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- EMDB-20351: VC-Tn6677 multisubunit CRISPR/Cas effector, close conformation. -

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Basic information

Entry
Database: EMDB / ID: EMD-20351
TitleVC-Tn6677 multisubunit CRISPR/Cas effector, close conformation.
Map datapost-processed map with B-factor automatically calculated by Relion3
Sample
  • Complex: VC-Tn667 transposon encoded CRISPR/Cas effector
    • Protein or peptide: cas7 type I-F CRISPR-associated protein Csy3
    • Protein or peptide: cas5_8 naturally occurring fusion protein
    • Protein or peptide: type I-F CRISPR-associated endoribonuclease Cas6/Csy4
    • Protein or peptide: TniQ monomer I
    • Protein or peptide: TniQ monomer 2
    • RNA: guide RNA
    • DNA: Targeting strand ssDNA
    • DNA: Non-targeting strand ssDNA
KeywordsCRISPR/Cas / Cascade / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex
Biological speciesVibrio cholerae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 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

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6pij
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20351.png

Downloads & links

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Map

FileDownload / File: emd_20351.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpost-processed map with B-factor automatically calculated by Relion3
Voxel sizeX=Y=Z: 1.0605 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.07761144 - 0.17915863
Average (Standard dev.)-0.000006529219 (±0.0049957177)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 424.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.06051.06051.0605
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z424.200424.200424.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0780.179-0.000

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Supplemental data

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Mask #1

Fileemd_20351_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram (log scale)

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Additional map: unsharpened map

Fileemd_20351_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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

Fileemd_20351_additional_2.map
Annotationmasked post-processed map with B-factor automatically calculated by Relion3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_20351_half_map_1.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_20351_half_map_2.map
Annotationhalf map 1
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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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
    • Protein or peptide: cas7 type I-F CRISPR-associated protein Csy3
    • Protein or peptide: cas5_8 naturally occurring fusion protein
    • Protein or peptide: type I-F CRISPR-associated endoribonuclease Cas6/Csy4
    • Protein or peptide: TniQ monomer I
    • Protein or peptide: TniQ monomer 2
    • RNA: guide RNA
    • DNA: Targeting strand ssDNA
    • DNA: Non-targeting strand ssDNA

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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

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Macromolecule #1: cas7 type I-F CRISPR-associated protein Csy3

MacromoleculeName: cas7 type I-F CRISPR-associated protein Csy3 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 39.588664 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 KEAAAAAAAA TQ NSRVFQS TTIDGERSPI LGAFKTGAAI ATIDDWYPEA TEPLRVGRFG VHREDVTCYR HPSTGKDFFS ILQQAEHYIE VLS ANKTPA QETINDMHFL MANLIKGGMF QHKGD

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Macromolecule #2: cas5_8 naturally occurring fusion protein

MacromoleculeName: cas5_8 naturally occurring fusion protein / type: protein_or_peptide / ID: 2 / 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

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Macromolecule #3: type I-F CRISPR-associated endoribonuclease Cas6/Csy4

MacromoleculeName: type I-F CRISPR-associated endoribonuclease Cas6/Csy4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 22.813025 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 ENSFQPVPL

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Macromolecule #4: TniQ monomer I

MacromoleculeName: TniQ monomer I / type: protein_or_peptide / ID: 4 / 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

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Macromolecule #5: TniQ monomer 2

MacromoleculeName: TniQ monomer 2 / type: protein_or_peptide / ID: 5 / 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

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Macromolecule #6: guide RNA

MacromoleculeName: guide RNA / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 19.237338 KDa
SequenceString:
CUGAUAACUU ACAGGACGCU UUGGCUUCAU UGCUUUUCAG GUGAACUGCC GAGUAGGUAG

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Macromolecule #7: Targeting strand ssDNA

MacromoleculeName: Targeting strand ssDNA / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 8.391409 KDa
SequenceString:
(DA)(DT)(DG)(DA)(DA)(DG)(DC)(DC)(DA)(DA) (DG)(DG)(DC)(DG)(DT)(DC)(DC)(DT)(DG)(DT) (DA)(DA)(DG)(DG)(DC)(DG)(DG)

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Macromolecule #8: Non-targeting strand ssDNA

MacromoleculeName: Non-targeting strand ssDNA / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 2.65175 KDa
SequenceString:
(DC)(DC)(DG)(DC)(DC)(DT)(DT)(DA)(DC)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

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

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Electron microscopy

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

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 74366
FSC plot (resolution estimation)

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