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- EMDB-27972: V-K CAST Transpososome from Scytonema hofmanni, minor configuration -

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Basic information

Entry
Database: EMDB / ID: EMD-27972
TitleV-K CAST Transpososome from Scytonema hofmanni, minor configuration
Map data
Sample
  • Complex: Sh_CAST Transpososome
    • Protein or peptide: x 5 types
    • DNA: x 6 types
    • RNA: x 1 types
  • Ligand: x 2 types
Function / homology
Function and homology information


rRNA binding / ribosome / structural constituent of ribosome / ribonucleoprotein complex / translation / cytoplasm
Similarity search - Function
Bacterial TniB / Bacterial TniB protein / TniQ / TniQ / Ribosomal protein S15, bacterial-type / Ribosomal_S15 / Ribosomal protein S15 signature. / Ribosomal protein S15 / Ribosomal protein S15 / S15/NS1, RNA-binding / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
TnsC / TniQ (Homology model) / Cas12k / Small ribosomal subunit protein uS15
Similarity search - Component
Biological species[Scytonema hofmanni] UTEX 2349 (bacteria) / Scytonema hofmannii (bacteria) / Escherichia coli (E. coli) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsRizo AR / Park J-U / Tsai AW / Kellogg EK
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RO1GM144566 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD030470-01 United States
CitationJournal: Nature / Year: 2023
Title: Structures of the holo CRISPR RNA-guided transposon integration complex.
Authors: Jung-Un Park / Amy Wei-Lun Tsai / Alexandrea N Rizo / Vinh H Truong / Tristan X Wellner / Richard D Schargel / Elizabeth H Kellogg /
Abstract: CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR ...CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.
History
DepositionAug 27, 2022-
Header (metadata) releaseNov 30, 2022-
Map releaseNov 30, 2022-
UpdateFeb 8, 2023-
Current statusFeb 8, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27972.png

Downloads & links

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Map

FileDownload / File: emd_27972.map.gz / Format: CCP4 / Size: 600.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.067 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.35947353 - 0.88481677
Average (Standard dev.)8.475471e-05 (±0.021786025)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions540540540
Spacing540540540
CellA=B=C: 576.18 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27972_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_27972_additional_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27972_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27972_half_map_2.map
Projections & Slices
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Sample components

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Entire : Sh_CAST Transpososome

EntireName: Sh_CAST Transpososome
Components
  • Complex: Sh_CAST Transpososome
    • Protein or peptide: TnsC
    • Protein or peptide: Cas12k
    • Protein or peptide: TniQ
    • Protein or peptide: 30S ribosomal protein S15
    • Protein or peptide: TnsB
    • DNA: Target-LE
    • DNA: LE_R
    • DNA: Non-target_R
    • DNA: RE_F
    • DNA: RE_R1
    • DNA: RE_R2
    • RNA: sg_RNA
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Sh_CAST Transpososome

SupramoleculeName: Sh_CAST Transpososome / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#12
Source (natural)Organism: [Scytonema hofmanni] UTEX 2349 (bacteria)

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Macromolecule #1: TnsC

MacromoleculeName: TnsC / type: protein_or_peptide / ID: 1 / Number of copies: 13 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 31.444617 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTV PVVYIRPHQK CGPKDLFKKI TEYLKYRVTK GTVSDFRDRT IEVLKGCGVE MLIIDEADRL KPETFADVRD I AEDLGIAV ...String:
MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTV PVVYIRPHQK CGPKDLFKKI TEYLKYRVTK GTVSDFRDRT IEVLKGCGVE MLIIDEADRL KPETFADVRD I AEDLGIAV VLVGTDRLDA VIKRDEQVLE RFRAHLRFGK LSGEDFKNTV EMWEQMVLKL PVSSNLKSKE MLRILTSATE GY IGRLDEI LREAAIRSLS RGLKKIDKAV LQEVAKEYK

