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- EMDB-51543: Tn7016 PseCAST QCascade -

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Basic information

Entry
Database: EMDB / ID: EMD-51543
TitleTn7016 PseCAST QCascade
Map data
Sample
  • Complex: Tn7016 PseCAST QCascade
    • RNA: crRNA
    • DNA: T-DNA
    • DNA: NT-DNA
    • Protein or peptide: Cas8
    • Protein or peptide: Cas7.1
    • Protein or peptide: Cas6
    • Protein or peptide: TniQ.1
KeywordsCRISPR / transposon / genome / RNA
Biological speciesPseudoalteromonas aestuariivivens (bacteria) / RNA satellites (virus) / DNA molecule (others) / Pseudoalteromonas (bacteria) / Pseudoalteromonas agarivorans S816 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsLampe GD / Liang AR / Zhang DJ / Fernandez IS / Sternberg SH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: bioRxiv / Year: 2024
Title: Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells.
Authors: George D Lampe / Ashley R Liang / Dennis J Zhang / Israel S Fernández / Samuel H Sternberg /
Abstract: Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in a heterogeneous mixture of undesirable editing ...Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in a heterogeneous mixture of undesirable editing outcomes. We recently leveraged a type I-F CRISPR-associated transposase (CAST) from the Tn transposon (CAST) for DSB-free, RNA-guided DNA integration in human cells, taking advantage of its programmability and large payload capacity. CAST is the only characterized CAST system that has achieved human genomic DNA insertions, but multiple lines of evidence suggest that DNA binding may be a critical bottleneck that limits high-efficiency activity. Here we report structural determinants of target DNA recognition by the CAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), which revealed novel subtype-specific interactions and RNA-DNA heteroduplex features. By combining our structural data with target DNA library screens and rationally engineered protein mutations, we uncovered CAST variants that exhibit increased integration efficiency and modified PAM stringency. Structure predictions of key interfaces in the transpososome holoenzyme also revealed opportunities for the design of hybrid CASTs, which we leveraged to build chimeric systems that combine high-activity DNA binding and DNA integration modules. Collectively, our work provides unique structural insights into type I-F CAST systems while showcasing multiple diverse strategies to investigate and engineer new RNA-guided transposase architectures for human genome editing applications.
History
DepositionSep 13, 2024-
Header (metadata) releaseOct 16, 2024-
Map releaseOct 16, 2024-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51543.png

Downloads & links

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Map

FileDownload / File: emd_51543.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.25 Å/pix.
x 300 pix.
= 373.53 Å		1.25 Å/pix.
x 300 pix.
= 373.53 Å		1.25 Å/pix.
x 300 pix.
= 373.53 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2451 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.027326064 - 0.09501792
Average (Standard dev.)0.0000753994 (±0.0018897874)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 373.53 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_51543_msk_1.map
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Additional map: Post-processed map, Auto-Bfactor Relion

Fileemd_51543_additional_1.map
AnnotationPost-processed map, Auto-Bfactor Relion
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Additional map: Composite map built joining global map and local refined maps

Fileemd_51543_additional_2.map
AnnotationComposite map built joining global map and local refined maps
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Additional map: Local refined map around cas8 protein

Fileemd_51543_additional_3.map
AnnotationLocal refined map around cas8 protein
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Additional map: Local refined map around TniQ dimer

Fileemd_51543_additional_4.map
AnnotationLocal refined map around TniQ dimer
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Half map: Global half map 1

Fileemd_51543_half_map_1.map
AnnotationGlobal half map 1
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Half map: Global half map 2

Fileemd_51543_half_map_2.map
AnnotationGlobal half map 2
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Sample components

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Entire : Tn7016 PseCAST QCascade

EntireName: Tn7016 PseCAST QCascade
Components
  • Complex: Tn7016 PseCAST QCascade
    • RNA: crRNA
    • DNA: T-DNA
    • DNA: NT-DNA
    • Protein or peptide: Cas8
    • Protein or peptide: Cas7.1
    • Protein or peptide: Cas6
    • Protein or peptide: TniQ.1

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Supramolecule #1: Tn7016 PseCAST QCascade

SupramoleculeName: Tn7016 PseCAST QCascade / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudoalteromonas aestuariivivens (bacteria)

