[English] 日本語
Yorodumi
- EMDB-20350: VC-Tn6677 multisubunit CRISPR/Cas effector, open conformation. -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-20350
TitleVC-Tn6677 multisubunit CRISPR/Cas effector, open conformation.
Map datapost-processed map using B-factor automatically calculated by Relion
Sample
  • Complex: VC-Tn667 transposon encoded CRISPR/Cas effector
    • RNA: RNA (60-MER)
    • Protein or peptide: cas7 type I-F CRISPR-associated protein Csy3
    • Protein or peptide: cas5_8 naturally occurring fusion protein from Vibrio cholerae transposon Tn6677
    • Protein or peptide: type I-F CRISPR-associated endoribonuclease Cas6/Csy4
    • Protein or peptide: TniQ monomer 1
    • Protein or peptide: TniQ monomer 2
KeywordsCRISPR/Cas / Cascade / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Biological speciesVibrio cholerae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsHalpin-Healy T / Klompe S
CitationJournal: Nature / Year: 2020
Title: Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system.
Authors: Tyler S Halpin-Healy / Sanne E Klompe / Samuel H Sternberg / Israel S Fernández /
Abstract: Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements. Type I CRISPR-Cas systems typically ...Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements. Type I CRISPR-Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas3, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3' end of the CRISPR RNA (crRNA). The natural Cas8-Cas5 fusion protein binds the 5' crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications.
History
DepositionJun 26, 2019-
Header (metadata) releaseOct 2, 2019-
Map releaseOct 2, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.022
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6pig
  • Surface level: 0.022
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20350.png

Downloads & links

-
Map

FileDownload / File: emd_20350.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpost-processed map using B-factor automatically calculated by Relion
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 300 pix.
= 285. Å		0.95 Å/pix.
x 300 pix.
= 285. Å		0.95 Å/pix.
x 300 pix.
= 285. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.95 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.022
Minimum - Maximum-0.08457878 - 0.12968093
Average (Standard dev.)-0.000010695795 (±0.0054820883)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 285.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.950.950.95
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z285.000285.000285.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0850.130-0.000

-
Supplemental data

-
Mask #1

Fileemd_20350_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Additional map: masked post-processed map using B-factor automatically calculated by...

Fileemd_20350_additional_1.map
Annotationmasked post-processed map using B-factor automatically calculated by Relion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Additional map: unsharpened map

Fileemd_20350_additional_2.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: half map 1 from Relion3

Fileemd_20350_half_map_1.map
Annotationhalf map 1 from Relion3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: half map 2 from Relion3

Fileemd_20350_half_map_2.map
Annotationhalf map 2 from Relion3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : VC-Tn667 transposon encoded CRISPR/Cas effector

EntireName: VC-Tn667 transposon encoded CRISPR/Cas effector
Components
  • Complex: VC-Tn667 transposon encoded CRISPR/Cas effector
    • RNA: RNA (60-MER)
    • Protein or peptide: cas7 type I-F CRISPR-associated protein Csy3
    • Protein or peptide: cas5_8 naturally occurring fusion protein from Vibrio cholerae transposon Tn6677
    • Protein or peptide: type I-F CRISPR-associated endoribonuclease Cas6/Csy4
    • Protein or peptide: TniQ monomer 1
    • Protein or peptide: TniQ monomer 2

-
Supramolecule #1: VC-Tn667 transposon encoded CRISPR/Cas effector

SupramoleculeName: VC-Tn667 transposon encoded CRISPR/Cas effector / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 600 KDa

-
Macromolecule #1: RNA (60-MER)

MacromoleculeName: RNA (60-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 19.237338 KDa
SequenceString:
CUGAUAACUU ACAGGACGCU UUGGCUUCAU UGCUUUUCAG GUGAACUGCC GAGUAGGUAG

-
Macromolecule #2: cas7 type I-F CRISPR-associated protein Csy3

MacromoleculeName: cas7 type I-F CRISPR-associated protein Csy3 / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 39.020035 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: KLPTNLAYER SIDPSDVCFF VVWPDDRKTP LTYNSRTLLG QMEAKSLAYD VSGQPIKSAT AEALAQGNPH QVDFCHVPYG ASHIECSFS VSFSSELRQP YKCNSSKVKQ TLVQLVELYE TKIGWTELAT RYLMNICNGK WLWKNTRKAY CWNIVLTPWP W NGEKVGFE ...String:
KLPTNLAYER SIDPSDVCFF VVWPDDRKTP LTYNSRTLLG QMEAKSLAYD VSGQPIKSAT AEALAQGNPH QVDFCHVPYG ASHIECSFS VSFSSELRQP YKCNSSKVKQ TLVQLVELYE TKIGWTELAT RYLMNICNGK WLWKNTRKAY CWNIVLTPWP W NGEKVGFE DIRTNYTSRQ DFKNNKNWSA IVEMIKTAFS STDGLAIFEV RATLHLPTNA MVRPSQVFTE KETQNSRVFQ ST TIDGERS PILGAFKTGA AIATIDDWYP EATEPLRVGR FGVHREDVTC YRHPSTGKDF FSILQQAEHY IEVLSANKTP AQE TINDMH FLMANLIKGG MFQHKGD

-
Macromolecule #3: cas5_8 naturally occurring fusion protein from Vibrio cholerae tr...