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Macromolecule #2: Cas12k

MacromoleculeName: Cas12k / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 73.280719 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSQITIQARL ISFESNRQQL WKLMADLNTP LINELLCQLG QHPDFEKWQQ KGKLPSTVVS QLCQPLKTDP RFAGQPSRLY MSAIHIVDY IYKSWLAIQK RLQQQLDGKT RWLEMLNSDA ELVELSGDTL EAIRVKAAEI LAIAMPASES DSASPKGKKG K KEKKPSSS ...String:
MSQITIQARL ISFESNRQQL WKLMADLNTP LINELLCQLG QHPDFEKWQQ KGKLPSTVVS QLCQPLKTDP RFAGQPSRLY MSAIHIVDY IYKSWLAIQK RLQQQLDGKT RWLEMLNSDA ELVELSGDTL EAIRVKAAEI LAIAMPASES DSASPKGKKG K KEKKPSSS SPKRSLSKTL FDAYQETEDI KSRSAISYLL KNGCKLTDKE EDSEKFAKRR RQVEIQIQRL TEKLISRMPK GR DLTNAKW LETLLTATTT VAEDNAQAKR WQDILLTRSS SLPFPLVFET NEDMVWSKNQ KGRLCVHFNG LSDLIFEVYC GNR QLHWFQ RFLEDQQTKR KSKNQHSSGL FTLRNGHLVW LEGEGKGEPW NLHHLTLYCC VDNRLWTEEG TEIVRQEKAD EITK FITNM KKKSDLSDTQ QALIQRKQST LTRINNSFER PSQPLYQGQS HILVGVSLGL EKPATVAVVD AIANKVLAYR SIKQL LGDN YELLNRQRRQ QQYLSHERHK AQKNFSPNQF GASELGQHID RLLAKAIVAL ARTYKAGSIV LPKLGDMREV VQSEIQ AIA EQKFPGYIEG QQKYAKQYRV NVHRWSYGRL IQSIQSKAAQ TGIVIEEGKQ PIRGSPHDKA KELALSAYNL RLTRRS

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Macromolecule #3: TniQ

MacromoleculeName: TniQ / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 19.01124 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MIEAPDVKPW LFLIKPYEGE SLSHFLGRFR RANHLSASGL GTLAGIGAIV ARWERFHFNP RPSQQELEAI ASVVEVDAQR LAQMLPPAG VGMQHEPIRL CGACYAESPC HRIEWQYKSV WKCDRHQLKI LAKCPNCQAP FKMPALWEDG CCHRCRMPFA E MAKLQKV

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Macromolecule #4: 30S ribosomal protein S15

MacromoleculeName: 30S ribosomal protein S15 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 10.290816 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSLSTEATAK IVSEFGRDAN DTGSTEVQVA LLTAQINHLQ GHFAEHKKDH HSRRGLLRMV SQRRKLLDYL KRKDVARYTQ LIERLGLRR

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Macromolecule #5: TnsB

MacromoleculeName: TnsB / type: protein_or_peptide / ID: 5 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 66.63707 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNSQQNPDLA VHPLAIPMEG LLGESATTLE KNVIATQLSE EAQVKLEVIQ SLLEPCDRTT YGQKLREAAE KLNVSLRTVQ RLVKNWEQD GLVGLTQTSR ADKGKHRIGE FWENFITKTY KEGNKGSKRM TPKQVALRVE AKARELKDSK PPNYKTVLRV L APILEKQQ ...String:
MNSQQNPDLA VHPLAIPMEG LLGESATTLE KNVIATQLSE EAQVKLEVIQ SLLEPCDRTT YGQKLREAAE KLNVSLRTVQ RLVKNWEQD GLVGLTQTSR ADKGKHRIGE FWENFITKTY KEGNKGSKRM TPKQVALRVE AKARELKDSK PPNYKTVLRV L APILEKQQ KAKSIRSPGW RGTTLSVKTR EGKDLSVDYS NHVWQCDHTR VDVLLVDQHG EILSRPWLTT VIDTYSRCIM GI NLGFDAP SSGVVALALR HAILPKRYGS EYKLHCEWGT YGKPEHFYTD GGKDFRSNHL SQIGAQLGFV CHLRDRPSEG GVV ERPFKT LNDQLFSTLP GYTGSNVQER PEDAEKDARL TLRELEQLLV RYIVDRYNQS IDARMGDQTR FERWEAGLPT VPVP IPERD LDICLMKQSR RTVQRGGCLQ FQNLMYRGEY LAGYAGETVN LRFDPRDITT ILVYRQENNQ EVFLTRAHAQ GLETE QLAL DEAEAASRRL RTAGKTISNQ SLLQEVVDRD ALVATKKSRK ERQKLEQTVL RSAAVDESNR ESLPSQIVEP DEVEST ETV HSQYEDIEVW DYEQLREEYG F

