[English] 日本語
Yorodumi
- PDB-8s9x: CRISPR-Cas type III-D effector complex bound to self-target RNA i... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 8s9x
TitleCRISPR-Cas type III-D effector complex bound to self-target RNA in a post-cleavage state
Components
  • CRISPR RNA
  • Cas10
  • Cas7-2x
  • Cas7-Cas5-Cas11
  • Self-target RNA
  • TIGR03984 family CRISPR-associated protein
  • TIGR03986 family CRISPR-associated RAMP protein
KeywordsRNA BINDING PROTEIN/RNA / CRISPR / CRISPR-Cas / type III / complex / RNA / crRNA / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


defense response to virus
Similarity search - Function
CRISPR-associated protein A791736 / CRISPR-associated RAMP BGP1436 / CRISPR type III-associated protein / RAMP superfamily / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
RNA / RNA (> 10) / GGDEF domain-containing protein / DUF324 domain-containing protein / DUF324 domain-containing protein / TIGR03984 family CRISPR-associated protein / DUF324 domain-containing protein
Similarity search - Component
Biological speciesSynechocystis sp. PCC 6803 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.44 Å
AuthorsSchwartz, E.A. / Taylor, D.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
CitationJournal: Nat Commun / Year: 2024
Title: RNA targeting and cleavage by the type III-Dv CRISPR effector complex.
Authors: Evan A Schwartz / Jack P K Bravo / Mohd Ahsan / Luis A Macias / Caitlyn L McCafferty / Tyler L Dangerfield / Jada N Walker / Jennifer S Brodbelt / Giulia Palermo / Peter C Fineran / Robert D ...Authors: Evan A Schwartz / Jack P K Bravo / Mohd Ahsan / Luis A Macias / Caitlyn L McCafferty / Tyler L Dangerfield / Jada N Walker / Jennifer S Brodbelt / Giulia Palermo / Peter C Fineran / Robert D Fagerlund / David W Taylor /
Abstract: CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular ...CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular intervals is characteristic of type III effector complexes. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.
History
DepositionMar 30, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Cas7-Cas5-Cas11
B: TIGR03984 family CRISPR-associated protein
C: Cas10
D: Cas7-2x
E: TIGR03986 family CRISPR-associated RAMP protein
F: CRISPR RNA
G: Self-target RNA


Theoretical massNumber of molelcules
Total (without water)352,1437
Polymers352,1437
Non-polymers00
Water0
1


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

-
Components

-
Protein , 5 types, 5 molecules ABCDE

#1: Protein Cas7-Cas5-Cas11


Mass: 87558.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: sll7066 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZED2
#2: Protein TIGR03984 family CRISPR-associated protein


Mass: 21899.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: sll7064 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZED4
#3: Protein Cas10


Mass: 64335.309 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: sll7067 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZED1
#4: Protein Cas7-2x


Mass: 56820.832 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: sll7065 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZED3
#5: Protein TIGR03986 family CRISPR-associated RAMP protein


Mass: 90334.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: sll7063 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZED5

-
RNA chain , 2 types, 2 molecules FG

#6: RNA chain CRISPR RNA /


Mass: 11927.167 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Production host: Escherichia coli (E. coli)
#7: RNA chain Self-target RNA


Mass: 19265.455 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Synechocystis sp. PCC 6803 (bacteria)

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

-
Sample preparation

ComponentName: CRISPR-Cas type III-D effector complex / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 0.332 MDa / Experimental value: YES
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES-NaOH1
2100 mMPotassium ChlorideKCl1
35 %Glycerol1
41 mMDTT1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Particles were monodisperse and homogeneous.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

-
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 magnification: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

-
Processing

Software
NameVersionClassification
phenix.real_space_refine1.18rc7_3834refinement
PHENIX1.18rc7_3834refinement
EM softwareName: cryoSPARC / Version: 3 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 41202 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 87.83 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003122985
ELECTRON MICROSCOPYf_angle_d0.651631392
ELECTRON MICROSCOPYf_chiral_restr0.04493487
ELECTRON MICROSCOPYf_plane_restr0.00563905
ELECTRON MICROSCOPYf_dihedral_angle_d24.19418775

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more