+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-37653 | ||||||||||||
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Title | Cryo-EM structure of DiCas7-11 mutant in complex with crRNA | ||||||||||||
Map data | |||||||||||||
Sample |
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Keywords | CRISPR-Cas / Gene editing / IMMUNE SYSTEM | ||||||||||||
Function / homology | : / CRISPR type III-associated protein / RAMP superfamily / defense response to virus / CRISPR-associated RAMP family protein Function and homology information | ||||||||||||
Biological species | Escherichia coli (E. coli) / Desulfonema ishimotonii (bacteria) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.53 Å | ||||||||||||
Authors | Ma HY / Tang XD | ||||||||||||
Funding support | China, 3 items
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Citation | Journal: Signal Transduct Target Ther / Year: 2024 Title: Structural basis of negative regulation of CRISPR-Cas7-11 by TPR-CHAT. Authors: Tian Hong / Qinghua Luo / Haiyun Ma / Xin Wang / Xinqiong Li / Chongrong Shen / Jie Pang / Yan Wang / Yuejia Chen / Changbin Zhang / Zhaoming Su / Haohao Dong / Xiaodi Tang / Abstract: CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as ...CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as a regulatory protein that interacts with CRISPR RNA (crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex (Craspase). However, the precise modulation of Cas7-11's nuclease activity by TPR-CHAT to enhance its utility requires further study. Here, we report cryo-electron microscopy (cryo-EM) structures of Desulfonema ishimotonii (Di) Cas7-11-crRNA, complexed with or without the full length or the N-terminus of TPR-CHAT. These structures unveil the molecular features of the Craspase complex. Structural analysis, combined with in vitro nuclease assay and electrophoretic mobility shift assay, reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT (DiTPR-CHAT). Our work demonstrates that DiTPR-CHAT can function as a small unit of DiCas7-11 regulator, potentially enabling safe applications to prevent overcutting and off-target effects of the CRISPR‒Cas7-11 system. | ||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_37653.map.gz | 89.8 MB | EMDB map data format | |
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Header (meta data) | emd-37653-v30.xml emd-37653.xml | 17.1 KB 17.1 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_37653_fsc.xml | 9.7 KB | Display | FSC data file |
Images | emd_37653.png | 122.4 KB | ||
Masks | emd_37653_msk_1.map | 95 MB | Mask map | |
Filedesc metadata | emd-37653.cif.gz | 6.6 KB | ||
Others | emd_37653_half_map_1.map.gz emd_37653_half_map_2.map.gz | 88 MB 88 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-37653 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-37653 | HTTPS FTP |
-Related structure data
Related structure data | 8wmiMC 8wm4C 8wmcC 8wmlC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_37653.map.gz / Format: CCP4 / Size: 95 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 0.85 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_37653_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_37653_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_37653_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Binary complex of DiCas7-11 mutant with crRNA
Entire | Name: Binary complex of DiCas7-11 mutant with crRNA |
