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Yorodumi- EMDB-29896: Exploiting Activation and Inactivation Mechanisms in Type I-C CRI... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-29896 | |||||||||
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Title | Exploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications | |||||||||
Map data | ||||||||||
Sample |
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Keywords | CRISPR / type I-C / cascade / anti-CRISPR / HYDROLASE-RNA complex | |||||||||
Function / homology | Function and homology information maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / RNA binding Similarity search - Function | |||||||||
Biological species | Neisseria lactamica (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.64 Å | |||||||||
Authors | Hu C / Nam KH / Ke A | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Mol Cell / Year: 2024 Title: Exploiting activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications. Authors: Chunyi Hu / Mason T Myers / Xufei Zhou / Zhonggang Hou / Macy L Lozen / Ki Hyun Nam / Yan Zhang / Ailong Ke / Abstract: Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size ...Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here, we use four cryoelectron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9 that strongly inhibit Neisseria lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through allosteric inhibition, whereas AcrIC9 achieves so through direct competition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for type I CRISPR eukaryotic genome engineering. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_29896.map.gz | 79.1 MB | EMDB map data format | |
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Header (meta data) | emd-29896-v30.xml emd-29896.xml | 21.2 KB 21.2 KB | Display Display | EMDB header |
Images | emd_29896.png | 110.6 KB | ||
Filedesc metadata | emd-29896.cif.gz | 6.7 KB | ||
Others | emd_29896_half_map_1.map.gz emd_29896_half_map_2.map.gz | 79.1 MB 79.1 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-29896 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-29896 | HTTPS FTP |
-Validation report
Summary document | emd_29896_validation.pdf.gz | 915.8 KB | Display | EMDB validaton report |
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Full document | emd_29896_full_validation.pdf.gz | 915.4 KB | Display | |
Data in XML | emd_29896_validation.xml.gz | 11.8 KB | Display | |
Data in CIF | emd_29896_validation.cif.gz | 14.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-29896 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-29896 | HTTPS FTP |
-Related structure data
Related structure data | 8gafMC 8g9sC 8g9tC 8g9uC 8gamC 8ganC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_29896.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.4057 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_29896_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_29896_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-lo...
Entire | Name: Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-loop formation |
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Components |
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-Supramolecule #1: Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-lo...
Supramolecule | Name: Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-loop formation type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 |
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Source (natural) | Organism: Neisseria lactamica (bacteria) |
Molecular weight | Theoretical: 400 KDa |
-Macromolecule #1: Cas7
Macromolecule | Name: Cas7 / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO |
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Source (natural) | Organism: Neisseria lactamica (bacteria) |
