[English] 日本語
Yorodumi
- EMDB-20127: Bacillus halodurans Cas1-Cas2 complex in presence of target CRISPR DNA -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-20127
TitleBacillus halodurans Cas1-Cas2 complex in presence of target CRISPR DNA
Map dataBacillus halodurans Cas1-Cas2 complex in presence of target CRISPR DNA
Sample
  • Organelle or cellular component: Bacillus halodurans Cas1-Cas2 complex with target CRISPR hairpin DNA
    • Protein or peptide: Cas1
    • Protein or peptide: Cas2
    • DNA: hairpin CRISPR target
Biological speciesBacillus halodurans C-125 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 22.1 Å
AuthorsSashital DG / Lee H / Dhingra Y
CitationJournal: Elife / Year: 2019
Title: The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation.
Authors: Hayun Lee / Yukti Dhingra / Dipali G Sashital /
Abstract: CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional ...CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional protein Cas4 has been implicated in selection and processing of prespacer substrates for Cas1-Cas2, although this mechanism remains unclear. We show that Cas4 interacts directly with Cas1-Cas2 forming a Cas4-Cas1-Cas2 complex that captures and processes prespacers prior to integration. Structural analysis of the Cas4-Cas1-Cas2 complex reveals two copies of Cas4 that closely interact with the two integrase active sites of Cas1, suggesting a mechanism for substrate handoff following processing. We also find that the Cas4-Cas1-Cas2 complex processes single-stranded DNA provided in cis or in trans with a double-stranded DNA duplex. Cas4 cleaves precisely upstream of PAM sequences, ensuring the acquisition of functional spacers. Our results explain how Cas4 cleavage coordinates with Cas1-Cas2 integration and defines the exact cleavage sites and specificity of Cas4.
History
DepositionApr 19, 2019-
Header (metadata) releaseMay 8, 2019-
Map releaseMay 8, 2019-
UpdateMay 8, 2019-
Current statusMay 8, 2019Processing site: RCSB / Status: Released

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20127.png

Downloads & links

-
Map

FileDownload / File: emd_20127.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBacillus halodurans Cas1-Cas2 complex in presence of target CRISPR DNA
Voxel sizeX=Y=Z: 3.8 Å
Density
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.3
Minimum - Maximum-0.088741094 - 0.5017328
Average (Standard dev.)0.004869582 (±0.051874746)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 304.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.83.83.8
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z304.000304.000304.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.0890.5020.005

-
Supplemental data

-
Sample components

-
Entire : Bacillus halodurans Cas1-Cas2 complex with target CRISPR hairpin DNA

EntireName: Bacillus halodurans Cas1-Cas2 complex with target CRISPR hairpin DNA
Components
  • Organelle or cellular component: Bacillus halodurans Cas1-Cas2 complex with target CRISPR hairpin DNA
    • Protein or peptide: Cas1
    • Protein or peptide: Cas2
    • DNA: hairpin CRISPR target

-
Supramolecule #1: Bacillus halodurans Cas1-Cas2 complex with target CRISPR hairpin DNA

SupramoleculeName: Bacillus halodurans Cas1-Cas2 complex with target CRISPR hairpin DNA
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus halodurans C-125 (bacteria)
Molecular weightExperimental: 240 KDa

-
Macromolecule #1: Cas1

MacromoleculeName: Cas1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacillus halodurans C-125 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKKLLNTLYV TQPDTYLSLD GDNVVLLKEQ EKLGRLPLHN LEAIVGFGYT GASPALMGYC AERNISITFL TKNGRFLARV VGESRGNVVL RKTQYRISEN DQESTKIARN FITGKVYNSK WMLERMTREH PLRVNVEQFK ATSQLLSVMM QEIRNCDSLE SLRGWEGQAA ...String:
MKKLLNTLYV TQPDTYLSLD GDNVVLLKEQ EKLGRLPLHN LEAIVGFGYT GASPALMGYC AERNISITFL TKNGRFLARV VGESRGNVVL RKTQYRISEN DQESTKIARN FITGKVYNSK WMLERMTREH PLRVNVEQFK ATSQLLSVMM QEIRNCDSLE SLRGWEGQAA INYNKVFDQM ILQQKEEFAF HGRSRRPPKD NVNAMLSFAY TLLANDVAAA LETVGLDAYV GFMHQDRPGR ASLALDLMEE LRGLYADRFV LSLINRKEMT ADGFYKKENG AVLMTDEARK TFLKAWQTKK QEKITHPYLG EKMSWGLVPY VQALLLARFL RGDLDEYPPF LWK

-
Macromolecule #2: Cas2

MacromoleculeName: Cas2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Bacillus halodurans C-125 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MLVLITYDVQ TSSMGGTKRL RKVAKACQNY GQRVQNSVFE CIVDSTQLTS LKLELTSLID EEKDSLRIYR LGNNYKTKVE HIGAKPSIDL EDPLIF

-
Macromolecule #3: hairpin CRISPR target

MacromoleculeName: hairpin CRISPR target / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: Bacillus halodurans C-125 (bacteria)
SequenceString:
GATTTTCGCT GTCGCACTCT TCATGGGTGC GTGGATTGAA ATATTGACGA TAGTCAATAT TTCAATCCAC GCACCCATGA AGAGTGCGAC AGCGAAAATC

-
Experimental details

-
Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

Concentration0.024 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
100.0 mMKClpotassium chloride
5.0 %C3H8O3glycerol
2.0 mMC4H10O2S2Dithiothreitol
2.0 mMMnCl2Manganese chloride
StainingType: NEGATIVE / Material: uranyl formate
Details: 3 uL of sample was applied to a glow-discharged copper 400-mesh continuous carbon grid for one minute at room temperature. The excess sample was blotted with Whatman filter paper, followed ...Details: 3 uL of sample was applied to a glow-discharged copper 400-mesh continuous carbon grid for one minute at room temperature. The excess sample was blotted with Whatman filter paper, followed by immediate application of 3 uL 2% (w/v) uranyl formate. The excess stain was blotted, followed by immediate application of 3 uL 2% uranyl formate. This step was repeated once more. The grids were allowed to dry for at least 5 minutes prior to imaging.
GridSupport film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: unspecified

-
Electron microscopy

MicroscopeJEOL 2100
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 200 / Average exposure time: 0.5 sec. / Average electron dose: 30.0 e/Å2

-
Image processing

Particle selectionNumber selected: 95669
CTF correctionSoftware - Name: CTFFIND (ver. 4.0)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4p6i


Details: low-pass filtered to 90 angstrom
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 22.1 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 5279

+
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