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- EMDB-19286: consensus map of the V-K CRISPR-associated Transposon Integration... -

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Basic information

Entry
Database: EMDB / ID: EMD-19286
Titleconsensus map of the V-K CRISPR-associated Transposon Integration Assembly
Map dataCAST V-K R1 global refinement, consensus map used as reference for composite map.
Sample
  • Complex: Type V-K CRISPR-associated transposon post-transposition state after transesterification (consensus map)
    • RNA: x 1 types
    • DNA: x 6 types
    • Protein or peptide: x 5 types
KeywordsCRISPR-associated Transposon genome editing transposition / DNA BINDING PROTEIN
Biological speciesScytonema hofmannii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.19 Å
AuthorsTenjo-Castano F / Mesa P / Montoya G
Funding support Denmark, European Union, 2 items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
European Research Council (ERC)101096548European Union
CitationJournal: Mol Cell / Year: 2024
Title: Conformational landscape of the type V-K CRISPR-associated transposon integration assembly.
Authors: Francisco Tenjo-Castaño / Nicholas Sofos / Luisa S Stutzke / Piero Temperini / Anders Fuglsang / Tillmann Pape / Pablo Mesa / Guillermo Montoya /
Abstract: CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site ...CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted ∼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and att sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications.
History
DepositionDec 31, 2023-
Header (metadata) releaseJun 19, 2024-
Map releaseJun 19, 2024-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19286.png

Downloads & links

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Map

FileDownload / File: emd_19286.map.gz / Format: CCP4 / Size: 107.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCAST V-K R1 global refinement, consensus map used as reference for composite map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 484 pix.
= 352.352 Å		0.73 Å/pix.
x 242 pix.
= 176.176 Å		0.73 Å/pix.
x 240 pix.
= 174.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.728 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.055
Minimum - Maximum-0.29208744 - 0.508315
Average (Standard dev.)0.003402445 (±0.027492493)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin314315152
Dimensions242240484
Spacing240242484
CellA: 174.72 Å / B: 176.176 Å / C: 352.352 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19286_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CAST V-K R1 global refinement, Half-A map.

Fileemd_19286_half_map_1.map
AnnotationCAST V-K R1 global refinement, Half-A map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CAST V-K R1 global refinement, Half-B map.

Fileemd_19286_half_map_2.map
AnnotationCAST V-K R1 global refinement, Half-B map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Type V-K CRISPR-associated transposon post-transposition state af...

EntireName: Type V-K CRISPR-associated transposon post-transposition state after transesterification (consensus map)
Components
  • Complex: Type V-K CRISPR-associated transposon post-transposition state after transesterification (consensus map)
    • RNA: sgRNA
    • DNA: Non-target strand - LE
    • DNA: Target strand - LE
    • DNA: LE
    • DNA: RE
    • DNA: Non-target strand - RE
    • DNA: Target strand
    • Protein or peptide: Cas12k
    • Protein or peptide: S15
    • Protein or peptide: TniQ
    • Protein or peptide: TnsC
    • Protein or peptide: TnsB

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Supramolecule #1: Type V-K CRISPR-associated transposon post-transposition state af...

SupramoleculeName: Type V-K CRISPR-associated transposon post-transposition state after transesterification (consensus map)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 1 MDa

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Macromolecule #1: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 1
Source (natural)Organism: Scytonema hofmannii (bacteria)
SequenceString: GGAUAUUAAU AGCGCCGCAA UUCAUGCUGC UUGCAGCCUC UGAAUUUUGU UAAAUGAGGG UUAGUUUGAC UGUAUAAAUA CAGUCUUGCU UUCUGACCCU GGUAGCUGCU CACCCUGAUG CUGCUGUCAA UAGACAGGAU AGGUGCGCUC CCAGCAAUAA GGGCGCGGAU ...String:
GGAUAUUAAU AGCGCCGCAA UUCAUGCUGC UUGCAGCCUC UGAAUUUUGU UAAAUGAGGG UUAGUUUGAC UGUAUAAAUA CAGUCUUGCU UUCUGACCCU GGUAGCUGCU CACCCUGAUG CUGCUGUCAA UAGACAGGAU AGGUGCGCUC CCAGCAAUAA GGGCGCGGAU GUACUGCUGU AGUGGCUACU GAAUCACCCC CGAUCAAGGG GGAACCCUCC AAAAGGUGGG UUGAAAGGAG AAGUCAUUUA AUAAGGCCAC U

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Macromolecule #2: Non-target strand - LE

MacromoleculeName: Non-target strand - LE / type: dna / ID: 2 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
SequenceString:
GtgaaggttC TCTTCAGTAT TAATAAGGCC ACTGTTAAAA CGTACTATAt agaCATCTCC ACAAAAGG

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Macromolecule #3: Target strand - LE

MacromoleculeName: Target strand - LE / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
SequenceString:
aattaaatag tcacaatgac attaatctgt caccgacgac agataatttg tcactgtaca ctacgCCTTT TGTGGAGATG tctaATATCT ACGTTTTAAC AGTGGCCTTA TTAAATGACT TCTCaacctt cac

