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- EMDB-0929: CryoEM structure of Cascade-TniQ-dsDNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-0929
TitleCryoEM structure of Cascade-TniQ-dsDNA complex
Map data
Sample
  • Complex: CryoEM structure of Cascade-TniQ-dsDNA
    • Protein or peptide: CRISPR-associated protein Cas7
    • Protein or peptide: CRISPR-associated protein Cas8
    • Protein or peptide: CRISPR-associated protein Cas6
    • Protein or peptide: Transposition protein TniQ
    • RNA: CRISPR RNA (60-MER)
    • DNA: Target DNA strand (51-MER)
    • DNA: Non-target DNA strand (51-MER)
KeywordsType I-F CRISPR-Cas system / Csy Cascade / Tn7-like transposons / RNA-guided tranposition / transposase subunit / IMMUNE SYSTEM
Biological speciesVibrio cholerae (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsWang B / Xu W
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31971135 China
CitationJournal: Cell Res / Year: 2020
Title: Structural basis of a Tn7-like transposase recruitment and DNA loading to CRISPR-Cas surveillance complex.
Authors: Beibei Wang / Wenhao Xu / Hui Yang /
History
DepositionDec 28, 2019-
Header (metadata) releaseFeb 19, 2020-
Map releaseFeb 19, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6lnb
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0929.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.0015 / Movie #1: 0.006
Minimum - Maximum-0.088798605 - 0.16237427
Average (Standard dev.)0.00021827052 (±0.0040957043)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z276.480276.480276.480
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0890.1620.000

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Supplemental data

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Sample components

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Entire : CryoEM structure of Cascade-TniQ-dsDNA

EntireName: CryoEM structure of Cascade-TniQ-dsDNA
Components
  • Complex: CryoEM structure of Cascade-TniQ-dsDNA
    • Protein or peptide: CRISPR-associated protein Cas7
    • Protein or peptide: CRISPR-associated protein Cas8
    • Protein or peptide: CRISPR-associated protein Cas6
    • Protein or peptide: Transposition protein TniQ
    • RNA: CRISPR RNA (60-MER)
    • DNA: Target DNA strand (51-MER)
    • DNA: Non-target DNA strand (51-MER)

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Supramolecule #1: CryoEM structure of Cascade-TniQ-dsDNA

SupramoleculeName: CryoEM structure of Cascade-TniQ-dsDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Vibrio cholerae (bacteria)

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Macromolecule #1: CRISPR-associated protein Cas7

MacromoleculeName: CRISPR-associated protein Cas7 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 40.030156 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSMKLPTNLA YERSIDPSDV CFFVVWPDDR KTPLTYNSRT LLGQMEAASL AYDVSGQPIK SATAEALAQG NPHQVDFCHV PYGASHIEC SFSVSFSSEL RQPYKCNSSK VKQTLVQLVE LYETKIGWTE LATRYLMNIC NGKWLWKNTR KAYCWNIVLT P WPWNGEKV ...String:
GSMKLPTNLA YERSIDPSDV CFFVVWPDDR KTPLTYNSRT LLGQMEAASL AYDVSGQPIK SATAEALAQG NPHQVDFCHV PYGASHIEC SFSVSFSSEL RQPYKCNSSK VKQTLVQLVE LYETKIGWTE LATRYLMNIC NGKWLWKNTR KAYCWNIVLT P WPWNGEKV GFEDIRTNYT SRQDFKNNKN WSAIVEMIKT AFSSTDGLAI FEVRATLHLP TNAMVRPSQV FTEKESGSKS KS KTQNSRV FQSTTIDGER SPILGAFKTG AAIATIDDWY PEATEPLRVG RFGVHREDVT CYRHPSTGKD FFSILQQAEH YIE VLSANK TPAQETINDM HFLMANLIKG GMFQHKGD

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Macromolecule #2: CRISPR-associated protein Cas8

MacromoleculeName: CRISPR-associated protein Cas8 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 72.29493 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MQTLKELIAS NPDDLTTELK RAFRPLTPHI AIDGNELDAL TILVNLTDKT DDQKDLLDRA KCKQKLRDEK WWASCINCVN YRQSHNPKF PDIRSEGVIR TQALGELPSF LLSSSKIPPY HWSYSHDSKY VNKSAFLTNE FCWDGEISCL GELLKDADHP L WNTLKKLG ...String:
MQTLKELIAS NPDDLTTELK RAFRPLTPHI AIDGNELDAL TILVNLTDKT DDQKDLLDRA KCKQKLRDEK WWASCINCVN YRQSHNPKF PDIRSEGVIR TQALGELPSF LLSSSKIPPY HWSYSHDSKY VNKSAFLTNE FCWDGEISCL GELLKDADHP L WNTLKKLG CSQKTCKAMA KQLADITLTT INVTLAPNYL TQISLPDSDT SYISLSPVAS LSMQSHFHQR LQDENRHSAI TR FSRTTNM GVTAMTCGGA FRMLKSGAKF SSPPHHRLNS KRSWLTSEHV QSLKQYQRLN KSLIPENSRI ALRRKYKIEL QNM VRSWFA MQDHTLDSNI LIQHLNHDLS YLGATKRFAY DPAMTKLFTE LLKRELSNSI NNGEQHTNGS FLVLPNIRVC GATA LSSPV TVGIPSLTAF FGFVHAFERN INRTTSSFRV ESFAICVHQL HVEKRGLTAE FVEKGDGTIS APATRDDWQC DVVFS LILN TNFAQHIDQD TLVTSLPKRL ARGSAKIAID DFKHINSFST LETAIESLPI EAGRWLSLYA QSNNNLSDLL AAMTED HQL MASCVGYHLL EEPKDKPNSL RGYKHAIAEC IIGLINSITF SSETDPNTIF WSLKNYQNYL VVQPRSINDE TTDKSSL