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Macromolecule #6: Target-LE

MacromoleculeName: Target-LE / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 43.44084 KDa
SequenceString: (DG)(DT)(DC)(DA)(DC)(DA)(DA)(DT)(DG)(DA) (DC)(DA)(DT)(DT)(DA)(DA)(DT)(DC)(DT)(DG) (DT)(DC)(DA)(DC)(DC)(DG)(DA)(DC)(DG) (DA)(DC)(DA)(DG)(DA)(DT)(DA)(DA)(DT)(DT) (DT) (DG)(DT)(DC)(DA)(DC)(DT) ...String:
(DG)(DT)(DC)(DA)(DC)(DA)(DA)(DT)(DG)(DA) (DC)(DA)(DT)(DT)(DA)(DA)(DT)(DC)(DT)(DG) (DT)(DC)(DA)(DC)(DC)(DG)(DA)(DC)(DG) (DA)(DC)(DA)(DG)(DA)(DT)(DA)(DA)(DT)(DT) (DT) (DG)(DT)(DC)(DA)(DC)(DT)(DG)(DT) (DA)(DC)(DA)(DG)(DT)(DA)(DG)(DA)(DA)(DT) (DA)(DT) (DA)(DG)(DA)(DT)(DG)(DC)(DG) (DC)(DA)(DT)(DC)(DT)(DA)(DT)(DA)(DT)(DA) (DG)(DA)(DT) (DG)(DC)(DA)(DA)(DA)(DT) (DT)(DG)(DA)(DG)(DT)(DG)(DG)(DC)(DC)(DT) (DT)(DA)(DT)(DT) (DA)(DA)(DA)(DT)(DG) (DA)(DC)(DT)(DT)(DC)(DT)(DC)(DA)(DA)(DC) (DC)(DA)(DG)(DT)(DC) (DA)(DG)(DC)(DA) (DC)(DG)(DC)(DC)(DC)(DA)(DG)(DA)(DC)(DC) (DA)(DG)(DG)(DG)(DC)(DA) (DC)

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Macromolecule #7: LE_R

MacromoleculeName: LE_R / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.775137 KDa
SequenceString:
(DT)(DG)(DT)(DA)(DC)(DA)(DG)(DT)(DG)(DA) (DC)(DA)(DA)(DA)(DT)(DT)(DA)(DT)(DC)(DT) (DG)(DT)(DC)(DG)(DT)(DC)(DG)(DG)(DT) (DG)(DA)(DC)(DA)(DG)(DA)(DT)(DT)(DA)(DA) (DT) (DG)(DT)(DC)(DA)(DT)(DT)(DG)(DT) (DG)(DA)(DC)

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Macromolecule #8: Non-target_R

MacromoleculeName: Non-target_R / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 21.814982 KDa
SequenceString: (DG)(DC)(DG)(DT)(DG)(DC)(DT)(DG)(DA)(DC) (DT)(DG)(DG)(DT)(DT)(DC)(DT)(DC)(DT)(DT) (DC)(DA)(DG)(DT)(DA)(DT)(DT)(DA)(DA) (DT)(DA)(DA)(DG)(DG)(DC)(DC)(DA)(DC)(DT) (DC) (DA)(DA)(DT)(DT)(DT)(DG) ...String:
(DG)(DC)(DG)(DT)(DG)(DC)(DT)(DG)(DA)(DC) (DT)(DG)(DG)(DT)(DT)(DC)(DT)(DC)(DT)(DT) (DC)(DA)(DG)(DT)(DA)(DT)(DT)(DA)(DA) (DT)(DA)(DA)(DG)(DG)(DC)(DC)(DA)(DC)(DT) (DC) (DA)(DA)(DT)(DT)(DT)(DG)(DC)(DA) (DT)(DC)(DT)(DA)(DT)(DA)(DT)(DA)(DG)(DA) (DT)(DG) (DC)(DG)(DC)(DA)(DT)(DC)(DT) (DA)(DT)(DA)(DT)

