAGCACUUU AUCACCGUGU CCCCAAUCUG GAUAUUUUGU GUGUGUCCAA ACCAUUGAUG CCGUAAGGCG U

GENBANK: GENBANK: CP007544.1

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Macromolecule #2: Target DNA

MacromoleculeName: Target DNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.390262 KDa
SequenceString:
(DA)(DT)(DA)(DA)(DA)(DC)(DA)(DT)(DG)(DG) (DA)

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Macromolecule #3: Non target DNA

MacromoleculeName: Non target DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.705797 KDa
SequenceString:
(DT)(DC)(DC)(DA)(DT)(DG)(DT)(DT)(DA)

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Macromolecule #4: CRISPR associated protein Cas5

MacromoleculeName: CRISPR associated protein Cas5 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Synechocystis sp. PCC 6714 (bacteria) / Strain: PCC 6714
Molecular weightTheoretical: 26.592424 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MAQLALALDT VTRYLRLKAP FAAFRPFQSG SFRSTTPVPS FSAVYGLLLN LAGIEQRQEV EGKVTLIKPK AELPKLAIAI GQVKPSSTS LINQQLHNYP VGNSGKEFAS RTFGSKYWIA PVRREVLVNL DLIIGLQSPV EFWQKLDQGL KGETVINRYG L PFAGDNNF ...String:
MAQLALALDT VTRYLRLKAP FAAFRPFQSG SFRSTTPVPS FSAVYGLLLN LAGIEQRQEV EGKVTLIKPK AELPKLAIAI GQVKPSSTS LINQQLHNYP VGNSGKEFAS RTFGSKYWIA PVRREVLVNL DLIIGLQSPV EFWQKLDQGL KGETVINRYG L PFAGDNNF LFDEIYPIEK PDLASWYCPL EPDTRPNQGA CRLTLWIDRE NNTQTTIKVF SPSDFRLEPP AKAWQQLPG

UniProtKB: Uncharacterized protein

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Macromolecule #5: CRISPR associated protein Cas7

MacromoleculeName: CRISPR associated protein Cas7 / type: protein_or_peptide / ID: 5 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Synechocystis sp. PCC 6714 (bacteria) / Strain: PCC 6714
Molecular weightTheoretical: 33.789074 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MSNLNLFATI LTYPAPASNY RGESEENRSV IQKILKDGQK YAIISPESMR NALREMLIEL GQPNNRTRLH SEDQLAVEFK EYPNPDKFA DDFLFGYMVA QTNDAKEMKK LNRPAKRDSI FRCNMAVAVN PYKYDTVFYQ SPLNAGDSAW KNSTSSALLH R EVTHTAFQ ...String:
MSNLNLFATI LTYPAPASNY RGESEENRSV IQKILKDGQK YAIISPESMR NALREMLIEL GQPNNRTRLH SEDQLAVEFK EYPNPDKFA DDFLFGYMVA QTNDAKEMKK LNRPAKRDSI FRCNMAVAVN PYKYDTVFYQ SPLNAGDSAW KNSTSSALLH R EVTHTAFQ YPFALAGKDC AAKPEWVKAL LQAIAELNGV AGGHARAYYE FAPRSVVARL TPKLVAGYQT YGFDAEGNWL EL SRLTATD SDNLDLPANE FWLGGELVRK MDQEQKAQLE AMGAHLYANP EKLFADLADS FLGV

UniProtKB: Fruiting body developmental protein R-like protein

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Macromolecule #6: CRISPR associated protein Cas8

MacromoleculeName: CRISPR associated protein Cas8 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Synechocystis sp. PCC 6714 (bacteria) / Strain: PCC 6714
Molecular weightTheoretical: 69.287305 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MGSSHHHHHH HHSQWSHPQF EKGGGSGGGS GGSAWSHPQF EKLEVLFQGP GSMPKTQAEI LTLDFNLAEL PSAQHRAGLA GLILMIREL KKWPWFKIRQ KEKDVLLSIE NLDQYGASIQ LNLEGLIALF DLAYLSFTEE RKSKSKIKDF KRVDEIEIEE N GKNKIQKY ...String:
MGSSHHHHHH HHSQWSHPQF EKGGGSGGGS GGSAWSHPQF EKLEVLFQGP GSMPKTQAEI LTLDFNLAEL PSAQHRAGLA GLILMIREL KKWPWFKIRQ KEKDVLLSIE NLDQYGASIQ LNLEGLIALF DLAYLSFTEE RKSKSKIKDF KRVDEIEIEE N GKNKIQKY YFYDVITPQG GFLAGWDKSD GQIWLRIWRD MFWSIIKGVP ATRNPFNNRC GLNLNAGDSF SKDVESVWKS LQ NAEKTTG QSGAFYLGAM AVNAENVSTD DLIKWQFLLH FWAFVAQVYC PYILDKDGKR NFNGYVIVIP DIANLEDFCD ILP DVLSNR NSKAFGFRPQ ESVIDVPEQG ALELLNLIKQ RIAKKAGSGL LSDLIVGVEV IHAEKQGNSI KLHSVSYLQP NEES VDDYN AIKNSYYCPW FRRQLLLNLV NPKFDLASQS WLKRHPWYGF GDLLSRIPQR WLKENNSYFS HDARQLFTQK GDFDM TVAT TKTREYAEIV YKIAQGFVLS KLSSKHDLQW SKCKGNPKLE REYNDKKEKV VNEAFLAIRS RTEKQAFIDY FVSTLY PHV RQDEFVDFAQ KLFQDTDEIR SLTLLALSSQ YPIKRQGETE

UniProtKB: Type I-MYXAN CRISPR-associated protein Cmx8

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Macromolecule #7: CRISPR associated protein Cas11b

MacromoleculeName: CRISPR associated protein Cas11b / type: protein_or_peptide / ID: 7 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Synechocystis sp. PCC 6714 (bacteria) / Strain: PCC 6714
Molecular weightTheoretical: 14.5385 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MTVATTKTRE YAEIVYKIAQ GFVLSKLSSK HDLQWSKCKG NPKLEREYND KKEKVVNEAF LAIRSRTEKQ AFIDYFVSTL YPHVRQDEF VDFAQKLFQD TDEIRSLTLL ALSSQYPIKR QGETE

UniProtKB: Type I-MYXAN CRISPR-associated protein Cmx8

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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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.1 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 33011
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: OTHER

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