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- PDB-8d1v: Cryo-EM structure of guide RNA and target RNA bound Cas7-11 -

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Basic information

Entry
Database: PDB / ID: 8d1v
TitleCryo-EM structure of guide RNA and target RNA bound Cas7-11
Components
  • CRISPR RNA (34-MER)
  • CRISPR-associated RAMP family protein
  • SS target RNA (5'-R(P*AP*GP*CP*UP*UP*GP*GP*UP*UP*CP*AP*AP*AP*GP*AP*AP*CP*G)-3')
KeywordsRNA BINDING PROTEIN/RNA / Crispr / type III-E Cas7-11 / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homologyCRISPR type III-associated protein / RAMP superfamily / defense response to virus / RNA / RNA (> 10) / CRISPR-associated RAMP family protein
Function and homology information
Biological speciesDesulfonema ishimotonii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.82 Å
AuthorsRai, J. / Goswami, H. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM R01 101343 United States
CitationJournal: Elife / Year: 2022
Title: Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA.
Authors: Hemant N Goswami / Jay Rai / Anuska Das / Hong Li /
Abstract: Cas7-11 is a Type III-E CRISPR Cas effector that confers programmable RNA cleavage and has potential applications in RNA interference. Cas7-11 encodes a single polypeptide containing four Cas7- and ...Cas7-11 is a Type III-E CRISPR Cas effector that confers programmable RNA cleavage and has potential applications in RNA interference. Cas7-11 encodes a single polypeptide containing four Cas7- and one Cas11-like segments that obscures the distinction between the multi-subunit Class 1 and the single-subunit Class-2 CRISPR Cas systems. We report a cryo-EM (cryo-electron microscopy) structure of the active Cas7-11 from (DiCas7-11) that reveals the molecular basis for RNA processing and interference activities. DiCas7-11 arranges its Cas7- and Cas11-like domains in an extended form that resembles the backbone made up by four Cas7 and one Cas11 subunits in the multi-subunit enzymes. Unlike the multi-subunit enzymes, however, the backbone of DiCas7-11 contains evolutionarily different Cas7 and Cas11 domains, giving rise to their unique functionality. The first Cas7-like domain nearly engulfs the last 15 direct repeat nucleotides in processing and recognition of the CRISPR RNA, and its free-standing fragment retains most of the activity. Both the second and the third Cas7-like domains mediate target RNA cleavage in a metal-dependent manner. The structure and mutational data indicate that the long variable insertion to the fourth Cas7 domain has little impact on RNA processing or targeting, suggesting the possibility for engineering a compact and programmable RNA interference tool.
History
DepositionMay 27, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 2, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated RAMP family protein
J: CRISPR RNA (34-MER)
N: SS target RNA (5'-R(P*AP*GP*CP*UP*UP*GP*GP*UP*UP*CP*AP*AP*AP*GP*AP*AP*CP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)200,8297
Polymers200,5673
Non-polymers2624
Water0
1


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

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Components

#1: Protein CRISPR-associated RAMP family protein


Mass: 183933.000 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfonema ishimotonii (bacteria) / Gene: DENIS_1082 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A401FT36
#2: RNA chain CRISPR RNA (34-MER)


Mass: 10837.447 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfonema ishimotonii (bacteria) / Production host: Escherichia coli (E. coli)
#3: RNA chain SS target RNA (5'-R(P*AP*GP*CP*UP*UP*GP*GP*UP*UP*CP*AP*AP*AP*GP*AP*AP*CP*G)-3')


Mass: 5796.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Desulfonema ishimotonii (bacteria) / Production host: Escherichia coli (E. coli)
#4: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn

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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: CryoEM structure of type III-E Cas7-11 from Desulfonema ishimotonii
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Desulfonema ishimotonii (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 FIELDBright-field microscopy / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.82 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 226320 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00611327
ELECTRON MICROSCOPYf_angle_d0.72315507
ELECTRON MICROSCOPYf_dihedral_angle_d14.7371963
ELECTRON MICROSCOPYf_chiral_restr0.0431673
ELECTRON MICROSCOPYf_plane_restr0.0051829

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