MacromoleculeName: cas5_8 naturally occurring fusion protein from Vibrio cholerae transposon Tn6677
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 57.064711 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: LKEIIASNPD DLTTELKRAF RPLTPHIAID GNEIDALTIL VNLTDKAKCK QKLRDEKWWA SCINCVNYRQ SHNPKFPDIR SEGVIRTQA LGELPSFLLS SSKIPPYHWS YSHDSKYVNK SAFLTNEFCW DGEISCLGEL LKDADHPLWN TLKKLGCSQK T CKAMAKQL ...String:
LKEIIASNPD DLTTELKRAF RPLTPHIAID GNEIDALTIL VNLTDKAKCK QKLRDEKWWA SCINCVNYRQ SHNPKFPDIR SEGVIRTQA LGELPSFLLS SSKIPPYHWS YSHDSKYVNK SAFLTNEFCW DGEISCLGEL LKDADHPLWN TLKKLGCSQK T CKAMAKQL ADITLTTINV TLAPNYLTQI SLPDSDTSYI SLSPVASLSM QSHFHQRLQD ENRHSAITRF SRTTNMGVTA MT CGGAFRM LKSGAKFSSP PHHRLNNGSF LVLPNIRVCG ATALSSPVTV GIPSLTAFFG FVHAFERNIN RTTSSFRVES FAI CVHQLH VEKRGLTAEF VEKGDGTISA PATRDDWQCD VVFSLILNTN FAQHIDQDTL VTSLPKRLAR GSAKIAIDDF KHIN SFSTL ETAIESLPIE AGRWLSLYAQ SNNNLSDLLA AMTEDHQLMA SCVGYHLLEE PKDKPNSLRG YKHAIAECII GLINS ITFS SETDPNTIFW SLKNYQNYLV VQPRSIN

-
Macromolecule #4: type I-F CRISPR-associated endoribonuclease Cas6/Csy4

MacromoleculeName: type I-F CRISPR-associated endoribonuclease Cas6/Csy4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 22.926184 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: KWYYKTITFL PELCNNESLA AKCLRVLHGF NYQYETRNIG VSFPLWCDAT VGKKISFVSK NKIELDLLLK QHYFVQMEQL QYFHISNTV LVPEDCTYVS FRRCQSIDKL TAAGLARKIR RLEKRALSRG EAFDPSSFAQ KEHTAIAHYH SLGESSKQTN R NFRLNIRM ...String:
KWYYKTITFL PELCNNESLA AKCLRVLHGF NYQYETRNIG VSFPLWCDAT VGKKISFVSK NKIELDLLLK QHYFVQMEQL QYFHISNTV LVPEDCTYVS FRRCQSIDKL TAAGLARKIR RLEKRALSRG EAFDPSSFAQ KEHTAIAHYH SLGESSKQTN R NFRLNIRM LSEQPREGNS IFSSYGLANS ENSFQPVPLI

-
Macromolecule #5: TniQ monomer 1

MacromoleculeName: TniQ monomer 1 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 41.51716 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: AMFLQRPKPY SDESLESFFI RVANKNGYGD VHRFLEATKR FLQDIDHNGY QTFPTDITRI NPYSAKNSSS ARTASFLKLA QLTFNEPPE LLGLAINRTN MKYSPSTSAV VRGAEVFPRS LLRTHSIPCC PLCLRENGYA SYLWHFQGYE YCHSHNVPLI T TCSGHEAA ...String:
AMFLQRPKPY SDESLESFFI RVANKNGYGD VHRFLEATKR FLQDIDHNGY QTFPTDITRI NPYSAKNSSS ARTASFLKLA QLTFNEPPE LLGLAINRTN MKYSPSTSAV VRGAEVFPRS LLRTHSIPCC PLCLRENGYA SYLWHFQGYE YCHSHNVPLI T TCSGHEAA CTVSNWLAGH ESKPLPNLPK SYRWGLVHWW MGIKDSDHFS FVQFFSNWPR SFHSIIEDEV EFNLEHAVVS TS ELRLKDL LGRLFFGSIR LPERNLQHNI ILGELLCYLE NRLWQDKGLI ANLKMNALEA TVMLNCSLDQ IASMVEQRIL KPN RKSKDV TDYLFHFGDI FCLWLAEFQS DEFNRSFYVS RW

-
Macromolecule #6: TniQ monomer 2

MacromoleculeName: TniQ monomer 2 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 42.315031 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: AMFLQRPKPY SDESLESFFI RVANKNGYGD VHRFLEATKR FLQDIDHNGY QTFPTDITRI NPYSAKNSSS ARTASFLKLA QLTFNEPPE LLGLAINRTN MKYSPSTSAV VRGAEVFPRS LLRTHSIPCC PLCLRENGYA SYLWHFQGYE YCHSHNVPLI T TCSCGKEF ...String:
AMFLQRPKPY SDESLESFFI RVANKNGYGD VHRFLEATKR FLQDIDHNGY QTFPTDITRI NPYSAKNSSS ARTASFLKLA QLTFNEPPE LLGLAINRTN MKYSPSTSAV VRGAEVFPRS LLRTHSIPCC PLCLRENGYA SYLWHFQGYE YCHSHNVPLI T TCSCGKEF DYRVSEAACT VSNWLAGHES KPLPNLPKSY RWGLVHWWMG IKDDHFSFVQ FFSNWPRSFH SIIEDEVEFN LE HAVVSTS ELRLKDLLGR LFFGSIRLPE RNLQHNIILG ELLCYLENRL WQDKGLIANL KMNALEATVM LNCSLDQIAS MVE QRILKP NAAAAAAAAA DVTDYLFHFG DIFCLWLAAF QSDEFNRSFY VSR

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

BufferpH: 7.2
Component:
ConcentrationFormulaName
20.0 mMhepeshepes-koh
200.0 mMNaClsodium chloride
2.0 mMDTTditiotreitol
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK II

-
Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

+
Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 87000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
FSC plot (resolution estimation)

+
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