[English] 日本語
Yorodumi
- PDB-7odf: Structure of the mini-RNA-guided endonuclease CRISPR-Cas_phi3 -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 7odf
TitleStructure of the mini-RNA-guided endonuclease CRISPR-Cas_phi3
Components
  • Cas_phi3
  • DNA (5'-D(P*GP*G)-3')
  • DNA (5'-D(P*GP*TP*AP*AP*TP*TP*CP*AP*G)-3')
  • DNA (5'-D(P*GP*TP*AP*TP*CP*CP*CP*AP*TP*TP*AP*CP*CP*AP*GP*CP*TP*GP*AP*AP*TP*TP*AP*C)-3')
  • RNA (43-MER)
KeywordsRNA BINDING PROTEIN / CRISPR-Cas / RNA-guided endonuclease / R-loop
Function / homologyNICKEL (II) ION / DNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesPhage #D (virus)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsCarabias del Rey, A. / Fugilsang, A. / Temperini, P. / Pape, T. / Sofos, N. / Stella, S. / Erledsson, S. / Montoya, G.
Funding support Denmark, 4items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
Novo Nordisk FoundationNNF0024386 Denmark
Novo Nordisk FoundationNNF17SA0030214 Denmark
Novo Nordisk FoundationNNF18OC0055061 Denmark
CitationJournal: Nat Commun / Year: 2021
Title: Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3.
Authors: Arturo Carabias / Anders Fuglsang / Piero Temperini / Tillmann Pape / Nicholas Sofos / Stefano Stella / Simon Erlendsson / Guillermo Montoya /
Abstract: CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome ...CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.
History
DepositionApr 29, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 21, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 11, 2021Group: Database references / Source and taxonomy
Category: citation / citation_author ...citation / citation_author / database_2 / entity_src_gen
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity_src_gen.pdbx_gene_src_scientific_name
Revision 1.2Jul 10, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / refine
Item: _em_admin.last_update / _refine.ls_d_res_high / _refine.ls_d_res_low

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-12827
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Cas_phi3
E: DNA (5'-D(P*GP*TP*AP*TP*CP*CP*CP*AP*TP*TP*AP*CP*CP*AP*GP*CP*TP*GP*AP*AP*TP*TP*AP*C)-3')
G: DNA (5'-D(P*GP*TP*AP*AP*TP*TP*CP*AP*G)-3')
C: DNA (5'-D(P*GP*G)-3')
F: RNA (43-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)112,0427
Polymers111,9185
Non-polymers1242
Water905
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, assay for oligomerization, mass photometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15320 Å2
ΔGint-95 kcal/mol
Surface area41180 Å2

-
Components

-
DNA chain , 3 types, 3 molecules EGC

#2: DNA chain DNA (5'-D(P*GP*TP*AP*TP*CP*CP*CP*AP*TP*TP*AP*CP*CP*AP*GP*CP*TP*GP*AP*AP*TP*TP*AP*C)-3')


Mass: 7288.730 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(P*GP*TP*AP*AP*TP*TP*CP*AP*G)-3')


Mass: 2754.835 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (5'-D(P*GP*G)-3')


Mass: 613.454 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

-
Protein / RNA chain , 2 types, 2 molecules AF

#1: Protein Cas_phi3


Mass: 87411.664 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Phage #D (virus) / Production host: Escherichia coli (E. coli)
#5: RNA chain RNA (43-MER)


Mass: 13849.232 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

-
Non-polymers , 3 types, 7 molecules

#6: Chemical ChemComp-NI / NICKEL (II) ION


Mass: 58.693 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ni / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Formula: H2O

-
Details

Has ligand of interestY

-
Experimental details

-
Experiment

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

-
Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1cas_phi/R-loop complexCOMPLEXcrispr-cas rna-guided endonuclease#1-#50RECOMBINANT
2Cas_phi3COMPLEX#11RECOMBINANT
3DNA and RNACOMPLEX#2-#51RECOMBINANT
Molecular weightValue: 0.14 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Phage (virus)77920
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD
VitrificationCryogen name: ETHANE-PROPANE

-
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: OTHER
Electron lensMode: OTHER / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.05 sec. / Electron dose: 42 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

-
Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameCategory
1Warpparticle selection
2EPUimage acquisition
4WarpCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3504102
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 178412 / Algorithm: FOURIER SPACE / Symmetry type: POINT
RefinementResolution: 2.66→2.66 Å / Cor.coef. Fo:Fc: 0.811 / SU B: 12.457 / SU ML: 0.229 / ESU R: 0.342
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.40225 --
obs0.40225 96543 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 95.528 Å2
Baniso -1Baniso -2Baniso -3
1--2.59 Å20.44 Å2-0.51 Å2
2--4.1 Å20.51 Å2
3----1.51 Å2
Refinement stepCycle: 1 / Total: 6846
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027146
ELECTRON MICROSCOPYf_angle_d0.44910057
ELECTRON MICROSCOPYf_dihedral_angle_d16.9451613
ELECTRON MICROSCOPYf_chiral_restr0.0351185
ELECTRON MICROSCOPYf_plane_restr0.0031014
LS refinement shellResolution: 2.7→2.77 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork3.055 7140 -
obs--100 %

+
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://pdbjlvh1.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