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- PDB-7ynb: Cryo-EM structure of Cas7-11-crRNA bound to target RNA-2 -

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Basic information

Entry
Database: PDB / ID: 7ynb
TitleCryo-EM structure of Cas7-11-crRNA bound to target RNA-2
Components
  • CRISPR-associated RAMP family protein
  • Target RNA-2 (28-MER)
  • crRNA (38-MER)
KeywordsRNA BINDING PROTEIN / Cas7-11 / crRNA / CRISPR-Cas / Csx29
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: 3.46 Å
AuthorsHuo, Y. / Dong, Q. / Zhao, H. / Jiang, T.
Funding support China, 1items
OrganizationGrant numberCountry
Chinese Academy of SciencesXDB37010301 China
CitationJournal: Nat Microbiol / Year: 2023
Title: Cryo-EM structure and protease activity of the type III-E CRISPR-Cas effector.
Authors: Yangao Huo / Hongshen Zhao / Qinghua Dong / Tao Jiang /
Abstract: The recently discovered type III-E CRISPR-Cas effector Cas7-11 shows promise when used as an RNA manipulation tool, but its structure and the mechanisms underlying its function remain unclear. Here ...The recently discovered type III-E CRISPR-Cas effector Cas7-11 shows promise when used as an RNA manipulation tool, but its structure and the mechanisms underlying its function remain unclear. Here we present four cryo-EM structures of Desulfonema ishimotonii Cas7-11-crRNA complex in pre-target and target RNA-bound states, and the cryo-EM structure of DiCas7-11-crRNA bound to its accessory protein DiCsx29. These data reveal structural elements for pre-crRNA processing, target RNA cleavage and regulation. Moreover, a 3' seed region of crRNA is involved in regulating RNA cleavage activity of DiCas7-11-crRNA-Csx29. Our analysis also shows that both the minimal mismatch of 4 nt to the 5' handle of crRNA and the minimal matching of the first 12 nt of the spacer by the target RNA are essential for triggering the protease activity of DiCas7-11-crRNA-Csx29 towards DiCsx30. Taken together, we propose that target RNA recognition and cleavage regulate and fine-tune the protease activity of DiCas7-11-crRNA-Csx29, thus preventing auto-immune responses.
History
DepositionJul 30, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 1, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 8, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 15, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated RAMP family protein
B: crRNA (38-MER)
C: Target RNA-2 (28-MER)


Theoretical massNumber of molelcules
Total (without water)214,0063
Polymers214,0063
Non-polymers00
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 / Cas7-11


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 crRNA (38-MER)


Mass: 15094.032 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 Target RNA-2 (28-MER)


Mass: 14978.832 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)

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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: The complex of Cas7-11-crRNA bound to target RNA-2 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Desulfonema ishimotonii (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
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: 2500 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 849013 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 65.37 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004814307
ELECTRON MICROSCOPYf_angle_d0.705219593
ELECTRON MICROSCOPYf_chiral_restr0.04662099
ELECTRON MICROSCOPYf_plane_restr0.0062318
ELECTRON MICROSCOPYf_dihedral_angle_d23.71865679

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