[English] 日本語
Yorodumi
- PDB-8rc2: DNA bound type IV-A3 CRISPR effector complex from K. pneumoniae -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 8rc2
TitleDNA bound type IV-A3 CRISPR effector complex from K. pneumoniae
Components
  • (CRISPR type AFERR-associated protein ...) x 3
  • Non-Target Strand (NTS)-DNA
  • Target Strand (TS)-DNA
  • crRNA
KeywordsGENE REGULATION / CRISPR / crRNA / DNA binding / type IV CRISPR-Cas / CRISPRi / nuclease deficient
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR type AFERR-associated protein Csf2 / Uncharacterized protein / Uncharacterized protein
Function and homology information
Biological speciesKlebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsMiksys, A. / Cepaite, R. / Malinauskaite, L. / Pausch, P.
Funding supportEuropean Union, 3items
OrganizationGrant numberCountry
Other government01.2.2-CPVA-V-716-01-0001
European Molecular Biology Organization (EMBO)5342-2023European Union
Other governmentS-MIP-22-10
CitationJournal: Nat Commun / Year: 2024
Title: Structural variation of types IV-A1- and IV-A3-mediated CRISPR interference.
Authors: R Čepaitė / N Klein / A Mikšys / S Camara-Wilpert / V Ragožius / F Benz / A Skorupskaitė / H Becker / G Žvejytė / N Steube / G K A Hochberg / L Randau / R Pinilla-Redondo / L ...Authors: R Čepaitė / N Klein / A Mikšys / S Camara-Wilpert / V Ragožius / F Benz / A Skorupskaitė / H Becker / G Žvejytė / N Steube / G K A Hochberg / L Randau / R Pinilla-Redondo / L Malinauskaitė / P Pausch /
Abstract: CRISPR-Cas mediated DNA-interference typically relies on sequence-specific binding and nucleolytic degradation of foreign genetic material. Type IV-A CRISPR-Cas systems diverge from this general ...CRISPR-Cas mediated DNA-interference typically relies on sequence-specific binding and nucleolytic degradation of foreign genetic material. Type IV-A CRISPR-Cas systems diverge from this general mechanism, using a nuclease-independent interference pathway to suppress gene expression for gene regulation and plasmid competition. To understand how the type IV-A system associated effector complex achieves this interference, we determine cryo-EM structures of two evolutionarily distinct type IV-A complexes (types IV-A1 and IV-A3) bound to cognate DNA-targets in the presence and absence of the type IV-A signature DinG effector helicase. The structures reveal how the effector complexes recognize the protospacer adjacent motif and target-strand DNA to form an R-loop structure. Additionally, we reveal differences between types IV-A1 and IV-A3 in DNA interactions and structural motifs that allow for in trans recruitment of DinG. Our study provides a detailed view of type IV-A mediated DNA-interference and presents a structural foundation for engineering type IV-A-based genome editing tools.
History
DepositionDec 5, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 6, 2024Provider: repository / Type: Initial release

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: CRISPR type AFERR-associated protein Csf2
B: CRISPR type AFERR-associated protein Csf2
C: CRISPR type AFERR-associated protein Csf2
D: CRISPR type AFERR-associated protein Csf2
E: CRISPR type AFERR-associated protein Csf2
F: CRISPR type AFERR-associated protein Csf3
G: CRISPR type AFERR-associated protein Csf1
H: crRNA
I: Target Strand (TS)-DNA
J: Non-Target Strand (NTS)-DNA
L: CRISPR type AFERR-associated protein Csf2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)343,84112
Polymers343,77611
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area57520 Å2
ΔGint-310 kcal/mol
Surface area96710 Å2
MethodPISA

-
Components

-
CRISPR type AFERR-associated protein ... , 3 types, 8 molecules ABCDELFG

#1: Protein
CRISPR type AFERR-associated protein Csf2 / Csf2 (Cas7)


Mass: 38654.406 Da / Num. of mol.: 6 / Mutation: 6xHis-tag
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: AI2781V1_5179 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A333ESG5
#2: Protein CRISPR type AFERR-associated protein Csf3 / Csf3 (Cas5)


Mass: 25474.385 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: EAO17_29825 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A8G1XN67
#3: Protein CRISPR type AFERR-associated protein Csf1 / Csf1 (Cas8)


Mass: 29762.467 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: G4V31_29130 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A7Z7WW72

-
DNA chain , 2 types, 2 molecules IJ

#5: DNA chain Target Strand (TS)-DNA


Mass: 18196.562 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Klebsiella pneumoniae (bacteria)
#6: DNA chain Non-Target Strand (NTS)-DNA


Mass: 18743.031 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Klebsiella pneumoniae (bacteria)

-
RNA chain / Non-polymers , 2 types, 2 molecules H

#4: RNA chain crRNA


Mass: 19672.641 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Star
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn

-
Details

Has ligand of interestN
Has protein modificationN

-
Experimental details

-
Experiment

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

-
Sample preparation

ComponentName: Type IV-A3 CRISPR-Cas effector complex from Klebsiella pneumoniae bound to crRNA and target DNA
Type: COMPLEX
Details: The Cas6 subunit found in the type IVA3 system from Klebsiella pneumoniae was present during expression, purification and complex assembly, but was not resolved due to high flexibility
Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 0.332 MDa / Experimental value: YES
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Star
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2150 mMNaCl1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 20 mA current / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

-
Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 92000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 48 sec. / Electron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1120
Image scansWidth: 4096 / Height: 4096

-
Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2particle selection
2EPUimage acquisition
4cryoSPARC4.2CTF correction
7Coot0.9.8model fitting
8ISOLDE1.4model fitting
10PHENIX1.19.2-4158model refinement
11cryoSPARC4.2initial Euler assignment
12cryoSPARC4.2final Euler assignment
13cryoSPARC4.2classification
14cryoSPARC4.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1082094
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 104035 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00620347
ELECTRON MICROSCOPYf_angle_d0.59927948
ELECTRON MICROSCOPYf_dihedral_angle_d17.3263451
ELECTRON MICROSCOPYf_chiral_restr0.0443136
ELECTRON MICROSCOPYf_plane_restr0.0053336

+
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