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Components |
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-Supramolecule #1: Binary complex of DiCas7-11 mutant with crRNA
Supramolecule | Name: Binary complex of DiCas7-11 mutant with crRNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: Escherichia coli (E. coli) |
-Macromolecule #1: crRNA (39-MER)
Macromolecule | Name: crRNA (39-MER) / type: rna / ID: 1 / Number of copies: 1 |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 12.463339 KDa |
Sequence | String: UUGAUGUCAC GGAACCUUUG UUGUCUUCGA CAUGGGUAA |
-Macromolecule #2: CRISPR-associated RAMP family protein
Macromolecule | Name: CRISPR-associated RAMP family protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Desulfonema ishimotonii (bacteria) |
Molecular weight | Theoretical: 183.844984 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MTTTMKISIE FLEPFRMTKW QESTRRNKNN KEFVRGQAFA RWHRNKKDNT KGRPYITGTL LRSAVIRSAE NLLTLSDGKI SEKTCCPGK FDTEDKDRLL QLRQRSTLRW TDKNPCPDNA ETYCPFCELL GRSGNDGKKA EKKDWRFRIH FGNLSLPGKP D FDGPKAIG ...String: MTTTMKISIE FLEPFRMTKW QESTRRNKNN KEFVRGQAFA RWHRNKKDNT KGRPYITGTL LRSAVIRSAE NLLTLSDGKI SEKTCCPGK FDTEDKDRLL QLRQRSTLRW TDKNPCPDNA ETYCPFCELL GRSGNDGKKA EKKDWRFRIH FGNLSLPGKP D FDGPKAIG SQRVLNRVDF KSGKAHDFFK AYEVDHTRFP RFEGEITIDN KVSAEARKLL CDSLKFTDRL CGALCVIRFD EY TPAADSG KQTENVQAEP NANLAEKTAE QIISILDDNK KTEYTRLLAD AIRSLRRSSK LVAGLPKDHD GKDDHYLWDI GKK KKDENS VTIRQILTTS ADTKELKNAG KWREFCEKLG EALYLKSKDM SGGLKITRRI LGDAEFHGKP DRLEKSRSVS IGSV LKETV VCGELVAKTP FFFGAIDEDA KQTALQVLLT PDNKYRLPRS AVRGILRRDL QTYFDSPCNA ELGGRPCMCK TCRIM RGIT VMDARSEYNA PPEIRHRTRI NPFTGTVAEG ALFNMEVAPE GIVFPFQLRY RGSEDGLPDA LKTVLKWWAE GQAFMS GAA STGKGRFRME NAKYETLDLS DENQRNDYLK NWGWRDEKGL EELKKRLNSG LPEPGNYRDP KWHEINVSIE MASPFIN GD PIRAAVDKRG TAVVTFVKYK AEGEEAKPVC AYKAESFRGV IRSAVARIHM EDGVPLTELT HSDCECLLCQ IFGSEYEA G KIRFEDLVFE SDPEPVTFDH VAIDRFTGGA ADKKKFDDSP LPGSPARPLM LKGSFWIRRD VLEDEEYCKA LGKALADVN NGLYPLGGKS AIGYGQVKSL GIKGDDKRIS RLMNPAFDET DVAVPEKPKT DAEVRIEAEK VYYPHYFVEP HKKVEREEKP CGHQKFHEG RLTGKIRCKL ITKTPLIVPD TSNDDFFRPA DKEARKEKDE YHKSYAFFRL HKQIMIPGSE LRGMVSSVYE T VTNSCFRI FDETKRLSWR MDADHQNVLQ DFLPGRVTAD GKHIQKFSET ARVPFYDKTQ KHFDILDEQE IAGEKPVRMW VK RFIKRLS LVDPAKHPQK KQDNKWKRRK EGIATFIEQK NGSYYFNVVT NNGCTSFHLW HKPDNFDQEK LEGIQNGEKL DCW VRDSRY QKAFQEIPEN DPDGWECKEG YLHVVGPSKV EFSDKKGDVI NNFQGTLPSV PNDWKTIRTN DFKNRKRKNE PVFC CEDDK GNYYTMAKYC ETFFFDLKEN EEYEIPEKAR IKYKELLRVY NNNPQAVPES VFQSRVAREN VEKLKSGDLV YFKHN EKYV EDIVPVRISR TVDDRMIGKR MSADLRPCHG DWVEDGDLSA LNAYPEKRLL LRHPKGLCPA CRLFGTGSYK GRVRFG FAS LENDPEWLIP GKNPGDPFHG GPVMLSLLER PRPTWSIPGS DNKFKVPGRK FYVHHHAWKT IKDGNHPTTG KAIEQSP NN RTVEALAGGN SFSFEIAFEN LKEWELGLLI HSLQLEKGLA HKLGMAKSMG FGSVEIDVES VRLRKDWKQW RNGNSEIP N WLGKGFAKLK EWFRDELDFI ENLKKLLWFP EGDQAPRVCY PMLRKKDDPN GNSGYEELKD GEFKKEDRQK KLTTPWTPW A UniProtKB: CRISPR-associated RAMP family protein |
-Macromolecule #3: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 3 / Number of copies: 4 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | cell |
-Sample preparation
Concentration | 0.8 mg/mL |
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Buffer | pH: 8 |
Vitrification | Cryogen name: NITROGEN |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 56.6 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.1 µm / Nominal defocus min: 0.8 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |