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- EMDB-35629: Cryo-EM structure of type I-B Cascade bound to a PAM-containing d... -
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Open data
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Basic information
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Title | Cryo-EM structure of type I-B Cascade bound to a PAM-containing dsDNA target at 3.6 angstrom resolution | |||||||||
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![]() | Type I-B / CRISPR-Cas / Cascade / RNA BINDING PROTEIN/RNA/DNA / RNA BINDING PROTEIN-RNA-DNA complex | |||||||||
Function / homology | CRISPR 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![]() | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.6 Å | |||||||||
![]() | Xiao Y / Lu M / Yu C / Zhang Y | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 52.1 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 21.4 KB 21.4 KB | Display Display | ![]() |
Images | ![]() | 52.3 KB | ||
Filedesc metadata | ![]() | 6.7 KB | ||
Others | ![]() ![]() | 95.7 MB 95.7 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8ip0MC ![]() 8h67C M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 1.32 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_35629_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_35629_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Cryo-EM structure of Synechocystis sp. PCC6714 Cascade bound to a...
Entire | Name: Cryo-EM structure of Synechocystis sp. PCC6714 Cascade bound to a PAM-containing dsDNA target presented as full R-loop formed state at 3.6 angstrom resolution |
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Components |
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-Supramolecule #1: Cryo-EM structure of Synechocystis sp. PCC6714 Cascade bound to a...
Supramolecule | Name: Cryo-EM structure of Synechocystis sp. PCC6714 Cascade bound to a PAM-containing dsDNA target presented as full R-loop formed state at 3.6 angstrom resolution type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8 |
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Source (natural) | Organism: ![]() ![]() |
-Macromolecule #1: Type I-MYXAN CRISPR-associated protein Cmx8
Macromolecule | Name: Type I-MYXAN CRISPR-associated protein Cmx8 / type: protein_or_peptide / ID: 1 / Details: GenBank ID WP_071880939 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 63.703344 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MPKTQAEILT LDFNLAELPS AQHRAGLAGL ILMIRELKKW PWFKIRQKEK DVLLSIENLD QYGASIQLNL EGLIALFDLA YLSFTEERK SKSKIKDFKR VDEIEIEENG KNKIQKYYFY DVITPQGGFL AGWDKSDGQI WLRIWRDMFW SIIKGVPATR N PFNNRCGL ...String: MPKTQAEILT LDFNLAELPS AQHRAGLAGL ILMIRELKKW PWFKIRQKEK DVLLSIENLD QYGASIQLNL EGLIALFDLA YLSFTEERK SKSKIKDFKR VDEIEIEENG KNKIQKYYFY DVITPQGGFL AGWDKSDGQI WLRIWRDMFW SIIKGVPATR N PFNNRCGL NLNAGDSFSK DVESVWKSLQ NAEKTTGQSG AFYLGAMAVN AENVSTDDLI KWQFLLHFWA FVAQVYCPYI LD KDGKRNF NGYVIVIPDI ANLEDFCDIL PDVLSNRNSK AFGFRPQESV IDVPEQGALE LLNLIKQRIA KKAGSGLLSD LIV GVEVIH AEKQGNSIKL HSVSYLQPNE ESVDDYNAIK NSYYCPWFRR QLLLNLVNPK FDLASQSWLK RHPWYGFGDL LSRI PQRWL KENNSYFSHD ARQLFTQKGD FDMTVATTKT REYAEIVYKI AQGFVLSKLS SKHDLQWSKC KGNPKLEREY NDKKE KVVN EAFLAIRSRT EKQAFIDYFV STLYPHVRQD EFVDFAQKLF QDTDEIRSLT LLALSSQYPI KRQGETE UniProtKB: Type I-MYXAN CRISPR-associated protein Cmx8 |
-Macromolecule #2: Type I-MYXAN CRISPR-associated protein Cas5/Cmx5/DevS
Macromolecule | Name: Type I-MYXAN CRISPR-associated protein Cas5/Cmx5/DevS / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 26.592424 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #3: Fruiting body developmental protein R-like protein
Macromolecule | Name: Fruiting body developmental protein R-like protein / type: protein_or_peptide / ID: 3 / Number of copies: 7 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 33.789074 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #8: CRISPR associated protein Cas11b
Macromolecule | Name: CRISPR associated protein Cas11b / type: protein_or_peptide / ID: 8 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 14.5385 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MTVATTKTRE YAEIVYKIAQ GFVLSKLSSK HDLQWSKCKG NPKLEREYND KKEKVVNEAF LAIRSRTEKQ AFIDYFVSTL YPHVRQDEF VDFAQKLFQD TDEIRSLTLL ALSSQYPIKR QGETE UniProtKB: Type I-MYXAN CRISPR-associated protein Cmx8 |
-Macromolecule #4: DNA (5'-D(P*TP*CP*CP*AP*TP*GP*TP*TP*TP*AP*TP*CP*AP*CP*C)-3')
Macromolecule | Name: DNA (5'-D(P*TP*CP*CP*AP*TP*GP*TP*TP*TP*AP*TP*CP*AP*CP*C)-3') type: dna / ID: 4 / Details: PAM proximal non target DNA / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 4.494934 KDa |
Sequence | String: (DT)(DC)(DC)(DA)(DT)(DG)(DT)(DT)(DT)(DA) (DT)(DC)(DA)(DC)(DC) |
-Macromolecule #6: DNA (41-MER)
Macromolecule | Name: DNA (41-MER) / type: dna / ID: 6 / Details: Target DNA / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 12.725236 KDa |
Sequence | String: (DA)(DC)(DA)(DC)(DA)(DA)(DA)(DA)(DT)(DA) (DT)(DC)(DC)(DA)(DG)(DA)(DT)(DT)(DG)(DG) (DG)(DG)(DA)(DC)(DA)(DC)(DG)(DG)(DT) (DG)(DA)(DT)(DA)(DA)(DA)(DC)(DA)(DT)(DG) (DG) (DA) |
-Macromolecule #7: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
Macromolecule | Name: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3') / type: dna / ID: 7 / Details: PAM distal non target DNA / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 3.08711 KDa |
Sequence | String: (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) |
-Macromolecule #5: RNA (44-MER)
Macromolecule | Name: RNA (44-MER) / type: rna / ID: 5 / Details: CRISPR RNA / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 13.975212 KDa |
Sequence | String: AGAGCACUUU UAUCACCGUG UCCCCAAUCU GGAUAUUUUG UGUG |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination 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 model | Type of model: NONE |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 92454 |
Initial angle assignment | Type: NOT APPLICABLE |
Final angle assignment | Type: OTHER |