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- EMDB-26348: I-F3b Cascade-TniQ full R-loop complex -

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Entry
Database: EMDB / ID: EMD-26348
TitleI-F3b Cascade-TniQ full R-loop complex
Map datacombined map of local-resolution filtered maps from full refinement and focused refinement
Sample
  • Complex: I-F3b Cascade-TniQ Full R-loop complex
    • RNA: crRNA
    • DNA: Target strand DNA
    • DNA: Non-target strand DNA
    • Protein or peptide: Cas8/5
    • Protein or peptide: Cas7
    • Protein or peptide: Cas6
    • Protein or peptide: TniQ
KeywordsCRISPR-Cas / Transposon / CAST / Cascade / I-F / I-F3 / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA complex
Biological speciesAeromonas salmonicida (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsPark JU / Mehrotra E / Kellogg EH
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00-GM124463 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM144566 United States
CitationJournal: Mol Cell / Year: 2023
Title: Multiple adaptations underly co-option of a CRISPR surveillance complex for RNA-guided DNA transposition.
Authors: Jung-Un Park / Michael T Petassi / Shan-Chi Hsieh / Eshan Mehrotra / Gabriel Schuler / Jagat Budhathoki / Vinh H Truong / Summer B Thyme / Ailong Ke / Elizabeth H Kellogg / Joseph E Peters /
Abstract: CRISPR-associated transposons (CASTs) are natural RNA-directed transposition systems. We demonstrate that transposon protein TniQ plays a central role in promoting R-loop formation by RNA-guided DNA- ...CRISPR-associated transposons (CASTs) are natural RNA-directed transposition systems. We demonstrate that transposon protein TniQ plays a central role in promoting R-loop formation by RNA-guided DNA-targeting modules. TniQ residues, proximal to CRISPR RNA (crRNA), are required for recognizing different crRNA categories, revealing an unappreciated role of TniQ to direct transposition into different classes of crRNA targets. To investigate adaptations allowing CAST elements to utilize attachment sites inaccessible to CRISPR-Cas surveillance complexes, we compared and contrasted PAM sequence requirements in both I-F3b CAST and I-F1 CRISPR-Cas systems. We identify specific amino acids that enable a wider range of PAM sequences to be accommodated in I-F3b CAST elements compared with I-F1 CRISPR-Cas, enabling CAST elements to access attachment sites as sequences drift and evade host surveillance. Together, this evidence points to the central role of TniQ in facilitating the acquisition of CRISPR effector complexes for RNA-guided DNA transposition.
History
DepositionMar 2, 2022-
Header (metadata) releaseJun 21, 2023-
Map releaseJun 21, 2023-
UpdateJun 21, 2023-
Current statusJun 21, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26348.png

Downloads & links

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Map

FileDownload / File: emd_26348.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcombined map of local-resolution filtered maps from full refinement and focused refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 420 pix.
= 366.66 Å		0.87 Å/pix.
x 420 pix.
= 366.66 Å		0.87 Å/pix.
x 420 pix.
= 366.66 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.873 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.007
Minimum - Maximum-0.028453883 - 0.108551964
Average (Standard dev.)0.00016127276 (±0.0017559092)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 366.66 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: unsharpened map of the full structure

Fileemd_26348_additional_1.map
Annotationunsharpened map of the full structure
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map from focused refinement

Fileemd_26348_additional_2.map
AnnotationUnsharpened map from focused refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: halfmap 1 from focused refinement

Fileemd_26348_additional_3.map
Annotationhalfmap 1 from focused refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: halfmap 2 from focused refinement

Fileemd_26348_additional_4.map
Annotationhalfmap 2 from focused refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap 2 of the full structure

Fileemd_26348_half_map_1.map
AnnotationHalfmap 2 of the full structure
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap 1 of the full structure

Fileemd_26348_half_map_2.map
AnnotationHalfmap 1 of the full structure
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : I-F3b Cascade-TniQ Full R-loop complex

EntireName: I-F3b Cascade-TniQ Full R-loop complex
Components
  • Complex: I-F3b Cascade-TniQ Full R-loop complex
    • RNA: crRNA
    • DNA: Target strand DNA
    • DNA: Non-target strand DNA
    • Protein or peptide: Cas8/5
    • Protein or peptide: Cas7
    • Protein or peptide: Cas6
    • Protein or peptide: TniQ

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Supramolecule #1: I-F3b Cascade-TniQ Full R-loop complex

SupramoleculeName: I-F3b Cascade-TniQ Full R-loop complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Aeromonas salmonicida (bacteria)
Molecular weightTheoretical: 450 KDa

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Macromolecule #1: crRNA

MacromoleculeName: crRNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Aeromonas salmonicida (bacteria)
Molecular weightTheoretical: 19.399604 KDa
SequenceString:
CCAAGAAAAG GACUGGAAGA AAUCAUCCAA GUUGGGGACU AUUUUCUGCC GUAUAGGCAG

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Macromolecule #2: Target strand DNA