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Macromolecule #3: CRISPR-associated protein Cas6

MacromoleculeName: CRISPR-associated protein Cas6 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 23.098365 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: VKWYYKTITF LPELCNNESL AAKCLRVLHG FNYQYETRNI GVSFPLWCDA TVGKKISFVS KNKIELDLLL KQHYFVQMEQ LQYFHISNT VLVPEDCTYV SFRRCQSIDK LTAAGLARKI RRLEKRALSR GEQFDPSSFA QKEHTAIAHY HSLGESSKQT N RNFRLNIR ...String:
VKWYYKTITF LPELCNNESL AAKCLRVLHG FNYQYETRNI GVSFPLWCDA TVGKKISFVS KNKIELDLLL KQHYFVQMEQ LQYFHISNT VLVPEDCTYV SFRRCQSIDK LTAAGLARKI RRLEKRALSR GEQFDPSSFA QKEHTAIAHY HSLGESSKQT N RNFRLNIR MLSEQPREGN SIFSSYGLSN SENSFQPVPL I

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Macromolecule #4: Transposition protein TniQ

MacromoleculeName: Transposition protein TniQ / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 45.597867 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MFLQRPKPYS DESLESFFIR VANKNGYGDV HRFLEATKRF LQDIDHNGYQ TFPTDITRIN PYSAKNSSSA RTASFLKLAQ LTFNEPPEL LGLAINRTNM KYSPSTSAVV RGAEVFPRSL LRTHSIPCCP LCLRENGYAS YLWHFQGYEY CHSHNVPLIT T CSCGKEFD ...String:
MFLQRPKPYS DESLESFFIR VANKNGYGDV HRFLEATKRF LQDIDHNGYQ TFPTDITRIN PYSAKNSSSA RTASFLKLAQ LTFNEPPEL LGLAINRTNM KYSPSTSAVV RGAEVFPRSL LRTHSIPCCP LCLRENGYAS YLWHFQGYEY CHSHNVPLIT T CSCGKEFD YRVSGLKGIC CKCKEPITLT SRENGHEAAC TVSNWLAGHE SKPLPNLPKS YRWGLVHWWM GIKDSEFDHF SF VQFFSNW PRSFHSIIED EVEFNLEHAV VSTSELRLKD LLGRLFFGSI RLPERNLQHN IILGELLCYL ENRLWQDKGL IAN LKMNAL EATVMLNCSL DQIASMVEQR ILKPNRKSKP NSPLDVTDYL FHFGDIFCLW LAEFQSDEFN RSFYVSRW

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Macromolecule #5: CRISPR RNA (60-MER)

MacromoleculeName: CRISPR RNA (60-MER) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 19.305428 KDa
SequenceString:
CUGAUAACUU CACGGCGGGC UUGAUGUCCG CGUCUACCUG GUGAACUGCC GAGUAGGUAG

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Macromolecule #6: Target DNA strand (51-MER)

MacromoleculeName: Target DNA strand (51-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 15.856158 KDa
SequenceString:
(DA)(DA)(DA)(DC)(DC)(DT)(DT)(DG)(DG)(DG) (DA)(DG)(DG)(DT)(DA)(DG)(DA)(DC)(DG)(DC) (DG)(DG)(DA)(DC)(DA)(DT)(DC)(DA)(DA) (DG)(DC)(DC)(DC)(DG)(DC)(DC)(DG)(DT)(DG) (DA) (DA)(DG)(DG)(DT)(DC)(DT)(DT)(DA) (DG)(DG)(DG)

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Macromolecule #7: Non-target DNA strand (51-MER)

MacromoleculeName: Non-target DNA strand (51-MER) / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 15.535982 KDa
SequenceString:
(DC)(DC)(DC)(DT)(DA)(DA)(DG)(DA)(DC)(DC) (DT)(DT)(DC)(DA)(DC)(DG)(DG)(DC)(DT)(DT) (DT)(DA)(DA)(DA)(DG)(DA)(DT)(DG)(DT) (DC)(DT)(DT)(DT)(DA)(DA)(DC)(DT)(DA)(DC) (DC) (DT)(DC)(DC)(DC)(DA)(DA)(DG)(DG) (DT)(DT)(DT)

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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
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 206913

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