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- PDB-7wah: Structure of Cas7-11 in complex with guide RNA and target RNA -

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Basic information

Entry
Database: PDB / ID: 7wah
TitleStructure of Cas7-11 in complex with guide RNA and target RNA
Components
  • CRISPR-associated RAMP family protein
  • crRNA (39-MER)
  • tgRNA (5'-R(P*UP*AP*CP*CP*CP*AP*UP*GP*UP*CP*GP*AP*AP*GP*AP*CP*AP*AP*CP*AP*AP*AP*G)-3')
KeywordsRNA BINDING PROTEIN/RNA / CRISPR / RNase / RNA BINDING PROTEIN-RNA COMPLEX
Function / homology: / CRISPR type III-associated protein / RAMP superfamily / defense response to virus / RNA / RNA (> 10) / CRISPR-associated RAMP family protein
Function and homology information
Biological speciesEscherichia coli (E. coli)
Desulfonema ishimotonii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.45 Å
AuthorsKato, K. / Okazaki, S. / Isayama, Y. / Nishizawa, T. / Nishimasu, H.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED) Japan
CitationJournal: Cell / Year: 2022
Title: Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex.
Authors: Kazuki Kato / Wenyuan Zhou / Sae Okazaki / Yukari Isayama / Tomohiro Nishizawa / Jonathan S Gootenberg / Omar O Abudayyeh / Hiroshi Nishimasu /
Abstract: The type III-E CRISPR-Cas effector Cas7-11, with dual RNase activities for precursor CRISPR RNA (pre-crRNA) processing and crRNA-guided target RNA cleavage, is a new platform for bacterial and ...The type III-E CRISPR-Cas effector Cas7-11, with dual RNase activities for precursor CRISPR RNA (pre-crRNA) processing and crRNA-guided target RNA cleavage, is a new platform for bacterial and mammalian RNA targeting. We report the 2.5-Å resolution cryoelectron microscopy structure of Cas7-11 in complex with a crRNA and its target RNA. Cas7-11 adopts a modular architecture comprising seven domains (Cas7.1-Cas7.4, Cas11, INS, and CTE) and four interdomain linkers. The crRNA 5' tag is recognized and processed by Cas7.1, whereas the crRNA spacer hybridizes with the target RNA. Consistent with our biochemical data, the catalytic residues for programmable cleavage in Cas7.2 and Cas7.3 neighbor the scissile phosphates before the flipped-out fourth and tenth nucleotides in the target RNA, respectively. Using structural insights, we rationally engineered a compact Cas7-11 variant (Cas7-11S) for single-vector AAV packaging for transcript knockdown in human cells, enabling in vivo Cas7-11 applications.
History
DepositionDec 14, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 15, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 6, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jun 26, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
R: crRNA (39-MER)
D: tgRNA (5'-R(P*UP*AP*CP*CP*CP*AP*UP*GP*UP*CP*GP*AP*AP*GP*AP*CP*AP*AP*CP*AP*AP*AP*G)-3')
A: CRISPR-associated RAMP family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)205,7517
Polymers205,4903
Non-polymers2624
Water00
1


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

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Components

#1: RNA chain crRNA (39-MER)


Mass: 12502.379 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
#2: RNA chain tgRNA (5'-R(P*UP*AP*CP*CP*CP*AP*UP*GP*UP*CP*GP*AP*AP*GP*AP*CP*AP*AP*CP*AP*AP*AP*G)-3')


Mass: 7377.513 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
#3: Protein CRISPR-associated RAMP family protein


Mass: 185609.875 Da / Num. of mol.: 1 / Mutation: D429A, D654A, D753A
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
#4: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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
IDNameTypeEntity IDParent-IDSource
1Cas7-11 in complex with a crRNA and its target RNACOMPLEX#1-#30MULTIPLE SOURCES
2crRNA and target RNACOMPLEX#1-#21RECOMBINANT
3Cas7-11COMPLEX#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Escherichia coli (E. coli)562
22Desulfonema ishimotonii (bacteria)45657
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Escherichia coli (E. coli)562
22Escherichia coli (E. coli)562
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD
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: 1500 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 76.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20_4459: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.45 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 127452 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00213651
ELECTRON MICROSCOPYf_angle_d0.47818696
ELECTRON MICROSCOPYf_dihedral_angle_d11.565377
ELECTRON MICROSCOPYf_chiral_restr0.0392010
ELECTRON MICROSCOPYf_plane_restr0.0042203

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