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- PDB-8g9u: Exploiting Activation and Inactivation Mechanisms in Type I-C CRI... -

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Basic information

Entry
Database: PDB / ID: 8g9u
TitleExploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications
Components
  • (CRISPR-associated protein, ...) x 2
  • Cas3
  • Distal nontarget DNA (5'-D(P*TP*TP*AP*TP*AP*TP*TP*AP*AP*TP*AP*TP*TP*AP*TP*AP*TP*TP*TP*A)-3')
  • Phage associated protein
  • Traget strand DNA (53-MER)
  • crRNA (43-MER)
  • pre-crRNA processing endonuclease
  • proximal nontarget strand DNA (5'-D(P*AP*TP*GP*AP*AP*CP*TP*TP*CP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
KeywordsDNA BINDING PROTEIN / CRISPR / type I-C / Cascade / Anti-CRISPR
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 Csd2 / CRISPR-associated protein Cas7, subtype I-B/I-C / CRISPR-associated protein Cas7 / 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
PHOSPHATE ION / : / DNA / DNA (> 10) / RNA / RNA (> 10) / pre-crRNA processing endonuclease / CRISPR-associated protein, Csd1 family / CRISPR-associated protein, Csd2 family
Similarity search - Component
Biological speciesNeisseria lactamica (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsHu, C. / Nam, K.H. / Ke, A.
Funding support United States, 1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
P: Cas3
A: CRISPR-associated protein, Csd2 family
B: CRISPR-associated protein, Csd2 family
D: CRISPR-associated protein, Csd2 family
C: CRISPR-associated protein, Csd2 family
E: CRISPR-associated protein, Csd2 family
F: CRISPR-associated protein, Csd2 family
I: Phage associated protein
G: Phage associated protein
H: CRISPR-associated protein, Csd1 family
J: Phage associated protein
K: crRNA (43-MER)
L: Traget strand DNA (53-MER)
M: CRISPR-associated protein, Csd2 family
N: pre-crRNA processing endonuclease
O: proximal nontarget strand DNA (5'-D(P*AP*TP*GP*AP*AP*CP*TP*TP*CP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
Q: Distal nontarget DNA (5'-D(P*TP*TP*AP*TP*AP*TP*TP*AP*AP*TP*AP*TP*TP*AP*TP*AP*TP*TP*TP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)484,28023
Polymers483,85217
Non-polymers4286
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 5 molecules PIGJN

#1: Protein Cas3


Mass: 84572.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria lactamica (bacteria) / Production host: Escherichia coli (E. coli)
#3: Protein Phage associated protein


Mass: 14245.184 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria lactamica (bacteria) / Gene: NCTC10618_01085 / Production host: Escherichia coli (E. coli)
#7: Protein pre-crRNA processing endonuclease


Mass: 23870.451 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria lactamica (bacteria) / Gene: cas5d, NEILACOT_03978 / Production host: Escherichia coli (E. coli)
References: UniProt: D0W8X4, Hydrolases; Acting on ester bonds

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CRISPR-associated protein, ... , 2 types, 8 molecules ABDCEFMH

#2: Protein
CRISPR-associated protein, Csd2 family


Mass: 32208.111 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria lactamica (bacteria) / Gene: NEILACOT_03980 / Production host: Escherichia coli (E. coli) / References: UniProt: D0W8X6
#4: Protein CRISPR-associated protein, Csd1 family


Mass: 64718.613 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria lactamica (bacteria) / Gene: csd1, NEILACOT_03979 / Production host: Escherichia coli (E. coli) / References: UniProt: D0W8X5

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RNA chain , 1 types, 1 molecules K

#5: RNA chain crRNA (43-MER)


Mass: 13816.202 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria lactamica (bacteria) / Production host: Escherichia coli (E. coli)

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DNA chain , 3 types, 3 molecules LOQ

#6: DNA chain Traget strand DNA (53-MER)


Mass: 16410.494 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Neisseria lactamica (bacteria) / References: GenBank: 1394359882
#8: DNA chain proximal nontarget strand DNA (5'-D(P*AP*TP*GP*AP*AP*CP*TP*TP*CP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')


Mass: 6160.087 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Neisseria lactamica (bacteria)
#9: DNA chain Distal nontarget DNA (5'-D(P*TP*TP*AP*TP*AP*TP*TP*AP*AP*TP*AP*TP*TP*AP*TP*AP*TP*TP*TP*A)-3')


Mass: 6111.011 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria lactamica (bacteria) / Production host: Escherichia coli (E. coli)

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Non-polymers , 2 types, 6 molecules

#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical
ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ternary complex of Cas3 with dsDNA bound type I-C Cascade at R-loop formation
Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT
Molecular weightValue: 0.4 MDa / Experimental value: YES
Source (natural)Organism: Neisseria lactamica (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 7.5 / Details: 25mM Tris pH 7.5, 150mM NaCl
Buffer componentConc.: 150 mM / Name: sodium chloride / Formula: NaCl
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal magnification: 67000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 2500 nm / Calibrated defocus min: 1500 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 70 K
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1798 / Num. of real images: 1798
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2EPU2image acquisition
4cryoSPARC12CTF correction
12cryoSPARCclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 307088 / Num. of class averages: 6 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01134482
ELECTRON MICROSCOPYf_angle_d1.0547158
ELECTRON MICROSCOPYf_dihedral_angle_d34.6085644
ELECTRON MICROSCOPYf_chiral_restr0.0575145
ELECTRON MICROSCOPYf_plane_restr0.0065737

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