Molecular weight | Theoretical: 32.208111 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: TIEKRYDFVF LFDVQDGNPN GDPDAGNLPR IDPQTGEGLV TDVCLKRKVR NFIQMTQNDE HHDIFIREKG ILNNLIDEAH EQENVKGKE KGEKTEAARQ YMCSRYYDIR TFGAVMTTGK NAGQVRGPVQ LTFSRSIDPI MTLEHSITRM AVTNEKDASE T GDNRTMGR ...String: TIEKRYDFVF LFDVQDGNPN GDPDAGNLPR IDPQTGEGLV TDVCLKRKVR NFIQMTQNDE HHDIFIREKG ILNNLIDEAH EQENVKGKE KGEKTEAARQ YMCSRYYDIR TFGAVMTTGK NAGQVRGPVQ LTFSRSIDPI MTLEHSITRM AVTNEKDASE T GDNRTMGR KFTVPYGLYR CHGFISTHFA KQTGFSENDL ELFWQALVNM FDHDHSAARG QMNARGLYVF EHSNNLGDAP AD SLFKRIQ VVKKDGVEVV RSFDDYLVSV DDKNLEETKL LRKLGG UniProtKB: UNIPROTKB: A0A378VEU0 |
-Macromolecule #2: Cas8
Macromolecule | Name: Cas8 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Neisseria lactamica (bacteria) |
Molecular weight | Theoretical: 64.715652 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MILHALTQYY QRKAESDGGI AQEGFENKEI PFIIVIDKQG NFIQLEDTRE LKVKKKVGRT FLVPKGLGRS GSKSYEVSNL LWDHYGYVL AYAGEKGQEQ ADKQHASFTA KVNELKQALP DDAGVTAVAA FLSSAEEKSK VMQAANWAEC AKVKGCNLSF R LVDEAVDL ...String: MILHALTQYY QRKAESDGGI AQEGFENKEI PFIIVIDKQG NFIQLEDTRE LKVKKKVGRT FLVPKGLGRS GSKSYEVSNL LWDHYGYVL AYAGEKGQEQ ADKQHASFTA KVNELKQALP DDAGVTAVAA FLSSAEEKSK VMQAANWAEC AKVKGCNLSF R LVDEAVDL VCQSKAVREY VSQANQTQSD NAQKGICLVT GKAAPIARLH NAVKGVNAKP APFASVNLSA FESYGKEQGF AF PIGEQAM FEYTTALNTL LAGENRFRIG DVTTVCWGAK RTPLEESLAS LINGGGKDKP DAHIDAVKAL YKSLYNGQYC KPD GEDKFY LLGLSPNSAR IVVRFWHETT VAALSESIAA WYDDLQMVRG ENSPYPEYMP LPRLLGNLVL DGKMENLPSD LIAQ ITDAA LNNRVLPVSL LQAALRRNKA EQKITYGRAS LLKAYINRAI RAGRLKNMKE LTMGLDRNRQ DIGYVLGRLF AVLEK IQAE ANPGLNATIA DRYFGSASST PIAVFGTLMR LLPHHLNKLE FEGRAVQLQW EIRQILEHCQ RFPNHLNLEQ QGLFAI GYY HETQFLFTKD ALKNLFNEA UniProtKB: Type I-C CRISPR-associated protein Cas8c/Csd1 |
-Macromolecule #3: Cas11
Macromolecule | Name: Cas11 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Neisseria lactamica (bacteria) |
Molecular weight | Theoretical: 14.245184 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: GLDRNRQDIG YVLGRLFAVL EKIQAEANPG LNATIADRYF GSASSTPIAV FGTLMRLLPH HLNKLEFEGR AVQLQWEIRQ ILEHCQRFP NHLNLEQQGL FAIGYYHETQ FLFTKDALKN LFNEA UniProtKB: UNIPROTKB: A0A378VF47 |
-Macromolecule #5: Cas5
Macromolecule | Name: Cas5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Neisseria lactamica (bacteria) |
Molecular weight | Theoretical: 23.870451 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: RFILEISGDL ACFTRSELKV ERVSYPVITP SAARNILMAI LWKPAIRWKV LKIEILKPIQ WTNIRRNEVG TKMSERSGSL YIEDNRQQR ASMLLKDVAY RIHADFDMTS EAGESDNYVK FAEMFKRRAK KGQYFHQPYL GCREFPCDFR LLEKAEDGLP L EDITQDFG ...String: RFILEISGDL ACFTRSELKV ERVSYPVITP SAARNILMAI LWKPAIRWKV LKIEILKPIQ WTNIRRNEVG TKMSERSGSL YIEDNRQQR ASMLLKDVAY RIHADFDMTS EAGESDNYVK FAEMFKRRAK KGQYFHQPYL GCREFPCDFR LLEKAEDGLP L EDITQDFG FMLYDMDFSK SDPRDSNNAE PMFYQCKAVN GVITVPP UniProtKB: pre-crRNA processing endonuclease |
-Macromolecule #4: crRNA (43-MER)
Macromolecule | Name: crRNA (43-MER) / type: rna / ID: 4 / Number of copies: 1 |
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Source (natural) | Organism: Neisseria lactamica (bacteria) |
Molecular weight | Theoretical: 13.816202 KDa |
Sequence | String: GUUGAAACAG GGUCAGCUUG CCGUAGGUGG CAUCGCCCUC GUC |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 7.5 / Component - Concentration: 150.0 mM / Component - Formula: NaCl / Component - Name: sodium chloride / Details: 25mM Tris pH 7.5, 150mM NaCl |
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Temperature | Min: 70.0 K / Max: 100.0 K |
Specialist optics | Energy filter - Name: GIF Bioquantum |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1798 / Number real images: 1798 / Average exposure time: 2.5 sec. / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 1.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 67000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Protocol: RIGID BODY FIT |
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Output model | PDB-8gaf: |