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- EMDB-29896: Exploiting Activation and Inactivation Mechanisms in Type I-C CRI... -

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Basic information

Entry
Database: EMDB / ID: EMD-29896
TitleExploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications
Map data
Sample
  • Complex: Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-loop formation
    • Protein or peptide: Cas7
    • Protein or peptide: Cas8Roberval (Air Saguenay) Water Aerodrome
    • Protein or peptide: Cas11
    • RNA: crRNA (43-MER)
    • Protein or peptide: Cas5
KeywordsCRISPR / 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
CRISPR-associated protein, Csd1-type / CRISPR-associated protein (Cas_Csd1) / CRISPR pre-crRNA endoribonuclease Cas5d / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) / CRISPR-associated protein Cas5, N-terminal
Similarity search - Domain/homology
Type I-C CRISPR-associated protein Cas8c/Csd1 / : / : / pre-crRNA processing endonuclease
Similarity search - Component
Biological speciesNeisseria lactamica (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.64 Å
AuthorsHu C / Nam KH / Ke A
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR)GM118174 United States
CitationJournal: 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
DepositionFeb 22, 2023-
Header (metadata) releaseMar 6, 2024-
Map releaseMar 6, 2024-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29896.png

Downloads & links

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Map

FileDownload / File: emd_29896.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.4057 Å
Density
Contour LevelBy AUTHOR: 0.119
Minimum - Maximum-0.42682198 - 0.9831813
Average (Standard dev.)-0.001205841 (±0.034581985)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 393.59598 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_29896_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_29896_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 : Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-lo...

EntireName: Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-loop formation
Components
  • Complex: Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-loop formation
    • Protein or peptide: Cas7
    • Protein or peptide: Cas8Roberval (Air Saguenay) Water Aerodrome
    • Protein or peptide: Cas11
    • RNA: crRNA (43-MER)
    • Protein or peptide: Cas5

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Supramolecule #1: Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-lo...

SupramoleculeName: 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
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 400 KDa

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Macromolecule #1: Cas7

MacromoleculeName: Cas7 / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 32.208111 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #2: Cas8

MacromoleculeName: Cas8 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 64.715652 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #3: Cas11

MacromoleculeName: Cas11 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 14.245184 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GLDRNRQDIG YVLGRLFAVL EKIQAEANPG LNATIADRYF GSASSTPIAV FGTLMRLLPH HLNKLEFEGR AVQLQWEIRQ ILEHCQRFP NHLNLEQQGL FAIGYYHETQ FLFTKDALKN LFNEA

UniProtKB: UNIPROTKB: A0A378VF47

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Macromolecule #5: Cas5

MacromoleculeName: Cas5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 23.870451 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #4: crRNA (43-MER)

MacromoleculeName: crRNA (43-MER) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Neisseria lactamica (bacteria)
Molecular weightTheoretical: 13.816202 KDa
SequenceString:
GUUGAAACAG GGUCAGCUUG CCGUAGGUGG CAUCGCCCUC GUC

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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

Concentration1 mg/mL
BufferpH: 7.5 / Component - Concentration: 150.0 mM / Component - Formula: NaClSodium chloride / Component - Name: sodium chloride / Details: 25mM Tris pH 7.5, 150mM NaCl
GridModel: 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.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 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
Specialist opticsEnergy filter - Name: GIF Bioquantum
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 100.0 K
Image recordingFilm 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
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 103405

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-8gaf:
Exploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications

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