[English] 日本語
Yorodumi
- PDB-7tr8: Cascade complex from type I-A CRISPR-Cas system -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 7tr8
TitleCascade complex from type I-A CRISPR-Cas system
Components
  • (CRISPR-associated ...) x 2
  • Cas11a
  • Cas5a
  • Cas7a
  • Cas8
  • crRNA (45-MER)
KeywordsRNA BINDING PROTEIN / CRISPR / cascade / Cas3 / type I-A / genome editing
Function / homology
Function and homology information


catalytic activity, acting on DNA / DNA conformation change / exonuclease activity / defense response to virus / endonuclease activity / nucleic acid binding / Hydrolases; Acting on ester bonds / ATP binding / metal ion binding
Similarity search - Function
CRISPR-associated protein, MJ0385 / CRISPR-associated protein (Cas_Csa4) / CRISPR-associated protein, Cas5a type / : / CRISPR-associated protein Cas7/Cst2/DevR, subtype I-a/Apern / CRISPR-associated protein Cas7/Cst2/DevR / : / CRISPR-associated negative auto-regulator DevR/Csa2 / Helicase Cas3, CRISPR-associated, core / Cas3, HD domain ...CRISPR-associated protein, MJ0385 / CRISPR-associated protein (Cas_Csa4) / CRISPR-associated protein, Cas5a type / : / CRISPR-associated protein Cas7/Cst2/DevR, subtype I-a/Apern / CRISPR-associated protein Cas7/Cst2/DevR / : / CRISPR-associated negative auto-regulator DevR/Csa2 / Helicase Cas3, CRISPR-associated, core / Cas3, HD domain / CRISPR-associated nuclease/helicase Cas3, C-terminal / CRISPR-associated Cas3-type HD domain / CRISPR-associated Cas3-type HD domain superfamily / HD Cas3-type domain profile. / CRISPR-associated protein Cas5, N-terminal / : / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
NICKEL (II) ION / RNA / RNA (> 10) / CRISPR-associated helicase Cas3 / Type I-A CRISPR-associated protein Cas5 / Uncharacterized protein / Type I-A CRISPR-associated protein Cas7/Csa2 / CRISPR-associated endonuclease Cas3-HD / Type I-A CRISPR-associated protein Cas8a2/Csa4
Similarity search - Component
Biological speciesPyrococcus furiosus DSM 3638 (archaea)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHu, C. / Ni, D. / Nam, K.H. / Majumdar, S. / McLean, J. / Stahlberg, H. / Terns, M. / Ke, A.
Funding support United States, Switzerland, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118117 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118160 United States
Swiss National Science FoundationNCCR Switzerland
CitationJournal: Mol Cell / Year: 2022
Title: Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools.
Authors: Chunyi Hu / Dongchun Ni / Ki Hyun Nam / Sonali Majumdar / Justin McLean / Henning Stahlberg / Michael P Terns / Ailong Ke /
Abstract: Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme ...Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme Cas3 is then recruited in trans for processive DNA degradation. Contrary to this model, here, we show that type I-A Cascade and Cas3 function as an integral effector complex. We provide four cryoelectron microscopy (cryo-EM) snapshots of the Pyrococcus furiosus (Pfu) type I-A effector complex in different stages of DNA recognition and degradation. The HD nuclease of Cas3 is autoinhibited inside the effector complex. It is only allosterically activated upon full R-loop formation, when the entire targeted region has been validated by the RNA guide. The mechanistic insights inspired us to convert Pfu Cascade-Cas3 into a high-sensitivity, low-background, and temperature-activated nucleic acid detection tool. Moreover, Pfu CRISPR-Cas3 shows robust bi-directional deletion-editing activity in human cells, which could find usage in allele-specific inactivation of disease-causing mutations.
History
DepositionJan 28, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 10, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 17, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: CRISPR-associated helicase Cas3
C: Cas8
D: Cas11a
E: Cas11a
F: Cas11a
G: Cas11a
H: Cas11a
I: Cas7a
J: Cas7a
K: Cas7a
L: Cas7a
M: Cas7a
N: Cas7a
O: Cas7a
P: Cas5a
Q: CRISPR-associated endonuclease Cas3-HD
R: crRNA (45-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)489,87819
Polymers489,76017
Non-polymers1172
Water00
1


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

-
Components

-
CRISPR-associated ... , 2 types, 2 molecules AQ

#1: Protein CRISPR-associated helicase Cas3


Mass: 61412.555 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1 / Gene: cas3, PFDSM3638_03195 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A5C0XNV5
#6: Protein CRISPR-associated endonuclease Cas3-HD / CRISPR-associated ss-nucleic acid exo- and endonuclease Cas3-HD


Mass: 26057.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1 / Gene: cas3, cas3'', PF0639 / Production host: Escherichia coli (E. coli)
References: UniProt: Q8U336, Hydrolases; Acting on ester bonds

-
Protein , 4 types, 14 molecules CDEFGHIJKLMNOP

#2: Protein Cas8


Mass: 38800.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1 / Gene: PF0637 / Production host: Escherichia coli (E. coli) / Strain (production host): DE3 / References: UniProt: Q8U338
#3: Protein
Cas11a


Mass: 12208.933 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1 / Gene: PF0643 / Production host: Escherichia coli (E. coli) / Strain (production host): DE3 / References: UniProt: Q8U332
#4: Protein
Cas7a


Mass: 36989.148 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1 / Gene: PF0642 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8U333
#5: Protein Cas5a


Mass: 29147.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Strain: ATCC 43587 / DSM 3638 / JCM 8422 / Vc1 / Gene: cas5a, PFDSM3638_03200 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A5C0XNV9

-
RNA chain / Non-polymers , 2 types, 3 molecules R

#7: RNA chain crRNA (45-MER)


Mass: 14373.537 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#8: Chemical ChemComp-NI / NICKEL (II) ION


Mass: 58.693 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ni / Feature type: SUBJECT OF INVESTIGATION

-
Details

Has ligand of interestY

-
Experimental details

-
Experiment

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

-
Sample preparation

ComponentName: Cas3 bound Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system
Type: COMPLEX / Details: Cascade alone / Entity ID: #1-#7 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.4 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Geobacter sulfurreducens (bacteria)243231
21Escherichia coli (E. coli)562
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer componentConc.: 150 mM / Name: sodium chloride / Formula: NaCl
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: 6 seconds

-
Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 822
EM imaging opticsPhase plate: VOLTA PHASE PLATE

-
Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2EMAN9image acquisition
4EMANCTF correction
12cryoSPARCclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83559 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00234323
ELECTRON MICROSCOPYf_angle_d0.51346728
ELECTRON MICROSCOPYf_dihedral_angle_d8.5925086
ELECTRON MICROSCOPYf_chiral_restr0.0415386
ELECTRON MICROSCOPYf_plane_restr0.0045827

+
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