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- EMDB-21146: Cryo-EM structure of Cascade-TniQ-dsDNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-21146
TitleCryo-EM structure of Cascade-TniQ-dsDNA ternary complex
Map dataCascade-TniQ-dsDNA ternary complex
Sample
  • Complex: Cascade-TniQ-dsDNA ternary complex
    • Complex: DNA/RNA
      • DNA: DNA (5'-D(*GP*CP*AP*GP*TP*CP*AP*TP*CP*AP*CP*CP*AP*AP*TP*TP*TP*AP*TP*TP*TP*A)-3')
      • DNA: DNA (100-MER)
      • Protein or peptide: Cas6
    • Complex: Cascade ternary complex
      • RNA: RNA (61-MER)
      • Protein or peptide: Cas8
      • Protein or peptide: Cas7
      • Protein or peptide: TniQ
  • Ligand: ZINC ION
KeywordsCRISPR-Cas system / Cascade- TniQ / IMMUNE SYSTEM
Biological speciesVibrio cholerae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsJia N / Patel DJ
CitationJournal: Cell Res / Year: 2020
Title: Structure-function insights into the initial step of DNA integration by a CRISPR-Cas-Transposon complex.
Authors: Ning Jia / Wei Xie / M Jason de la Cruz / Edward T Eng / Dinshaw J Patel /
History
DepositionDec 19, 2019-
Header (metadata) releaseJan 29, 2020-
Map releaseJan 29, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vbw
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21146.png

Downloads & links

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Map

FileDownload / File: emd_21146.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCascade-TniQ-dsDNA ternary complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 288 pix.
= 306.432 Å		1.06 Å/pix.
x 288 pix.
= 306.432 Å		1.06 Å/pix.
x 288 pix.
= 306.432 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.064 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.101571605 - 0.17907953
Average (Standard dev.)0.00019449755 (±0.0036486867)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 306.432 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0641.0641.064
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z306.432306.432306.432
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.1020.1790.000

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Supplemental data

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Sample components

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Entire : Cascade-TniQ-dsDNA ternary complex

EntireName: Cascade-TniQ-dsDNA ternary complex
Components
  • Complex: Cascade-TniQ-dsDNA ternary complex
    • Complex: DNA/RNA
      • DNA: DNA (5'-D(*GP*CP*AP*GP*TP*CP*AP*TP*CP*AP*CP*CP*AP*AP*TP*TP*TP*AP*TP*TP*TP*A)-3')
      • DNA: DNA (100-MER)
      • Protein or peptide: Cas6
    • Complex: Cascade ternary complex
      • RNA: RNA (61-MER)
      • Protein or peptide: Cas8
      • Protein or peptide: Cas7
      • Protein or peptide: TniQ
  • Ligand: ZINC ION

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Supramolecule #1: Cascade-TniQ-dsDNA ternary complex

SupramoleculeName: Cascade-TniQ-dsDNA ternary complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Molecular weightTheoretical: 400 KDa

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Supramolecule #2: DNA/RNA

SupramoleculeName: DNA/RNA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2, #6
Source (natural)Organism: Vibrio cholerae (bacteria)

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Supramolecule #3: Cascade ternary complex

SupramoleculeName: Cascade ternary complex / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#5, #7
Source (natural)Organism: Vibrio cholerae (bacteria)

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Macromolecule #1: DNA (5'-D(*GP*CP*AP*GP*TP*CP*AP*TP*CP*AP*CP*CP*AP*AP*TP*TP*TP*AP*...