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Macromolecule #1: crRNA

MacromoleculeName: crRNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: RNA satellites (virus)
Molecular weightTheoretical: 19.501658 KDa
SequenceString:
CUGAAAAUAC AGUGGGGCCA CUAGGGACAG GAUUGGUGAC GUGACCUGCC GUAUAGGCAG

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Macromolecule #2: T-DNA

MacromoleculeName: T-DNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 22.801545 KDa
SequenceString: (DT)(DT)(DT)(DT)(DG)(DG)(DC)(DC)(DG)(DT) (DC)(DA)(DA)(DG)(DG)(DC)(DG)(DA)(DA)(DG) (DG)(DT)(DC)(DA)(DC)(DC)(DA)(DA)(DT) (DC)(DC)(DT)(DG)(DT)(DC)(DC)(DC)(DT)(DA) (DG) (DT)(DG)(DG)(DC)(DC)(DC) ...String:
(DT)(DT)(DT)(DT)(DG)(DG)(DC)(DC)(DG)(DT) (DC)(DA)(DA)(DG)(DG)(DC)(DG)(DA)(DA)(DG) (DG)(DT)(DC)(DA)(DC)(DC)(DA)(DA)(DT) (DC)(DC)(DT)(DG)(DT)(DC)(DC)(DC)(DT)(DA) (DG) (DT)(DG)(DG)(DC)(DC)(DC)(DC)(DA) (DC)(DT)(DG)(DT)(DG)(DG)(DC)(DG)(DG)(DT) (DC)(DC) (DA)(DA)(DT)(DG)(DG)(DC)(DT) (DT)(DG)(DA)(DT)(DG)(DA)(DA)

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Macromolecule #3: NT-DNA

MacromoleculeName: NT-DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 3.296191 KDa
SequenceString:
(DA)(DA)(DC)(DC)(DG)(DC)(DC)(DA)(DA)(DA) (DC)

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Macromolecule #4: Cas8

MacromoleculeName: Cas8 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudoalteromonas (bacteria)
Molecular weightTheoretical: 79.290211 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MHLKELLEIT DTTERDRSLR RAFSPYTAMI DITGSEAVAL IILLNLTYRK NQVDDLLDKK LAKQALKSED HINKCIKEIA WFHTHNLKY PDIRVSKQNL AVEPPTLHSY VLSSANYPKA YGWSHNSAKV NFAKLFVSYF KWQNQVSWLA QVLATNSDNW K SAFTSLGL ...String:
MHLKELLEIT DTTERDRSLR RAFSPYTAMI DITGSEAVAL IILLNLTYRK NQVDDLLDKK LAKQALKSED HINKCIKEIA WFHTHNLKY PDIRVSKQNL AVEPPTLHSY VLSSANYPKA YGWSHNSAKV NFAKLFVSYF KWQNQVSWLA QVLATNSDNW K SAFTSLGL SVKAFKSLCV TVKNSLPEEA IPDSVDRYSR QIRMPYHDGY LAVTPVISHV VQSKIQQAAI DKRARFSNVE FT RPAAVSM LAASLGGVIN VLNYPPYIRS KYHGLSNSRA FKLNNGQTVF NVEALLKPEL IKALEGIIFS NNALALKQRR QQK VKNIKE LRNTLLEWFS PVFEWRLDAI ENGYDLEQLE SASERLEYKI LSLPDNELPS LTIPLFRLLN EMLGGVSMTQ RYAF HPKLM SPLKAALQWL LVNLTDQKHV LIEEDDEHYR YLHLSGIRVF DAQALSNPYC SGIPSLTAVW GMIHSYQRKL NEALG TNVR FTSFSWFIRN YSAVAGKKLP ELSLQGAQQS RLKRPGIIDG KYCDLVFDLI IHIDGYEDDL QAVDSKPDIL KAHFPS NFA GGVMHQPELN SNINWCCLYS NENQLFEKLR RLPLSGCWVM PTEHKIQDLD ELLLLLNSDS KLSPSMMGYM LLTEPMA RV GSLERLHCYA EPAIGVVKYE AATSVRLKGI GNYFNSAFWM LDAQEKFMLM KKV

