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- PDB-8z4l: Cryo-EM structure of CTR-bound type VII CRISPR-Cas complex at sub... -

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Basic information

Entry
Database: PDB / ID: 8z4l
TitleCryo-EM structure of CTR-bound type VII CRISPR-Cas complex at substrate-engaged state I
Components
  • (a protein) x 3
  • RNA (40-MER)
  • RNA (49-MER)
KeywordsANTIVIRAL PROTEIN / a protein complex
Function / homologyRNA / RNA (> 10)
Function and homology information
Biological speciesmetagenome (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsZhang, H. / Deng, Z. / Li, X.
Funding support China, 1items
OrganizationGrant numberCountry
Other government China
CitationJournal: Nature / Year: 2024
Title: Structural basis for the activity of the type VII CRISPR-Cas system.
Authors: Jie Yang / Xuzichao Li / Qiuqiu He / Xiaoshen Wang / Jingjing Tang / Tongyao Wang / Yi Zhang / Feiyang Yu / Shuqin Zhang / Zhikun Liu / Lingling Zhang / Fumeng Liao / Hang Yin / Haiyan Zhao ...Authors: Jie Yang / Xuzichao Li / Qiuqiu He / Xiaoshen Wang / Jingjing Tang / Tongyao Wang / Yi Zhang / Feiyang Yu / Shuqin Zhang / Zhikun Liu / Lingling Zhang / Fumeng Liao / Hang Yin / Haiyan Zhao / Zengqin Deng / Heng Zhang /
Abstract: The newly identified type VII CRISPR-Cas candidate system uses a CRISPR RNA-guided ribonucleoprotein complex formed by Cas5 and Cas7 proteins to target RNA. However, the RNA cleavage is executed by a ...The newly identified type VII CRISPR-Cas candidate system uses a CRISPR RNA-guided ribonucleoprotein complex formed by Cas5 and Cas7 proteins to target RNA. However, the RNA cleavage is executed by a dedicated Cas14 nuclease, which is distinct from the effector nucleases of the other CRISPR-Cas systems. Here we report seven cryo-electron microscopy structures of the Cas14-bound interference complex at different functional states. Cas14, a tetrameric protein in solution, is recruited to the Cas5-Cas7 complex in a target RNA-dependent manner. The N-terminal catalytic domain of Cas14 binds a stretch of the substrate RNA for cleavage, whereas the C-terminal domain is primarily responsible for tethering Cas14 to the Cas5-Cas7 complex. The biochemical cleavage assays corroborate the captured functional conformations, revealing that Cas14 binds to different sites on the Cas5-Cas7 complex to execute individual cleavage events. Notably, a plugged-in arginine of Cas7 sandwiched by a C-shaped clamp of C-terminal domain precisely modulates Cas14 binding. More interestingly, target RNA cleavage is altered by a complementary protospacer flanking sequence at the 5' end, but not at the 3' end. Altogether, our study elucidates critical molecular details underlying the assembly of the interference complex and substrate cleavage in the type VII CRISPR-Cas system, which may help rational engineering of the type VII CRISPR-Cas system for biotechnological applications.
History
DepositionApr 17, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 21, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 28, 2024Group: Data collection / Database references / Category: citation / citation_author / 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 / Structure summary / Category: em_admin / struct
Item: _em_admin.last_update / _em_admin.title / _struct.title
Revision 1.3Sep 25, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: a protein
B: a protein
C: a protein
D: a protein
E: a protein
F: a protein
G: a protein
H: a protein
I: a protein
J: a protein
K: a protein
L: a protein
M: RNA (49-MER)
N: RNA (40-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)503,89023
Polymers503,30114
Non-polymers5899
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 3 types, 12 molecules ABCDEFJGHIKL

#1: Protein
a protein


Mass: 22255.604 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) metagenome (others) / Production host: Escherichia coli (E. coli)
#2: Protein a protein


Mass: 27139.533 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) metagenome (others) / Production host: Escherichia coli (E. coli)
#3: Protein
a protein


Mass: 70468.898 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) metagenome (others) / Production host: Escherichia coli (E. coli)

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RNA chain , 2 types, 2 molecules MN

#4: RNA chain RNA (49-MER)


Mass: 19142.277 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) metagenome (others) / Production host: Escherichia coli (E. coli)
#5: RNA chain RNA (40-MER)


Mass: 19354.707 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) metagenome (others) / Production host: Escherichia coli (E. coli)

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Non-polymers , 1 types, 9 molecules

#6: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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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: a protein / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)Organism: metagenome (others)
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

MicroscopyModel: JEOL CRYO ARM 300
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: 500 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 96774 / Symmetry type: POINT

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