MacromoleculeName: DNA (5'-D(*GP*CP*AP*GP*TP*CP*AP*TP*CP*AP*CP*CP*AP*AP*TP*TP*TP*AP*TP*TP*TP*A)-3')
type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 6.685354 KDa
SequenceString:
(DG)(DC)(DA)(DG)(DT)(DC)(DA)(DT)(DC)(DA) (DC)(DC)(DA)(DA)(DT)(DT)(DT)(DA)(DT)(DT) (DT)(DA)

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Macromolecule #2: DNA (100-MER)

MacromoleculeName: DNA (100-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 30.901777 KDa
SequenceString: (DA)(DC)(DA)(DT)(DA)(DT)(DG)(DG)(DC)(DA) (DG)(DA)(DT)(DC)(DT)(DC)(DA)(DA)(DT)(DT) (DG)(DG)(DA)(DT)(DA)(DT)(DC)(DG)(DG) (DC)(DC)(DG)(DG)(DC)(DC)(DA)(DC)(DG)(DC) (DG) (DA)(DT)(DC)(DG)(DC)(DT) ...String:
(DA)(DC)(DA)(DT)(DA)(DT)(DG)(DG)(DC)(DA) (DG)(DA)(DT)(DC)(DT)(DC)(DA)(DA)(DT)(DT) (DG)(DG)(DA)(DT)(DA)(DT)(DC)(DG)(DG) (DC)(DC)(DG)(DG)(DC)(DC)(DA)(DC)(DG)(DC) (DG) (DA)(DT)(DC)(DG)(DC)(DT)(DG)(DA) (DC)(DG)(DT)(DT)(DT)(DC)(DA)(DC)(DC)(DT) (DG)(DA) (DA)(DA)(DA)(DG)(DC)(DA)(DA) (DT)(DG)(DA)(DA)(DG)(DC)(DC)(DA)(DA)(DA) (DG)(DC)(DG) (DT)(DC)(DC)(DT)(DG)(DT) (DA)(DA)(DG)(DG)(DT)(DG)(DA)(DT)(DG)(DA) (DC)(DT)(DG)(DC)

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Macromolecule #3: RNA (61-MER)

MacromoleculeName: RNA (61-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 19.566543 KDa
SequenceString:
CUGAUAACUU ACAGGACGCU UUGGCUUCAU UGCUUUUCAG GUGAACUGCC GAGUAGGUAG A

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Macromolecule #4: Cas8

MacromoleculeName: Cas8 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 72.29493 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 #5: Cas7

MacromoleculeName: Cas7 / type: protein_or_peptide / ID: 5 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 39.886031 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKLPTNLAYE RSIDPSDVCF FVVWPDDRKT PLTYNSRTLL GQMEAASLAY DVSGQPIKSA TAEALAQGNP HQVDFCHVPY GASHIECSF SVSFSSELRQ PYKCNSSKVK QTLVQLVELY ETKIGWTELA TRYLMNICNG KWLWKNTRKA YCWNIVLTPW P WNGEKVGF ...String:
MKLPTNLAYE RSIDPSDVCF FVVWPDDRKT PLTYNSRTLL GQMEAASLAY DVSGQPIKSA TAEALAQGNP HQVDFCHVPY GASHIECSF SVSFSSELRQ PYKCNSSKVK QTLVQLVELY ETKIGWTELA TRYLMNICNG KWLWKNTRKA YCWNIVLTPW P WNGEKVGF EDIRTNYTSR QDFKNNKNWS AIVEMIKTAF SSTDGLAIFE VRATLHLPTN AMVRPSQVFT EKESGSKSKS KT QNSRVFQ STTIDGERSP ILGAFKTGAA IATIDDWYPE ATEPLRVGRF GVHREDVTCY RHPSTGKDFF SILQQAEHYI EVL SANKTP AQETINDMHF LMANLIKGGM FQHKGD

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Macromolecule #6: Cas6

MacromoleculeName: Cas6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 23.13043 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKWYYKTITF LPELCNNESL AAKCLRVLHG FNYQYETRNI GVSFPLWCDA TVGKKISFVS KNKIELDLLL KQHYFVQMEQ LQYFHISNT VLVPEDCTYV SFRRCQSIDK LTAAGLARKI RRLEKRALSR GEQFDPSSFA QKEHTAIAHY HSLGESSKQT N RNFRLNIR ...String:
MKWYYKTITF 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 #7: TniQ

MacromoleculeName: TniQ / type: protein_or_peptide / ID: 7 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 45.597867 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration0.5 mg/mL
BufferpH: 7.5 / Component - Formula: Tris / Details: 20 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM DTT
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 2.16 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 55900
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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