[English] 日本語
Yorodumi
- PDB-6qxf: Cas1-Cas2-Csn2-DNA complex from the Type II-A CRISPR-Cas system -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 6qxf
TitleCas1-Cas2-Csn2-DNA complex from the Type II-A CRISPR-Cas system
Components
  • (CRISPR-associated ...) x 3
  • (DNA (25-MER)) x 2
KeywordsDNA BINDING PROTEIN / CRISPR / Cas / DNA binding / Nuclease / Adaptation / Spacer acquisition / Protein complex / Protein-DNA complex / Calcium / Metal-binding / Antiviral
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / RNA endonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / metal ion binding
Similarity search - Function
Rossmann fold - #11940 / Single alpha-helices involved in coiled-coils or other helix-helix interfaces - #2340 / CRISPR-associated protein, Csn2-type / CRISPR-associated protein Csn2 superfamily / CRISPR-associated protein (Cas_Csn2) / CRISPR-associated protein Cas1, NMENI subtype / CRISPR-associated endonuclease Cas2 / Virulence-associated protein D / CRISPR associated protein Cas2 / CRISPR associated protein Cas2 / : ...Rossmann fold - #11940 / Single alpha-helices involved in coiled-coils or other helix-helix interfaces - #2340 / CRISPR-associated protein, Csn2-type / CRISPR-associated protein Csn2 superfamily / CRISPR-associated protein (Cas_Csn2) / CRISPR-associated protein Cas1, NMENI subtype / CRISPR-associated endonuclease Cas2 / Virulence-associated protein D / CRISPR associated protein Cas2 / CRISPR associated protein Cas2 / : / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR associated protein Cas1 / Single alpha-helices involved in coiled-coils or other helix-helix interfaces / Helix non-globular / Special / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / CRISPR-associated endonuclease Cas1 / CRISPR-associated endoribonuclease Cas2 / CRISPR-associated protein Csn2
Similarity search - Component
Biological speciesStreptococcus thermophilus (bacteria)
Escherichia coli BL21 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWilkinson, M. / Drabavicius, G. / Silanskas, A. / Gasiunas, G. / Siksnys, V. / Wigley, D.B.
CitationJournal: Mol Cell / Year: 2019
Title: Structure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System.
Authors: Martin Wilkinson / Gediminas Drabavicius / Arunas Silanskas / Giedrius Gasiunas / Virginijus Siksnys / Dale B Wigley /
Abstract: CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR ...CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas1-Cas2-Csn2, referred to as monomer, contains ∼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA ∼30-bp duplexes within the channel. A filamentous form also comprises Cas1-Cas2-Csn2 units (typically 2-6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.
History
DepositionMar 7, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 8, 2019Provider: repository / Type: Initial release
Revision 1.1May 22, 2019Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.2Jul 24, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3May 15, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_conn.pdbx_dist_value / _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-4668
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: CRISPR-associated protein Csn2
B: CRISPR-associated protein Csn2
C: CRISPR-associated protein Csn2
D: CRISPR-associated protein Csn2
E: CRISPR-associated protein Csn2
F: CRISPR-associated protein Csn2
G: CRISPR-associated protein Csn2
H: CRISPR-associated protein Csn2
I: CRISPR-associated endonuclease Cas1
J: CRISPR-associated endonuclease Cas1
K: CRISPR-associated endoribonuclease Cas2
L: CRISPR-associated endonuclease Cas1
M: CRISPR-associated endonuclease Cas1
N: CRISPR-associated endoribonuclease Cas2
O: CRISPR-associated endonuclease Cas1
P: CRISPR-associated endonuclease Cas1
Q: CRISPR-associated endoribonuclease Cas2
R: CRISPR-associated endonuclease Cas1
S: CRISPR-associated endonuclease Cas1
T: CRISPR-associated endoribonuclease Cas2
X: DNA (25-MER)
Y: DNA (25-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)556,56730
Polymers556,24722
Non-polymers3218
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area59260 Å2
ΔGint-470 kcal/mol
Surface area189380 Å2
MethodPISA

-
Components

-
CRISPR-associated ... , 3 types, 20 molecules ABCDEFGHIJLMOPRSKNQT

#1: Protein
CRISPR-associated protein Csn2


Mass: 25533.473 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: csn2 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G3ECR4
#2: Protein
CRISPR-associated endonuclease Cas1


Mass: 35363.461 Da / Num. of mol.: 8 / Mutation: C-terminal Strep tag
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: cas1 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: G3ECR2, Hydrolases; Acting on ester bonds
#3: Protein
CRISPR-associated endoribonuclease Cas2


Mass: 13431.560 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: cas2 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: G3ECR3, Hydrolases; Acting on ester bonds

-
DNA chain , 2 types, 2 molecules XY

#4: DNA chain DNA (25-MER)


Mass: 7559.863 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: Ordered portion representing the mixed DNA fragments bound to the complex after sonicating cells and treating with DNaseI.
Source: (natural) Escherichia coli BL21(DE3) (bacteria)
#5: DNA chain DNA (25-MER)


Mass: 7785.214 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: Ordered portion representing the mixed DNA fragments bound to the complex after sonicating cells and treating with DNaseI.
Source: (natural) Escherichia coli BL21(DE3) (bacteria)

-
Non-polymers , 1 types, 8 molecules

#6: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca

-
Experimental details

-
Experiment

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

-
Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Cas1-Cas2-Csn2-DNA monomer complexCOMPLEX#1-#50MULTIPLE SOURCES
2Cas1-Cas2-Csn2COMPLEX#1-#31RECOMBINANT
3DNACOMPLEX#4-#51NATURAL
Molecular weightValue: 0.53 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Streptococcus thermophilus (bacteria)1308
33Escherichia coli BL21(DE3) (bacteria)469008
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: Buffer solution was filtered
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris1
2150 mMSodium chlorideNaCl1
31 mMTCEP1
SpecimenConc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was DNaseI treated followed by gel filtration. Peak fractions were concentrated prior to making grids.
Specimen supportDetails: Graphene oxide sheets were applied to the grid prior to sample application
Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K / Details: 15 s wait time, 1 s blot time.

-
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Calibrated magnification: 129032 X / Nominal defocus max: 2800 nm / Nominal defocus min: 1300 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
Image recordingAverage exposure time: 1 sec. / Electron dose: 92.8 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4908 / Details: Images were collected in movie mode with 39 frames
Image scansSampling size: 14 µm

-
Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
14Cootmodel refinement
15REFMACmodel refinement
16PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 853391
Details: Reproductions of an initial ab initio generated 3D reconstruction were used as templates for auto picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 306470 / Num. of class averages: 5 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Target criteria: Correlation coefficient and visual inspection
Details: Rounds of manual fitting in Coot, jelly-body refinement in REFMAC and real-space refinement in PHENIX
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
13V7F

3v7f

3V7F1
25XVN

5xvn

A5XVN2
35XVN

5xvn

B5XVN2
45XVN

5xvn

E5XVN2

+
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