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- PDB-7r21: elongated Cascade complex from type I-A CRISPR-Cas system -

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Basic information

Entry
Database: PDB / ID: 7r21
Titleelongated Cascade complex from type I-A CRISPR-Cas system
Components
  • Cas11a
  • Cas7a
  • CrRNA (62-MER)
  • Type I-A CRISPR-associated protein Cas5
KeywordsANTIVIRAL PROTEIN / elongated Cascade complex / cascade / type I-A / genome editing
Function / homology
Function and homology information


defense response to virus
Similarity search - Function
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 / CRISPR-associated protein Cas5, N-terminal
Similarity search - Domain/homology
RNA / RNA (> 10) / Type I-A CRISPR-associated protein Cas5 / Uncharacterized protein / Type I-A CRISPR-associated protein Cas7/Csa2
Similarity search - Component
Biological speciesPyrococcus furiosus DSM 3638 (archaea)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsHu, C. / Ni, D. / Nam, K.H. / 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
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
Revision 1.1Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cas11a
C: Cas11a
D: Cas11a
E: Cas11a
F: Cas11a
G: Cas11a
H: Cas11a
I: Cas7a
J: Cas7a
K: Cas7a
L: Cas7a
M: Cas7a
N: Cas7a
O: Cas7a
P: Type I-A CRISPR-associated protein Cas5
Q: Cas7a
R: CrRNA (62-MER)
S: Cas7a
T: Cas7a


Theoretical massNumber of molelcules
Total (without water)504,48119
Polymers504,48119
Non-polymers00
Water00
1


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

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Components

#1: 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) / Strain (production host): DE3 / References: UniProt: Q8U332
#2: 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) / Strain (production host): DE3 / References: UniProt: Q8U333
#3: 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) / Strain (production host): DE3 / References: UniProt: A0A5C0XNV9
#4: RNA chain CrRNA (62-MER)


Mass: 19979.840 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1elongated Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas systemCOMPLEXCascade aloneall0MULTIPLE SOURCES
2Cas11a, Cas7a and Type I-A CRISPR-associated protein Cas5COMPLEX#1-#31RECOMBINANT
3CrRNA (62-MER)COMPLEX#41NATURAL
Molecular weightValue: 0.55 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Pyrococcus furiosus DSM 3638 (archaea)186497
23Escherichia 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.: 0.8 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
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 FIELD / 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
1cryoSPARCparticle selection
2cryoSPARC2image acquisition
4cryoSPARCCTF correction
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 240851 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00335792
ELECTRON MICROSCOPYf_angle_d0.56848810
ELECTRON MICROSCOPYf_dihedral_angle_d9.6535430
ELECTRON MICROSCOPYf_chiral_restr0.0435602
ELECTRON MICROSCOPYf_plane_restr0.0056062

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