MacromoleculeName: Target strand DNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Aeromonas salmonicida (bacteria)
Molecular weightTheoretical: 35.61368 KDa
SequenceString: (DC)(DT)(DG)(DG)(DC)(DT)(DG)(DG)(DC)(DG) (DA)(DA)(DC)(DG)(DA)(DG)(DC)(DG)(DC)(DA) (DA)(DG)(DG)(DT)(DG)(DG)(DT)(DG)(DG) (DC)(DC)(DC)(DC)(DA)(DT)(DC)(DA)(DG)(DC) (DC) (DA)(DC)(DA)(DT)(DC)(DC) ...String:
(DC)(DT)(DG)(DG)(DC)(DT)(DG)(DG)(DC)(DG) (DA)(DA)(DC)(DG)(DA)(DG)(DC)(DG)(DC)(DA) (DA)(DG)(DG)(DT)(DG)(DG)(DT)(DG)(DG) (DC)(DC)(DC)(DC)(DA)(DT)(DC)(DA)(DG)(DC) (DC) (DA)(DC)(DA)(DT)(DC)(DC)(DC)(DG) (DG)(DC)(DA)(DC)(DT)(DC)(DG)(DA)(DA)(DG) (DT)(DC) (DC)(DC)(DC)(DA)(DA)(DC)(DT) (DT)(DG)(DG)(DA)(DT)(DG)(DA)(DT)(DT)(DT) (DC)(DT)(DT) (DC)(DC)(DA)(DG)(DT)(DC) (DC)(DT)(DG)(DG)(DT)(DA)(DA)(DG)(DC)(DA) (DC)(DC)(DC)(DG) (DA)(DA)(DT)(DC)(DA) (DT)(DC)(DC)(DT)(DC)(DT)(DT)(DG)(DC)(DG) (DG)

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Macromolecule #3: Non-target strand DNA

MacromoleculeName: Non-target strand DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Aeromonas salmonicida (bacteria)
Molecular weightTheoretical: 35.737723 KDa
SequenceString: (DC)(DC)(DG)(DC)(DA)(DA)(DG)(DA)(DG)(DG) (DA)(DT)(DG)(DA)(DT)(DT)(DC)(DG)(DG)(DG) (DT)(DG)(DC)(DT)(DT)(DA)(DC)(DC)(DT) (DC)(DC)(DT)(DG)(DA)(DC)(DC)(DT)(DT)(DC) (DT) (DT)(DT)(DA)(DG)(DT)(DA) ...String:
(DC)(DC)(DG)(DC)(DA)(DA)(DG)(DA)(DG)(DG) (DA)(DT)(DG)(DA)(DT)(DT)(DC)(DG)(DG)(DG) (DT)(DG)(DC)(DT)(DT)(DA)(DC)(DC)(DT) (DC)(DC)(DT)(DG)(DA)(DC)(DC)(DT)(DT)(DC) (DT) (DT)(DT)(DA)(DG)(DT)(DA)(DG)(DG) (DT)(DT)(DC)(DA)(DA)(DC)(DC)(DC)(DC)(DT) (DG)(DA) (DT)(DC)(DG)(DA)(DG)(DT)(DG) (DC)(DC)(DG)(DG)(DG)(DA)(DT)(DG)(DT)(DG) (DG)(DC)(DT) (DG)(DA)(DT)(DG)(DG)(DG) (DG)(DC)(DC)(DA)(DC)(DC)(DA)(DC)(DC)(DT) (DT)(DG)(DC)(DG) (DC)(DT)(DC)(DG)(DT) (DT)(DC)(DG)(DC)(DC)(DA)(DG)(DC)(DC)(DA) (DG)

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Macromolecule #4: Cas8/5

MacromoleculeName: Cas8/5 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Aeromonas salmonicida (bacteria)
Molecular weightTheoretical: 78.728367 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVTIMHIEEL LDIEDHGERD RQLRRYLAPY SAEIGVDGAE KMALVVLLNL TLKRDRVESL CDEGLARQLL SDEGHITNCL HTVRWLHTH NLKYPDARVS GERLIINAPP LIPGVISSAG LPMRMGWAHD SSDINLAKLF GTSFRYRDDS TNLALQLVAR S KTWEQALI ...String:
MVTIMHIEEL LDIEDHGERD RQLRRYLAPY SAEIGVDGAE KMALVVLLNL TLKRDRVESL CDEGLARQLL SDEGHITNCL HTVRWLHTH NLKYPDARVS GERLIINAPP LIPGVISSAG LPMRMGWAHD SSDINLAKLF GTSFRYRDDS TNLALQLVAR S KTWEQALI GLGLTQQQLD IWCQLLASNL ENNTFPTVVS PFSKQVRFLY QGNYCVVTPV VSHALLAQLQ NVVHEKKLQC TY IHHDHPA SVGSLVGALG GKVAVLDYPP PVSPDKARSF SQARKHRLAN GQSLFDRSVF NDHVFIDALK HVISRPGLTR KQQ RQLRLS ALRYLRRQLA IWLGPIIEWR DEIVSSGRGE PGNLPSGGLE LELITQPKKM LPELMLQVAG RFHLELQNHS AGRR FAFHP ALMAPIKSQI LWLLRQLADD EEKDEPHPPT SCYYLHLSGL TVYDASALAN PYLCGIPSLS ALAGFCHDYE RRLQS LIGQ SVYFRGLAWY LGRYSLVTGK HLPEPSKSAD PKSVSAIRRP GLLDGRYCDL GMDLIIEVHI PTGGSLPFTT CLDLLR VAL PARFAGGCLH PPSLYEEYNW CTVYQDKSTL FTVLSRLPRY GCWIYPSDAD LRSFEELSEA LALDRRLRPV ATGFVFL EE PVERAGSIEG QHVYAESAIG TALCINPVEM RLAGKKRFFG AGFWQLNDAK GAILMNGSAN TG

