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Yorodumi- EMDB-29877: Exploiting Activation and Inactivation Mechanisms in Type I-C CRI... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-29877 | |||||||||
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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 / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding Similarity search - Function | |||||||||
Biological species | Neisseria lactamica (bacteria) / Rhodobacter phage RcNL1 (virus) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.4 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_29877.map.gz | 40.6 MB | EMDB map data format | |
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Header (meta data) | emd-29877-v30.xml emd-29877.xml | 23.9 KB 23.9 KB | Display Display | EMDB header |
Images | emd_29877.png | 124.3 KB | ||
Filedesc metadata | emd-29877.cif.gz | 6.7 KB | ||
Others | emd_29877_additional_1.map.gz emd_29877_half_map_1.map.gz emd_29877_half_map_2.map.gz | 27.5 MB 40.6 MB 40.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-29877 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-29877 | HTTPS FTP |
-Related structure data
Related structure data | 8g9sMC 8g9tC 8g9uC 8gafC 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_29877.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 1.4124 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: #1
File | emd_29877_additional_1.map | ||||||||||||
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Density Histograms |
-Half map: #2
File | emd_29877_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_29877_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Binary complex of AcrIC8 with crRNA bound type I-C Cascade
Entire | Name: Binary complex of AcrIC8 with crRNA bound type I-C Cascade |
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Components |
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-Supramolecule #1: Binary complex of AcrIC8 with crRNA bound type I-C Cascade
Supramolecule | Name: Binary complex of AcrIC8 with crRNA bound type I-C Cascade type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6 |
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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 BL21(DE3) (bacteria) |
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: Cas11
Macromolecule | Name: Cas11 / type: protein_or_peptide / ID: 2 / Number of copies: 4 / 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 #3: Cas5
Macromolecule | Name: Cas5 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Neisseria lactamica (bacteria) |
Molecular weight | Theoretical: 23.854451 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: RFILEISGDL ACFTRSELKV ERVSYPVITP AAARNILMAI LWKPAIRWKV LKIEILKPIQ WTNIRRNEVG TKMSERSGSL YIEDNRQQR ASMLLKDVAY RIHADFDMTS EAGESDNYVK FAEMFKRRAK KGQYFHQPYL GCREFPCDFR LLEKAEDGLP L EDITQDFG ...String: RFILEISGDL ACFTRSELKV ERVSYPVITP AAARNILMAI LWKPAIRWKV LKIEILKPIQ WTNIRRNEVG TKMSERSGSL YIEDNRQQR ASMLLKDVAY RIHADFDMTS EAGESDNYVK FAEMFKRRAK KGQYFHQPYL GCREFPCDFR LLEKAEDGLP L EDITQDFG FMLYDMDFSK SDPRDSNNAE PMFYQCKAVN GVITVPP UniProtKB: pre-crRNA processing endonuclease |
-Macromolecule #5: Cas8
Macromolecule | Name: Cas8 / 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: 45.864527 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MILHALTQYY QRKAESAQKG ICLVTGKAAP IARLHNAVKG VNAKPAPFAS VNLSAFESYG KEQGFAFPIG EQAMFEYTTA LNTLLAGEN RFRIGDVTTV CWGAKRTPLE ESLASMINGG GKDKPDEHID AVKTLYKSLY NGQYQKPDGK EKFYLLGLSP N SARIVVRF ...String: MILHALTQYY QRKAESAQKG ICLVTGKAAP IARLHNAVKG VNAKPAPFAS VNLSAFESYG KEQGFAFPIG EQAMFEYTTA LNTLLAGEN RFRIGDVTTV CWGAKRTPLE ESLASMINGG GKDKPDEHID AVKTLYKSLY NGQYQKPDGK EKFYLLGLSP N SARIVVRF WHETTVAALS ESIAAWYDDL QMVRGENSPY PEYMPLPRLL GNLVLDGKME NLPSDLIAQI TDAALNNRVL PV SLLQAAL RRNKAEQKIT YGRASLLKAY INRAIRAGRL KNMKELTMGL DRNRQDIGYV LGRLFAVLEK IQAEANPGLN ATI ADRYFG SASSTPIAVF GTLMRLLPHH LNKLEFEGRA VQLQWEIRQI LEHCQRFPNH LNLEQQGLFA IGYYHETQFL FTKD ALKNL FNEA UniProtKB: UNIPROTKB: A0A378VF47 |
-Macromolecule #6: AcrIC8
Macromolecule | Name: AcrIC8 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Rhodobacter phage RcNL1 (virus) |
Molecular weight | Theoretical: 8.190161 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: SMYAIRKIQF FYGPTDKKSY VGEEAGGRRE LFKTRAEAQA RIEDLEEGVY YLAHNESGRP DYKIVWVRGE |
-Macromolecule #4: RNA (42-MER)
Macromolecule | Name: RNA (42-MER) / type: rna / ID: 4 / Number of copies: 1 |
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Source (natural) | Organism: Neisseria lactamica (bacteria) |
Molecular weight | Theoretical: 13.495021 KDa |
Sequence | String: AUUGAAACAG GGUCAGCUUG CCGUAGGUGG CAUCGCCCUC GU |
-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. / Pretreatment - Pressure: 0.00039000000000000005 kPa |
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: 1200 / Number real images: 1200 / 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 |