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Macromolecule #4: LE

MacromoleculeName: LE / type: dna / ID: 4 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
SequenceString:
AAAAAAAAAA AAAAAtgtac agtgacaaat tatctgtcgt cggtgacaga ttaatgtcat tgtgactatt taatt

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Macromolecule #5: RE

MacromoleculeName: RE / type: dna / ID: 5 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
SequenceString:
AAAAAAAAAA AAAAAtgtac agtgactaat tatatgtcgt tgtgacaaat tattgtcatc agtaaaatcc ttat

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Macromolecule #6: Non-target strand - RE

MacromoleculeName: Non-target strand - RE / type: dna / ID: 6 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
SequenceString:
ataaggattt tactgatgac aataatttgt cacaacgaca tataattagt cactgtacac gtagAGACGT AGCAATGCT

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Macromolecule #7: Target strand

MacromoleculeName: Target strand / type: dna / ID: 7 / Classification: DNA
Source (natural)Organism: Scytonema hofmannii (bacteria)
SequenceString:
AGCATTGCTA CGTCT

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Macromolecule #8: Cas12k

MacromoleculeName: Cas12k / type: protein_or_peptide / ID: 8 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MASWSHPQFE KGGGSGGGSG GSAWSHPQFE KENLYFQGGS SQITIQARLI SFESNRQQLW KLMADLNTPL INELLCQLGQ HPDFEKWQQK GKLPSTVVSQ LCQPLKTDPR FAGQPSRLYM SAIHIVDYIY KSWLAIQKRL QQQLDGKTRW ...String:
MGSSHHHHHH SSGLVPRGSH MASWSHPQFE KGGGSGGGSG GSAWSHPQFE KENLYFQGGS SQITIQARLI SFESNRQQLW KLMADLNTPL INELLCQLGQ HPDFEKWQQK GKLPSTVVSQ LCQPLKTDPR FAGQPSRLYM SAIHIVDYIY KSWLAIQKRL QQQLDGKTRW LEMLNSDAEL VELSGDTLEA IRVKAAEILA IAMPASESDS ASPKGKKGKK EKKPSSSSPK RSLSKTLFDA YQETEDIKSR SAISYLLKNG CKLTDKEEDS EKFAKRRRQV EIQIQRLTEK LISRMPKGRD LTNAKWLETL LTATTTVAED NAQAKRWQDI LLTRSSSLPF PLVFETNEDM VWSKNQKGRL CVHFNGLSDL IFEVYCGNRQ LHWFQRFLED QQTKRKSKNQ HSSGLFTLRN GHLVWLEGEG KGEPWNLHHL TLYCCVDNRL WTEEGTEIVR QEKADEITKF ITNMKKKSDL SDTQQALIQR KQSTLTRINN SFERPSQPLY QGQSHILVGV SLGLEKPATV AVVDAIANKV LAYRSIKQLL GDNYELLNRQ RRQQQYLSHE RHKAQKNFSP NQFGASELGQ HIDRLLAKAI VALARTYKAG SIVLPKLGDM REVVQSEIQA IAEQKFPGYI EGQQKYAKQY RVNVHRWSYG RLIQSIQSKA AQTGIVIEEG KQPIRGSPHD KAKELALSAY NLRLTRRS

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Macromolecule #9: S15

MacromoleculeName: S15 / type: protein_or_peptide / ID: 9 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SMALTQERKQ EIIVNYQVHE TDTGSADVQV AMLTERINRL SLHLQANKKD HSSRRGLLKL IGQRKRLLAY IQKDSREKYQ ALIGRLGIRG

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Macromolecule #10: TniQ

MacromoleculeName: TniQ / type: protein_or_peptide / ID: 10 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MIEAPDVKPW LFLIKPYEGE SLSHFLGRFR RANHLSASGL GTLAGIGAIV ARWERFHFNP RPSQQELEAI ASVVEVDAQR LAQMLPPAGV GMQHEPIRLC GACYAESPCH RIEWQYKSVW KCDRHQLKIL AKCPNCQAPF KMPALWEDGC CHRCRMPFAE MAKLQKVGSE FELENLYFQ

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Macromolecule #11: TnsC

MacromoleculeName: TnsC / type: protein_or_peptide / ID: 11 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: STEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTVP VVYIRPHQKC GPKDLFKKIT EYLKYRVTKG TVSDFRDRTI EVLKGCGVEM LIIDEADRLK PETFADVRDI AEDLGIAVVL ...String:
STEAQAIAKQ 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 #12: TnsB

MacromoleculeName: TnsB / type: protein_or_peptide / ID: 12 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SNSQQNPDLA VHPLAIPMEG LLGESATTLE KNVIATQLSE EAQVKLEVIQ SLLEPCDRTT YGQKLREAAE KLNVSLRTVQ RLVKNWEQDG LVGLTQTSRA DKGKHRIGEF WENFITKTYK EGNKGSKRMT PKQVALRVEA KARELKDSKP PNYKTVLRVL APILEKQQKA ...String:
SNSQQNPDLA 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
GridModel: UltrAuFoil R0./1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.19 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 41000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementProtocol: AB INITIO MODEL

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