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Macromolecule #5: Cas7

MacromoleculeName: Cas7 / type: protein_or_peptide / ID: 5 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Aeromonas salmonicida (bacteria)
Molecular weightTheoretical: 39.105172 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MELCTHLSYS RSLSPGKAVF FYKTAESDFV PLRIEVAKIS GQKCGYTEGF DANLKPKNIE RYELAYSNPQ TIEACYVPPN VDELYCRFS LRVEANSMRP YVCSNPDVLR VMIGLAQAYQ RLGGYNELAR RYSANVLRGI WLWRNQYTQG TKIEIKTSLG S TYHIPDAR ...String:
MELCTHLSYS RSLSPGKAVF FYKTAESDFV PLRIEVAKIS GQKCGYTEGF DANLKPKNIE RYELAYSNPQ TIEACYVPPN VDELYCRFS LRVEANSMRP YVCSNPDVLR VMIGLAQAYQ RLGGYNELAR RYSANVLRGI WLWRNQYTQG TKIEIKTSLG S TYHIPDAR RLSWSGDWPE LEQKQLEQLT SEMAKALSQP DIFWFADVTA SLKTGFCQEI FPSQKFTERP DDHSVASRQL AT VECSDGQ LAACINPQKI GAALQKIDDW WANDADLPLR VHEYGANHEA LTALRHPATG QDFYHLLTKA EQFVTVLESS EGG GVELPG EVHYLMAVLV KGGLFQKGKG R

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Macromolecule #6: Cas6

MacromoleculeName: Cas6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Aeromonas salmonicida (bacteria)
Molecular weightTheoretical: 23.640832 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTENRYFFAI RYLSDDVDCG LLAGRCISIL HGFRQAHPGI QIGVAFPEWS DRDLGRSIAF VSTNKSLLER FRERSYFQVM QADNFFALS LVLEVPDTCQ NVRFIRNQNL AKLFVGERRR RLARAKRRAK ARGEAFQPHM PDETKVVGVF HSVFMQSASS G QSYILHIQ ...String:
MTENRYFFAI RYLSDDVDCG LLAGRCISIL HGFRQAHPGI QIGVAFPEWS DRDLGRSIAF VSTNKSLLER FRERSYFQVM QADNFFALS LVLEVPDTCQ NVRFIRNQNL AKLFVGERRR RLARAKRRAK ARGEAFQPHM PDETKVVGVF HSVFMQSASS G QSYILHIQ KHRYERSEDS GYSSYGLASN DLYTGYVPDL GAIFSTLF

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Macromolecule #7: TniQ

MacromoleculeName: TniQ / type: protein_or_peptide / ID: 7 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Aeromonas salmonicida (bacteria)
Molecular weightTheoretical: 46.366637 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MHLLVRPEPF ADEALESYFL RLSQENGFER YRIFSGSVQD WLHTTDHAAA GAFPLELSRL NIFHASRSSG LRVRALQLVD RLTDGAPFR LLQLALCHSA ISFGNHYKAV HRSGVDIPLS FIRVHQIPCC PDCLRESAYV RQCWHFKPYV GCHRHGGRLI Y SCPACGES ...String:
MHLLVRPEPF ADEALESYFL RLSQENGFER YRIFSGSVQD WLHTTDHAAA GAFPLELSRL NIFHASRSSG LRVRALQLVD RLTDGAPFR LLQLALCHSA ISFGNHYKAV HRSGVDIPLS FIRVHQIPCC PDCLRESAYV RQCWHFKPYV GCHRHGGRLI Y SCPACGES LNYLASESIN HCQCGFDLRT ASTVPAQPDE IQLSALAYGC SFESSNPLLA IGSLSARFGA LYWYQQRYLS DH EAVRDDR ALTKAIGHFT AWPDAFWREL QQMVDDALVR QTKPLNHTDF VDVFGSVVAD CRQIPMRNTG QNFILKNLIG FLT DLVARH PQCRVANVGD LLLSAVDAAT LLSTSVEQVR RLHHEGFLPL SIRPASRNTV SPHRAVFHLR HVVELRQARM QSHH DHSST YLPAW

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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

Concentration0.9 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chlorideSodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / 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 / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 53353
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6pif

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7u5d:
I-F3b Cascade-TniQ full R-loop complex

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