[English] 日本語
Yorodumi
- PDB-6c66: CRISPR RNA-guided surveillance complex, pre-nicking -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 6c66
TitleCRISPR RNA-guided surveillance complex, pre-nicking
Components
  • (CRISPR-associated protein, ...) x 4
  • CRISPR-associated helicase, Cas3 family
  • Nontarget strand
  • Target strand
  • Uncharacterized protein
  • crRNA
KeywordsDNA BINDING PROTEIN/DNA/RNA / CRISPR-Cas / Cascade / Cas3 / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / helicase activity / defense response to virus / hydrolase activity / RNA binding / ATP binding / identical protein binding / metal ion binding
Similarity search - Function
Cas3, C-terminal / Cas3 C-terminal domain / CRISPR-associated protein, CT1975 / CT1975-like protein / CRISPR-associated protein, CasD / Helicase Cas3, CRISPR-associated, core / CRISPR-associated protein Cse2 / Cse2 superfamily / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein Cse1 ...Cas3, C-terminal / Cas3 C-terminal domain / CRISPR-associated protein, CT1975 / CT1975-like protein / CRISPR-associated protein, CasD / Helicase Cas3, CRISPR-associated, core / CRISPR-associated protein Cse2 / Cse2 superfamily / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein Cse1 / CRISPR-associated protein Cse1 (CRISPR_cse1) / CRISPR-associated protein Cse3 / CRISPR associated protein / CRISPR_assoc / CRISPR-associated Cas3-type HD domain / CRISPR-associated Cas3-type HD domain superfamily / Cas3, HD domain / HD Cas3-type domain profile. / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) / AAA domain / CRISPR-associated protein Cas5, N-terminal / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Helicase, C-terminal / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
: / DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated helicase, Cas3 family / CRISPR-associated protein, Cse1 family / CRISPR-associated protein, Cse2 family / CRISPR-associated protein, Cse4 family / CRISPR-associated protein, Cas5e family / CRISPR-associated protein, Cse3 family
Similarity search - Component
Biological speciesThermobifida fusca (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsXiao, Y. / Luo, M. / Liao, M. / Ke, A.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118174 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM102543 United States
CitationJournal: Science / Year: 2018
Title: Structure basis for RNA-guided DNA degradation by Cascade and Cas3.
Authors: Yibei Xiao / Min Luo / Adam E Dolan / Maofu Liao / Ailong Ke /
Abstract: Type I CRISPR-Cas system features a sequential target-searching and degradation process on double-stranded DNA by the RNA-guided Cascade (CRISPR associated complex for antiviral defense) complex and ...Type I CRISPR-Cas system features a sequential target-searching and degradation process on double-stranded DNA by the RNA-guided Cascade (CRISPR associated complex for antiviral defense) complex and the nuclease-helicase fusion enzyme Cas3, respectively. Here, we present a 3.7-angstrom-resolution cryo-electron microscopy (cryo-EM) structure of the Type I-E Cascade/R-loop/Cas3 complex, poised to initiate DNA degradation. Cas3 distinguishes Cascade conformations and only captures the R-loop-forming Cascade, to avoid cleaving partially complementary targets. Its nuclease domain recruits the nontarget strand (NTS) DNA at a bulged region for the nicking of single-stranded DNA. An additional 4.7-angstrom-resolution cryo-EM structure captures the postnicking state, in which the severed NTS retracts to the helicase entrance, to be threaded for adenosine 5'-triphosphate-dependent processive degradation. These snapshots form the basis for understanding RNA-guided DNA degradation in Type I-E CRISPR-Cas systems.
History
DepositionJan 17, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 27, 2018Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Data collection / Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2Feb 20, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _pdbx_audit_support.funding_organization
Revision 1.4Nov 29, 2023Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_related / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_related.db_name / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-7347
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
G: CRISPR-associated helicase, Cas3 family
A: CRISPR-associated protein, Cse1 family
B: CRISPR-associated protein, Cse4 family
C: CRISPR-associated protein, Cse4 family
D: CRISPR-associated protein, Cse4 family
E: CRISPR-associated protein, Cse4 family
F: CRISPR-associated protein, Cse4 family
H: CRISPR-associated protein, Cse4 family
I: Uncharacterized protein
J: crRNA
K: Uncharacterized protein
L: Target strand
M: CRISPR-associated protein, Cas5e family
N: Nontarget strand
O: CRISPR-associated protein, Cse3 family
hetero molecules


Theoretical massNumber of molelcules
Total (without water)575,03517
Polymers574,92415
Non-polymers1122
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

-
Components

-
Protein , 2 types, 3 molecules GIK

#1: Protein CRISPR-associated helicase, Cas3 family


Mass: 104039.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1593 / Production host: Escherichia coli (E. coli) / References: UniProt: Q47PJ0
#4: Protein Uncharacterized protein


Mass: 27446.613 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1591 / Production host: Escherichia coli (E. coli) / References: UniProt: Q47PJ2

-
CRISPR-associated protein, ... , 4 types, 9 molecules ABCDEFHMO

#2: Protein CRISPR-associated protein, Cse1 family


Mass: 61433.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1592 / Production host: Escherichia coli (E. coli) / References: UniProt: Q47PJ1
#3: Protein
CRISPR-associated protein, Cse4 family


Mass: 41043.043 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1590 / Production host: Escherichia coli (E. coli) / References: UniProt: Q47PJ3
#7: Protein CRISPR-associated protein, Cas5e family


Mass: 28279.260 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1589 / Production host: Escherichia coli (E. coli) / References: UniProt: Q47PJ4
#9: Protein CRISPR-associated protein, Cse3 family


Mass: 26327.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermobifida fusca (strain YX) (bacteria)
Strain: YX / Gene: Tfu_1588 / Production host: Escherichia coli (E. coli) / References: UniProt: Q47PJ5

-
DNA chain , 2 types, 2 molecules LN

#6: DNA chain Target strand


Mass: 16801.725 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermobifida fusca (bacteria)
#8: DNA chain Nontarget strand


Mass: 17099.920 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermobifida fusca (bacteria)

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

#10: Chemical ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe
#5: RNA chain crRNA


Mass: 19790.793 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermobifida fusca (bacteria) / Production host: Escherichia coli (E. coli)

-
Experimental details

-
Experiment

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

-
Sample preparation

ComponentName: CRISPR RNA-guided surveillance complex with Cas3 bound in its pre-nicking state
Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT
Source (natural)Organism: Thermobifida fusca (strain YX) (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pcdf
Buffer solutionpH: 7.5 / Details: 10 mM HEPES pH 7.5, 150 mM NaCl, 5 mM DTT
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 85 %

-
Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 31000 X / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2700 nm / Cs: 2 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: GATAN LIQUID NITROGEN / Temperature (max): 105 K / Temperature (min): 80 K
Image recordingElectron dose: 8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1428

-
Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
1SPIDERparticle selection
2RELIONparticle selection
3UCSFImage44image acquisition
5RELION1.4CTF correction
8Coot0.8.1model fitting
10PHENIX1.11.1model refinement
11SPIDERinitial Euler assignment
12RELION1.4final Euler assignment
13RELION1.4classification
14RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 322268
3D reconstructionResolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51889 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00837324
ELECTRON MICROSCOPYf_angle_d1.09551376
ELECTRON MICROSCOPYf_dihedral_angle_d14.47721993
ELECTRON MICROSCOPYf_chiral_restr0.0575814
ELECTRON MICROSCOPYf_plane_restr0.0076200

+
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