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- PDB-8w1p: Structure of Selenomonas sp. Cascade (SsCascade) -

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Basic information

Entry
Database: PDB / ID: 8w1p
TitleStructure of Selenomonas sp. Cascade (SsCascade)
Components
  • Cas5
  • Cas6
  • Cas7
  • Cas8
  • Non-target strand DNA
  • Target strand DNA
  • crRNA
KeywordsIMMUNE SYSTEM / CRISPR-Cas / Genome engineering / RNA-guided DNA endonuclease / Non-coding RNA / HNH nuclease
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesSelenomonas sp. (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsHirano, S. / Zhang, F.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)5R01HG009761-07 United States
CitationJournal: Mol Cell / Year: 2024
Title: Structural determinants of DNA cleavage by a CRISPR HNH-Cascade system.
Authors: Seiichi Hirano / Han Altae-Tran / Soumya Kannan / Rhiannon K Macrae / Feng Zhang /
Abstract: Canonical prokaryotic type I CRISPR-Cas adaptive immune systems contain a multicomponent effector complex called Cascade, which degrades large stretches of DNA via Cas3 helicase-nuclease activity. ...Canonical prokaryotic type I CRISPR-Cas adaptive immune systems contain a multicomponent effector complex called Cascade, which degrades large stretches of DNA via Cas3 helicase-nuclease activity. Recently, a highly precise subtype I-F1 CRISPR-Cas system (HNH-Cascade) was found that lacks Cas3, the absence of which is compensated for by the insertion of an HNH endonuclease domain in the Cas8 Cascade component. Here, we describe the cryo-EM structure of Selenomonas sp. HNH-Cascade (SsCascade) in complex with target DNA and characterize its mechanism of action. The Cascade scaffold is complemented by the HNH domain, creating a ring-like structure in which the unwound target DNA is precisely cleaved. This structure visualizes a unique hybrid of two extensible biological systems-Cascade, an evolutionary platform for programmable DNA effectors, and an HNH nuclease, an adaptive domain with a spectrum of enzymatic activity.
History
DepositionFeb 16, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 7, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 28, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Sep 4, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.3Sep 11, 2024Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cas7
B: Cas7
C: Cas7
D: Cas7
E: Cas7
F: Cas7
G: Cas8
H: Cas5
I: Cas6
N: Non-target strand DNA
R: crRNA
T: Target strand DNA


Theoretical massNumber of molelcules
Total (without water)385,78012
Polymers385,78012
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 9 molecules ABCDEFGHI

#1: Protein
Cas7


Mass: 38700.172 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli (E. coli)
#2: Protein Cas8


Mass: 43706.289 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: This protein is histidine tagged at N-terminal region from residues 1 to 41. Residues 42 to 385 are derived from natural Cas8 protein sequences.
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli (E. coli)
#3: Protein Cas5


Mass: 28703.135 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli (E. coli)
#4: Protein Cas6


Mass: 20735.873 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli (E. coli)

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DNA chain , 2 types, 2 molecules NT

#5: DNA chain Non-target strand DNA


Mass: 20484.176 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain Target strand DNA


Mass: 20202.930 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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RNA chain , 1 types, 1 molecules R

#6: RNA chain crRNA


Mass: 19746.777 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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: Target DNA-bound SsCascade / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Selenomonas sp. (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN 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: 800 nm
Image recordingElectron dose: 1.05 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80908 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00624499
ELECTRON MICROSCOPYf_angle_d0.93333562
ELECTRON MICROSCOPYf_dihedral_angle_d15.6789499
ELECTRON MICROSCOPYf_chiral_restr0.0533726
ELECTRON MICROSCOPYf_plane_restr0.0093921

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