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- PDB-7r2k: elongated Cascade complex from type I-A CRISPR-Cas system -

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Basic information

Entry
Database: PDB / ID: 7r2k
Titleelongated Cascade complex from type I-A CRISPR-Cas system
Components
  • (CRISPR-associated ...) x 2
  • Cas11a
  • Cas7a
  • Cas8Roberval (Air Saguenay) Water Aerodrome
  • DNA (5'-D(P*TP*AP*CP*AP*AP*GP*GP*GP*A)-3')
  • DNA (5'-D(P*TP*CP*CP*C)-3')
  • Type I-A CRISPR-associated protein Cas5
  • crRNA (57-MER)
KeywordsANTIVIRAL PROTEIN / elongated Cascade complex / cascade / type I-A / genome editing
Function / homology
Function and homology information


exonuclease activity / helicase activity / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / nucleic acid binding / hydrolase activity / 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 / CRISPR-associated Cas3-type HD domain / CRISPR-associated Cas3-type HD domain superfamily / 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 / CRISPR-associated Cas3-type HD domain / CRISPR-associated Cas3-type HD domain superfamily / Cas3, HD domain / HD Cas3-type domain profile. / CRISPR-associated protein Cas5, N-terminal / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / Helicase conserved C-terminal domain / 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 / 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. / Terns, M. / Stahlberg, H. / 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
Swiss National Science Foundation(NCCR) TransCure Switzerland
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118160 United States
CitationJournal: To Be Published
Title: Structural snapshots for an atypic type I CRISPR-Cas system
Authors: Ni, D. / Hu, C. / Nam, K.H. / Terns, M. / Stahlberg, H. / Ke, A.
History
DepositionFeb 4, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 16, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated helicase Cas3
C: Cas11a
D: DNA (5'-D(P*TP*AP*CP*AP*AP*GP*GP*GP*A)-3')
E: Cas11a
F: Cas11a
G: Cas11a
H: Cas11a
I: Cas11a
J: Cas11a
K: Cas7a
L: Cas7a
M: Cas7a
N: Cas7a
O: Cas7a
P: Cas7a
Q: CRISPR-associated endonuclease Cas3-HD
R: Cas7a
S: Type I-A CRISPR-associated protein Cas5
T: Cas7a
U: crRNA (57-MER)
V: Cas7a
W: Cas7a
X: DNA (5'-D(P*TP*CP*CP*C)-3')
Y: Cas8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)647,33126
Polymers647,21324
Non-polymers1172
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
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: 74475.742 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
#5: Protein CRISPR-associated endonuclease Cas3-HD / CRISPR-associated ss-nucleic acid exo- and endonuclease Cas3-HD


Mass: 27130.014 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, 19 molecules CEFGHIJKLMNOPRTVWSY

#2: Protein
Cas11a


Mass: 12208.933 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: PF0643 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8U332
#4: Protein
Cas7a


Mass: 36989.148 Da / Num. of mol.: 10
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
#6: Protein Type I-A CRISPR-associated protein Cas5


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
#9: Protein Cas8 / Roberval (Air Saguenay) Water Aerodrome


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

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DNA chain , 2 types, 2 molecules DX

#3: DNA chain DNA (5'-D(P*TP*AP*CP*AP*AP*GP*GP*GP*A)-3')


Mass: 2788.862 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#8: DNA chain DNA (5'-D(P*TP*CP*CP*C)-3')


Mass: 1126.781 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)

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

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


Mass: 58.693 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ni
#7: RNA chain crRNA (57-MER)


Mass: 18389.883 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)

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Details

Has ligand of interestN

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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: elongated Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system
Type: COMPLEX / Details: Cascade alone / Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.55 MDa / Experimental value: YES
Buffer solutionpH: 7.5
Buffer componentConc.: 150 mM / Name: sodium chloride / Formula: NaClSodium chloride
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: 4s force 0

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 800 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
Image recordingElectron dose: 70 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 8500

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2cryoSPARC2image acquisition
4cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 49699 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00244930
ELECTRON MICROSCOPYf_angle_d0.63161222
ELECTRON MICROSCOPYf_dihedral_angle_d9.1476732
ELECTRON MICROSCOPYf_chiral_restr0.0447039
ELECTRON MICROSCOPYf_plane_restr0.0047613

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