EMDB-57739: Cryo-EM structure of the PseCascade-TniQ-TnsC complex bound to Ps...

Basic information

Entry

Database: EMDB / ID: EMD-57739

• Title: Cryo-EM structure of the PseCascade-TniQ-TnsC complex bound to PseTnsB-hook motifs

Sample

• Complex: PseCascade-TniQ-TnsC complex bound to PseTnsB-hook motifs
  • RNA: x 1 types
  • DNA: x 2 types
  • Protein or peptide: x 6 types
• Ligand: x 2 types

• Keywords: Transposase / DNA-binding / RNA-binding / CRISPR-Cas / CRISPR-associated transposon / DNA BINDING PROTEIN

Function / homology

Function:

maintenance of CRISPR repeat elements / detection of maltose stimulus / maltose transport complex / carbohydrate transport / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / outer membrane-bounded periplasmic space / endonuclease activity / periplasmic space / DNA damage response / membrane

Domain/homology:

TniQ / TniQ / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST superfamily / CRISPR-associated protein (Cas_Csy4) / CRISPR-associated protein Csy2 / CRISPR-associated protein (Cas_Csy2) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3) / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein

Component:

: / Cas8 / Cas7.1 / Cas6 / TniQ.1 / Maltose/maltodextrin-binding periplasmic protein

• Biological species: Pseudoalteromonas sp. S983 (bacteria)
• Method: single particle reconstruction / cryo EM / Resolution: 2.9 Å
• Authors: Finocchio G / Oberli S / Schmitz M / Jinek M

Funding support

European Union, Switzerland, 2 items

Organization	Grant number	Country
European Research Council (ERC)	820152	European Union
Swiss National Science Foundation	320030-228089	Switzerland

• Citation: Journal: bioRxiv / Year: 2026; Title: Structural basis of RNA-guided DNA integration by type I CRISPR-associated transposases.; Authors: Giada Finocchio / Seraina Oberli / George Lampe / Michael Schmitz / Samuel H Sternberg / Martin Jinek / ; Abstract: CRISPR-associated transposases (CASTs) achieve site-specific DNA integration by coupling the RNA-guided targeting action of a nuclease-deficient CRISPR-Cas system with the assembly of a Tn7-like transpososome complex. Understanding the detailed mechanisms of this elaborate process is paramount to engineering CAST systems into programmable genetic tools. The type I-F CAST (CAST) displays the highest activity in mammalian cells to date and has been the subject of extensive directed evolution, but efforts to rationally engineer further improvements have been hampered by critical gaps in our understanding of transpososome assembly and activation. Here we use cryo-EM structural analysis, validated by DNA transposition assays, to visualize the CAST system in a series of functional states that define the stepwise mechanism of RNA-guided DNA integration. The structure of a target DNA-bound Cascade-TniQ-TnsC complex reveals that conformational changes induced by R-loop formation are coupled to target DNA stabilization and TnsC heptamerization, which in turn recruits the TnsAB transposase via conserved interactions with its C-terminal tail. Finally, the structure of the 1.2 MDa CAST transpososome holocomplex reveals specific TnsC-TnsB and TnsB-target DNA interactions that drive allosteric remodelling of the TnsB catalytic site to activate donor DNA integration. Together, these findings establish a unified structural and mechanistic blueprint for RNA-guided DNA integration and lay the foundation for engineering next-generation DNA insertion systems for genome editing applications.

History

Deposition	Apr 23, 2026	-
Header (metadata) release	Jun 17, 2026	-
Map release	Jun 17, 2026	-
Update	Jun 17, 2026	-
Current status	Jun 17, 2026	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_57739.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 0.65 Å

Density

Contour Level	By AUTHOR: 4.3
Minimum - Maximum	-12.636056999999999 - 38.365414000000001
Average (Standard dev.)	0.0041612624 (±1.0804031)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order Z Y X Origin 0 0 0 Dimensions 600 600 600 Spacing 600 600 600
Cell	A=B=C: 390.0 Å α=β=γ: 90.0 °

Supplemental data

Sample components

Entire : PseCascade-TniQ-TnsC complex bound to PseTnsB-hook motifs

• Entire: Name: PseCascade-TniQ-TnsC complex bound to PseTnsB-hook motifs

Components

• Complex: PseCascade-TniQ-TnsC complex bound to PseTnsB-hook motifs
  • RNA: CRISPR RNA
  • DNA: Target DNA strand
  • DNA: Non-target DNA strand
  • Protein or peptide: Cas7.1
  • Protein or peptide: Cas8
  • Protein or peptide: Cas6
  • Protein or peptide: TniQ.1
  • Protein or peptide: AAA+ ATPase domain-containing protein
  • Protein or peptide: Maltose/maltodextrin-binding periplasmic protein,TnsA endonuclease N-terminal domain-containing protein,TnsB transposase
• Ligand: ADENOSINE-5'-TRIPHOSPHATE
• Ligand: MAGNESIUM ION