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Macromolecule #5: Cas7.1

MacromoleculeName: Cas7.1 / type: protein_or_peptide / ID: 5 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Pseudoalteromonas (bacteria)
Molecular weightTheoretical: 39.845145 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MELCNILKYD RSLYPGKAVF FYKTADSDFV PLEADINKIR GPKSGFTEAF TPQFSPKNIS PQDLTHNNIL TLEECYVPPN VEHIFCRFS LRVQANSLVP SGCSDPEVFS LLKELAETFK ECGGYKELAV RYCRNILIGT WLWRNQNTGN TQIEIKTSKG S CYLIDNTR ...String:
MELCNILKYD RSLYPGKAVF FYKTADSDFV PLEADINKIR GPKSGFTEAF TPQFSPKNIS PQDLTHNNIL TLEECYVPPN VEHIFCRFS LRVQANSLVP SGCSDPEVFS LLKELAETFK ECGGYKELAV RYCRNILIGT WLWRNQNTGN TQIEIKTSKG S CYLIDNTR KLAWESKWAS DDLKVLEELS NEIESALTDP NVFWSADITA KIEASFCQEI YPSQILNDKV KQGEASKQFV KA KCADGRY AVSFNSVKIG AALQSIDDWW DEDASKRLRV HEFGADKEIG VARRPPDSEQ NFYSIFKNTE WYLSALKNCI TNK NEKIDP AIYYLFSVLI KGGMFQKKAE AKKA

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Macromolecule #6: Cas6

MacromoleculeName: Cas6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudoalteromonas (bacteria)
Molecular weightTheoretical: 23.341666 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQRYYFTVHF LPKQANLALL TGRCISIMHG FILKHNIEGM GVTFPAWSDS SIGNEIAFVY TDKEILNTLK DQAYFVDMQD CGFFKVSQV LAVPDSCEEV RFIRNQAVAK IFTGESRRRL KRLQKRALAR GEDFNPKKIE APREIDIFHR VAMTSKSSQE D YILHIQKQ ...String:
MQRYYFTVHF LPKQANLALL TGRCISIMHG FILKHNIEGM GVTFPAWSDS SIGNEIAFVY TDKEILNTLK DQAYFVDMQD CGFFKVSQV LAVPDSCEEV RFIRNQAVAK IFTGESRRRL KRLQKRALAR GEDFNPKKIE APREIDIFHR VAMTSKSSQE D YILHIQKQ DVDCQAEPYF SNYGLASNEK FKGTVPDLSP SIDRN

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Macromolecule #7: TniQ.1

MacromoleculeName: TniQ.1 / type: protein_or_peptide / ID: 7 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudoalteromonas agarivorans S816 (bacteria)
Molecular weightTheoretical: 49.832406 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHHH HHGGSENLYF QSGMAFLFSP KARAFSDESL ESYLLRVVSE NFFDSYEGLS LAIREELHEL DFEAHGAFPV DLKRLNVYH AKHNSHFRMR ALGLLETLLD LPRYELQKLA LLKSDIKFNS SVALYNNGVD IPLRFIRHHA EEAVDSIPVC S QCLAEEAY ...String:
MGHHHHHHHH HHGGSENLYF QSGMAFLFSP KARAFSDESL ESYLLRVVSE NFFDSYEGLS LAIREELHEL DFEAHGAFPV DLKRLNVYH AKHNSHFRMR ALGLLETLLD LPRYELQKLA LLKSDIKFNS SVALYNNGVD IPLRFIRHHA EEAVDSIPVC S QCLAEEAY IKQSWHIKWV NACTKHQCAL LHNCPECYAP INYIENESIT HCSCGFELSC ASTSPVNTLS IEHLNKLLDK GE RNDSNPL FNNMTLTERF AALLWYQERY SQTDNFCLND AVNYFSKWPA VFNTELDELS KNAEMKLIDL FNKTEFKFIF GDA ILACPS TQKQSESHFI YRALLDYLVT LVESNPKTKK PNAADLLVSV LEAATLLGTS VEQVYRLYQN GILQTAFRHK MNQR INPYK GAFFLRHVIE YKTSFGNDKA RMYLSAW

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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 opticsC2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

20349

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 150000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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