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Macromolecule #9: RE_F

MacromoleculeName: RE_F / type: dna / ID: 9 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.08083 KDa
SequenceString: (DT)(DT)(DA)(DC)(DT)(DG)(DA)(DT)(DG)(DA) (DC)(DA)(DA)(DT)(DA)(DA)(DT)(DT)(DT)(DG) (DT)(DC)(DA)(DC)(DA)(DA)(DC)(DG)(DA) (DC)(DA)(DT)(DA)(DT)(DA)(DA)(DT)(DT)(DA) (DG) (DT)(DC)(DA)(DC)(DT)(DG) ...String:
(DT)(DT)(DA)(DC)(DT)(DG)(DA)(DT)(DG)(DA) (DC)(DA)(DA)(DT)(DA)(DA)(DT)(DT)(DT)(DG) (DT)(DC)(DA)(DC)(DA)(DA)(DC)(DG)(DA) (DC)(DA)(DT)(DA)(DT)(DA)(DA)(DT)(DT)(DA) (DG) (DT)(DC)(DA)(DC)(DT)(DG)(DT)(DA) (DC)(DA)(DT)(DC)(DT)(DA)(DC)(DG)(DA)(DT) (DA)(DC) (DG)(DT)(DA)(DG)(DC)(DG)(DG) (DC)(DC)(DG)(DA)(DC)(DG)(DC)(DG)

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Macromolecule #10: RE_R1

MacromoleculeName: RE_R1 / type: dna / ID: 10 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.443954 KDa
SequenceString:
(DT)(DG)(DT)(DA)(DC)(DA)(DG)(DT)(DG)(DA) (DC)(DT)(DA)(DA)(DT)(DT)(DA)(DT)(DA)(DT) (DG)(DT)(DC)(DG)(DT)(DT)(DG)(DT)(DG) (DA)(DC)(DA)(DA)(DA)(DT)(DT)(DA)(DT)(DT) (DG) (DT)(DC)(DA)(DT)(DC)(DA)(DG)(DT) (DA)(DA)

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Macromolecule #11: RE_R2

MacromoleculeName: RE_R2 / type: dna / ID: 11 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.086917 KDa
SequenceString:
(DC)(DG)(DC)(DG)(DT)(DC)(DG)(DG)(DC)(DC) (DG)(DC)(DT)(DA)(DC)(DG)(DT)(DA)(DT)(DC)

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Macromolecule #12: sg_RNA

MacromoleculeName: sg_RNA / type: rna / ID: 12 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 85.26125 KDa
SequenceString: AUAUUAAUAG CGCCGCAAUU CAUGCUGCUU GCAGCCUCUG AAUUUUGUUA AAUGAGGGUU AGUUUGACUG UAUAAAUACA GUCUUGCUU UCUGACCCUG GUAGCUGCUC ACCCUGAUGC UGCUGUCAAU AGACAGGAUA GGUGCGCUCC CAGCAAUAAG G GCGCGGAU ...String:
AUAUUAAUAG CGCCGCAAUU CAUGCUGCUU GCAGCCUCUG AAUUUUGUUA AAUGAGGGUU AGUUUGACUG UAUAAAUACA GUCUUGCUU UCUGACCCUG GUAGCUGCUC ACCCUGAUGC UGCUGUCAAU AGACAGGAUA GGUGCGCUCC CAGCAAUAAG G GCGCGGAU GUACUGCUGU AGUGGCUACU GAAUCACCCC CGAUCAAGGG GGAACCCUCC AAAAGGUGGG UUGAAAGGAG AA GUCAUUU AAUAAGGCCA CUGUUAAA

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Macromolecule #13: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 13 / Number of copies: 15 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #14: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 14 / Number of copies: 13 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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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: 7.4
VitrificationCryogen name: ETHANE

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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 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 67096
FSC plot (resolution estimation)

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