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- PDB-7tra: Cascade complex from type I-A CRISPR-Cas system -

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Basic information

Entry
Database: PDB / ID: 7tra
TitleCascade complex from type I-A CRISPR-Cas system
Components
  • (CRISPR-associated ...) x 2
  • Cas11a
  • Cas5a
  • Cas7a
  • Cas8a
  • Non-Target strand DNA (25-MER)
  • Target strand DNA (44-MER)
  • crRNA (44-MER)
KeywordsRNA BINDING PROTEIN / CRISPR / cascade / Cas3 / type I-A / genome editing / DNA targeting
Function / homology
Function and homology information


catalytic activity, acting on DNA / DNA conformation change / exonuclease activity / defense response to virus / endonuclease activity / nucleic acid binding / Hydrolases; Acting on ester bonds / ATP binding / metal ion binding
Similarity search - Function
CRISPR-associated protein, MJ0385 / CRISPR-associated protein (Cas_Csa4) / CRISPR-associated protein, Cas5a type / : / CRISPR-associated protein Cas7/Cst2/DevR, subtype I-a/Apern / CRISPR-associated protein Cas7/Cst2/DevR / : / CRISPR-associated negative auto-regulator DevR/Csa2 / Helicase Cas3, CRISPR-associated, core / Cas3, HD domain ...CRISPR-associated protein, MJ0385 / CRISPR-associated protein (Cas_Csa4) / CRISPR-associated protein, Cas5a type / : / CRISPR-associated protein Cas7/Cst2/DevR, subtype I-a/Apern / CRISPR-associated protein Cas7/Cst2/DevR / : / CRISPR-associated negative auto-regulator DevR/Csa2 / Helicase Cas3, CRISPR-associated, core / Cas3, HD domain / CRISPR-associated nuclease/helicase Cas3, C-terminal / CRISPR-associated Cas3-type HD domain / CRISPR-associated Cas3-type HD domain superfamily / HD Cas3-type domain profile. / CRISPR-associated protein Cas5, N-terminal / : / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
NICKEL (II) ION / DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated helicase Cas3 / Type I-A CRISPR-associated protein Cas5 / Uncharacterized protein / Type I-A CRISPR-associated protein Cas7/Csa2 / CRISPR-associated endonuclease Cas3-HD / Type I-A CRISPR-associated protein Cas8a2/Csa4
Similarity search - Component
Biological speciesPyrococcus furiosus DSM 3638 (archaea)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsHu, C. / Ni, D. / Nam, K.H. / Majumdar, S. / McLean, J. / Stahlberg, H. / Terns, M. / Ke, A.
Funding support United States, Switzerland, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118117 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118160 United States
Swiss National Science FoundationNCCR Switzerland
CitationJournal: Mol Cell / Year: 2022
Title: Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools.
Authors: Chunyi Hu / Dongchun Ni / Ki Hyun Nam / Sonali Majumdar / Justin McLean / Henning Stahlberg / Michael P Terns / Ailong Ke /
Abstract: Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme ...Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme Cas3 is then recruited in trans for processive DNA degradation. Contrary to this model, here, we show that type I-A Cascade and Cas3 function as an integral effector complex. We provide four cryoelectron microscopy (cryo-EM) snapshots of the Pyrococcus furiosus (Pfu) type I-A effector complex in different stages of DNA recognition and degradation. The HD nuclease of Cas3 is autoinhibited inside the effector complex. It is only allosterically activated upon full R-loop formation, when the entire targeted region has been validated by the RNA guide. The mechanistic insights inspired us to convert Pfu Cascade-Cas3 into a high-sensitivity, low-background, and temperature-activated nucleic acid detection tool. Moreover, Pfu CRISPR-Cas3 shows robust bi-directional deletion-editing activity in human cells, which could find usage in allele-specific inactivation of disease-causing mutations.
History
DepositionJan 28, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 10, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 17, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Oct 9, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated helicase Cas3
B: Cas8a
C: Cas11a
D: Cas11a
H: Cas7a
I: Cas7a
J: Cas7a
K: Cas7a
O: Cas5a
Q: CRISPR-associated endonuclease Cas3-HD
R: crRNA (44-MER)
S: Target strand DNA (44-MER)
T: Non-Target strand DNA (25-MER)
E: Cas11a
F: Cas11a
G: Cas11a
L: Cas7a
M: Cas7a
N: Cas7a
hetero molecules


Theoretical massNumber of molelcules
Total (without water)509,67321
Polymers509,55619
Non-polymers1172
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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CRISPR-associated ... , 2 types, 2 molecules AQ

#1: Protein CRISPR-associated helicase Cas3


Mass: 61412.555 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1 / Gene: cas3, PFDSM3638_03195 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A5C0XNV5
#6: Protein CRISPR-associated endonuclease Cas3-HD / CRISPR-associated ss-nucleic acid exo- and endonuclease Cas3-HD


Mass: 24778.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1 / Gene: cas3, cas3'', PF0639 / Production host: Escherichia coli (E. coli)
References: UniProt: Q8U336, Hydrolases; Acting on ester bonds

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Protein , 4 types, 14 molecules BCDEFGHIJKLMNO

#2: Protein Cas8a


Mass: 38800.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1 / Gene: PF0637 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8U338
#3: Protein
Cas11a


Mass: 12208.933 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1 / Gene: PF0643 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8U332
#4: Protein
Cas7a


Mass: 36989.148 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1 / Gene: PF0642 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8U333
#5: Protein Cas5a


Mass: 29147.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1 / Gene: cas5a, PFDSM3638_03200 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A5C0XNV9

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DNA chain , 2 types, 2 molecules ST

#8: DNA chain Target strand DNA (44-MER)


Mass: 13692.797 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#9: DNA chain Non-Target strand DNA (25-MER)


Mass: 7727.109 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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RNA chain / Non-polymers , 2 types, 3 molecules R

#10: Chemical ChemComp-NI / NICKEL (II) ION


Mass: 58.693 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ni / Feature type: SUBJECT OF INVESTIGATION
#7: RNA chain crRNA (44-MER)


Mass: 14028.332 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: DE3

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Details

Has ligand of interestY
Has protein modificationY

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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: full R-loop state of Cas3-Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system
Type: COMPLEX / Details: full R-loop state / Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.45 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Geobacter sulfurreducens (bacteria)243231
21Escherichia coli (E. coli)562
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
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: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: 6 seconds

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 1209
EM imaging opticsPhase plate: VOLTA PHASE PLATE

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC10particle selection
2EMAN9image acquisition
4EMANCTF correction
12cryoSPARCclassification
13RELION3D reconstruction
Image processingDetails: full R-loop state of type I-A CRISPR/cas complex
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 44773 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00235789
ELECTRON MICROSCOPYf_angle_d0.58948970
ELECTRON MICROSCOPYf_dihedral_angle_d14.6675678
ELECTRON MICROSCOPYf_chiral_restr0.0435603
ELECTRON MICROSCOPYf_plane_restr0.0045878

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