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- EMDB-41280: Non-targeted transpososome from ShCAST -

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Basic information

Entry
Database: EMDB / ID: EMD-41280
TitleNon-targeted transpososome from ShCAST
Map datacomposite map of focused refinements
Sample
  • Complex: Non-targeted transpososome from ShCAST
    • Protein or peptide: TnsB from ShCAST
    • Protein or peptide: TnsC from ShCAST
    • Protein or peptide: TniQ from ShCAST
KeywordsCAST / transposase / AAA+ ATPase / AAA+ / CRISPR / Cas / DNA BINDING PROTEIN-DNA complex / DNA BINDING PROTEIN
Biological species[Scytonema hofmanni] UTEX 2349 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.11 Å
AuthorsPark J / Kellogg EH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00-GM124463 United States
CitationJournal: Science / Year: 2023
Title: Mechanism of target site selection by type V-K CRISPR-associated transposases.
Authors: Jerrin Thomas George / Christopher Acree / Jung-Un Park / Muwen Kong / Tanner Wiegand / Yanis Luca Pignot / Elizabeth H Kellogg / Eric C Greene / Samuel H Sternberg /
Abstract: CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to catalyze RNA-guided transposition of large genetic payloads. Type V-K CASTs offer potential technology ...CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to catalyze RNA-guided transposition of large genetic payloads. Type V-K CASTs offer potential technology advantages but lack accuracy, and the molecular basis for this drawback has remained elusive. Here, we reveal that type V-K CASTs maintain an RNA-independent, "untargeted" transposition pathway alongside RNA-dependent integration, driven by the local availability of TnsC filaments. Using cryo-electron microscopy, single-molecule experiments, and high-throughput sequencing, we found that a minimal, CRISPR-less transpososome preferentially directs untargeted integration at AT-rich sites, with additional local specificity imparted by TnsB. By exploiting this knowledge, we suppressed untargeted transposition and increased type V-K CAST specificity up to 98.1% in cells without compromising on-target integration efficiency. These findings will inform further engineering of CAST systems for accurate, kilobase-scale genome engineering applications.
History
DepositionJul 19, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41280.png

Downloads & links

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Map

FileDownload / File: emd_41280.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcomposite map of focused refinements
Voxel sizeX=Y=Z: 1.33 Å
Density
Contour LevelBy AUTHOR: 0.9
Minimum - Maximum-0.25970194 - 5.675871
Average (Standard dev.)0.013970158 (±0.12413385)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 478.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Local-resolution filtered consensus map

Fileemd_41280_additional_1.map
AnnotationLocal-resolution filtered consensus map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Local-resolution filtered map from focused refinement of TnsC-TniQ...

Fileemd_41280_additional_2.map
AnnotationLocal-resolution filtered map from focused refinement of TnsC-TniQ region
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Local-resolution filtered map from focused refinement of TnsB region

Fileemd_41280_additional_3.map
AnnotationLocal-resolution filtered map from focused refinement of TnsB region
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Non-targeted transpososome from ShCAST

EntireName: Non-targeted transpososome from ShCAST
Components
  • Complex: Non-targeted transpososome from ShCAST
    • Protein or peptide: TnsB from ShCAST
    • Protein or peptide: TnsC from ShCAST
    • Protein or peptide: TniQ from ShCAST

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Supramolecule #1: Non-targeted transpososome from ShCAST

SupramoleculeName: Non-targeted transpososome from ShCAST / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
Molecular weightTheoretical: 400 KDa

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Macromolecule #1: TnsB from ShCAST

MacromoleculeName: TnsB from ShCAST / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNSQQNPDLA VHPLAIPMEG LLGESATTLE KNVIATQLSE EAQVKLEVIQ SLLEPCDRTT YGQKLREAAE KLNVSLRTVQ RLVKNWEQDG LVGLTQTSRA DKGKHRIGEF WENFITKTYK EGNKGSKRMT PKQVALRVEA KARELKDSKP PNYKTVLRVL APILEKQQKA ...String:
MNSQQNPDLA VHPLAIPMEG LLGESATTLE KNVIATQLSE EAQVKLEVIQ SLLEPCDRTT YGQKLREAAE KLNVSLRTVQ RLVKNWEQDG LVGLTQTSRA DKGKHRIGEF WENFITKTYK EGNKGSKRMT PKQVALRVEA KARELKDSKP PNYKTVLRVL APILEKQQKA KSIRSPGWRG TTLSVKTREG KDLSVDYSNH VWQCDHTRVD VLLVDQHGEI LSRPWLTTVI DTYSRCIMGI NLGFDAPSSG VVALALRHAI LPKRYGSEYK LHCEWGTYGK PEHFYTDGGK DFRSNHLSQI GAQLGFVCHL RDRPSEGGVV ERPFKTLNDQ LFSTLPGYTG SNVQERPEDA EKDARLTLRE LEQLLVRYIV DRYNQSIDAR MGDQTRFERW EAGLPTVPVP IPERDLDICL MKQSRRTVQR GGCLQFQNLM YRGEYLAGYA GETVNLRFDP RDITTILVYR QENNQEVFLT RAHAQGLETE QLALDEAEAA SRRLRTAGKT ISNQSLLQEV VDRDALVATK KSRKERQKLE QTVLRSAAVD ESNRESLPSQ IVEPDEVEST ETVHSQYEDI EVWDYEQLRE EYGF

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Macromolecule #2: TnsC from ShCAST

MacromoleculeName: TnsC from ShCAST / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTVP VVYIRPHQKC GPKDLFKKIT EYLKYRVTKG TVSDFRDRTI EVLKGCGVEM LIIDEADRLK PETFADVRDI AEDLGIAVVL ...String:
MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTVP VVYIRPHQKC GPKDLFKKIT EYLKYRVTKG TVSDFRDRTI EVLKGCGVEM LIIDEADRLK PETFADVRDI AEDLGIAVVL VGTDRLDAVI KRDEQVLERF RAHLRFGKLS GEDFKNTVEM WEQMVLKLPV SSNLKSKEML RILTSATEGY IGRLDEILRE AAIRSLSRGL KKIDKAVLQE VAKEYK

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Macromolecule #3: TniQ from ShCAST

MacromoleculeName: TniQ from ShCAST / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MIEAPDVKPW LFLIKPYEGE SLSHFLGRFR RANHLSASGL GTLAGIGAIV ARWERFHFNP RPSQQELEAI ASVVEVDAQR LAQMLPPAGV GMQHEPIRLC GACYAESPCH RIEWQYKSVW KCDRHQLKIL AKCPNCQAPF KMPALWEDGC CHRCRMPFAE MAKLQKV

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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

Concentration0.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
200.0 mMNaClSodium Chloride
1.0 mMC4H10O2S2Dithiothreitol
2.0 mMC10H16N5O13P3Adenosine triphosphate
10.0 mMMgCl2Magnesium Chloride
2.0 %C3H8O3Glycerol

Details: 25 mM HEPES pH 7.5, 200 mM NaCl, 1 mM DTT, 2 mM ATP, and 10 mM MgCl2 and 2% glycerol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 2 / Number real images: 3081 / Average exposure time: 3.2 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 63000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 8.11 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.3) / Number images used: 13392
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0.3)
Final 3D classificationNumber classes: 2 / Avg.num./class: 20000 / Software - Name: cryoSPARC (ver. 4.0.3)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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