[English] 日本語
Yorodumi
- PDB-8g9t: Exploiting Activation and Inactivation Mechanisms in Type I-C CRI... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 8g9t
TitleExploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications
Components
  • AcrIC9
  • Cas11
  • Cas5
  • Cas7
  • Cas8Roberval (Air Saguenay) Water Aerodrome
  • crRNA (43-MER)
KeywordsHYDROLASE/RNA / CRISPR / type I-C / cascade / anti-CRISPR / HYDROLASE-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding
Similarity search - Function
CRISPR pre-crRNA endoribonuclease Cas5d / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) / CRISPR-associated protein Cas5, N-terminal
Similarity search - Domain/homology
RNA / RNA (> 10) / : / : / pre-crRNA processing endonuclease / Uncharacterized protein
Similarity search - Component
Biological speciesRhodobacter phage RcNL1 (virus)
Neisseria lactamica (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHu, C. / Nam, K.H. / Ke, A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR)GM118174 United States
CitationJournal: Mol Cell / Year: 2024
Title: Exploiting activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications.
Authors: Chunyi Hu / Mason T Myers / Xufei Zhou / Zhonggang Hou / Macy L Lozen / Ki Hyun Nam / Yan Zhang / Ailong Ke /
Abstract: Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size ...Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here, we use four cryoelectron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9 that strongly inhibit Neisseria lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through allosteric inhibition, whereas AcrIC9 achieves so through direct competition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for type I CRISPR eukaryotic genome engineering.
History
DepositionFeb 22, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: AcrIC9
B: Cas7
C: Cas7
D: Cas7
E: Cas7
F: Cas7
G: Cas11
H: Cas7
I: Cas11
J: Cas11
K: Cas8
M: Cas7
N: Cas5
O: crRNA (43-MER)
L: Cas11


Theoretical massNumber of molelcules
Total (without water)393,28715
Polymers393,28715
Non-polymers00
Water0
1


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

-
Components

-
Protein , 5 types, 14 molecules ABCDEFHMGIJLKN

#1: Protein AcrIC9


Mass: 8247.822 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhodobacter phage RcNL1 (virus) / Gene: RHZG_00032 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: I3UM23
#2: Protein
Cas7


Mass: 32208.111 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria lactamica (bacteria) / Gene: NCTC10618_01084 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A378VEU0
#3: Protein
Cas11


Mass: 14245.184 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria lactamica (bacteria) / Gene: NCTC10618_01085 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A378VF47
#4: Protein Cas8 / Roberval (Air Saguenay) Water Aerodrome


Mass: 64860.832 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria lactamica (bacteria) / Gene: NCTC10618_01085 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A378VF47
#5: Protein Cas5


Mass: 23870.451 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria lactamica (bacteria) / Gene: cas5d / Production host: Escherichia coli (E. coli) / References: UniProt: D0W8X4

-
RNA chain , 1 types, 1 molecules O

#6: RNA chain crRNA (43-MER)


Mass: 13870.306 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Neisseria lactamica (bacteria)

-
Experimental details

-
Experiment

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

-
Sample preparation

ComponentName: Binary complex of AcrIC9 with crRNA bound type I-C Cascade
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.4 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Neisseria lactamica (bacteria)486
31Rhodobacter phage RcNL1 (virus)754047
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Escherichia coli (E. coli)562BL21
31Escherichia coli BL21 (bacteria)511693
Buffer solutionpH: 7.5 / Details: 25mM Tris pH 7.5, 150mM NaCl
Buffer componentConc.: 150 mM / Name: sodium chloride / Formula: NaClSodium chloride
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

-
Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal magnification: 67000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 2500 nm / Calibrated defocus min: 1500 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 70 K
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1200 / Num. of real images: 1200
EM imaging opticsEnergyfilter name: GIF Bioquantum

-
Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2EPU2image acquisition
4cryoSPARC12CTF correction
11cryoSPARCclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 200000 / Num. of class averages: 6 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01327430
ELECTRON MICROSCOPYf_angle_d1.28537194
ELECTRON MICROSCOPYf_dihedral_angle_d33.8774036
ELECTRON MICROSCOPYf_chiral_restr0.0654023
ELECTRON MICROSCOPYf_plane_restr0.0074752

+
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