Supramolecule #1: PseCascade-TniQ-TnsC complex bound to PseTnsB-hook motifs

• Supramolecule: Name: PseCascade-TniQ-TnsC complex bound to PseTnsB-hook motifs; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9; Details: PseCascade-TniQ-TnsC complex bound to seven C-terminal PseTnsB-hooks. The CRISPR RNA (crRNA) is base-paired with a double-stranded DNA target. The hook comprises residues 605-613 of TnsB (843-851 of the TnsA-TnsB fusion).
• Source (natural): Organism: Pseudoalteromonas sp. S983 (bacteria)
• Molecular weight: Theoretical: 740 KDa

Macromolecule #1: CRISPR RNA

• Macromolecule: Name: CRISPR RNA / type: rna / ID: 1 ; Details: CRISPR RNA (crRNA) from the type I-F PseCAST (Tn7016) Cascade complex of Pseudoalteromonas sp. S983.; Number of copies: 1
• Source (natural): Organism: Pseudoalteromonas sp. S983 (bacteria)
• Molecular weight: Theoretical: 30.196002 KDa

Sequence

String:

GUGACCUGCC GUAUAGGCAG CUGAAAAUAC AGUGGGGCCA CUAGGGACAG GAUUGGUGAC GUGACCUGCC GUAUAGGCAG CUGAAAAUG UAGC

Macromolecule #2: Target DNA strand

• Macromolecule: Name: Target DNA strand / type: dna / ID: 2 ; Details: Target DNA strand complementary to the crRNA spacer sequence.; Number of copies: 1 / Classification: DNA
• Source (natural): Organism: Pseudoalteromonas sp. S983 (bacteria)
• Molecular weight: Theoretical: 37.162676 KDa

Sequence

String:

(DG)(DC)(DC)(DT)(DA)(DA)(DC)(DA)(DG)(DG) (DA)(DG)(DG)(DT)(DG)(DC)(DG)(DC)(DG)(DT) (DT)(DA)(DG)(DG)(DC)(DC)(DC)(DA)(DA) (DT)(DA)(DT)(DC)(DA)(DG)(DG)(DA)(DG)(DA) (DC) (DT)(DA)(DG)(DG)(DA)(DA)(DG)(DG) (DA)(DG)(DG)(DA)(DG)(DG)(DC)(DC)(DT)(DA) (DA)(DG) (DG)(DA)(DT)(DG)(DC)(DG)(DC) (DC)(DT)(DT)(DT)(DT)(DC)(DT)(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)(DT)(DG)(DG)(DA)(DC) (DG)(DC)(DA)(DT)(DG)

Macromolecule #3: Non-target DNA strand

• Macromolecule: Name: Non-target DNA strand / type: dna / ID: 3 ; Details: Non-target DNA strand containing a +1 to +18 region identical to the target DNA strand, rather than complementary, to facilitate crRNA annealing to the protospacer.; Number of copies: 1 / Classification: DNA
• Source (natural): Organism: Pseudoalteromonas sp. S983 (bacteria)
• Molecular weight: Theoretical: 36.726367 KDa

Sequence

String:

(DC)(DA)(DT)(DG)(DC)(DG)(DT)(DC)(DC)(DA) (DC)(DG)(DC)(DC)(DT)(DG)(DT)(DC)(DA)(DC) (DC)(DC)(DC)(DG)(DG)(DT)(DG)(DA)(DT) (DC)(DC)(DC)(DA)(DC)(DA)(DG)(DG)(DA)(DT) (DT) (DG)(DG)(DT)(DG)(DA)(DC)(DA)(DG) (DA)(DA)(DA)(DA)(DG)(DG)(DC)(DG)(DC)(DA) (DT)(DC) (DC)(DT)(DT)(DA)(DG)(DG)(DC) (DC)(DT)(DC)(DC)(DT)(DC)(DC)(DT)(DT)(DC) (DC)(DT)(DA) (DG)(DT)(DC)(DT)(DC)(DC) (DT)(DG)(DA)(DT)(DA)(DT)(DT)(DG)(DG)(DG) (DC)(DC)(DT)(DA) (DA)(DC)(DG)(DC)(DG) (DC)(DA)(DC)(DC)(DT)(DC)(DC)(DT)(DG)(DT) (DT)(DA)(DG)(DG)(DC)

Macromolecule #4: Cas7.1

• Macromolecule: Name: Cas7.1 / type: protein_or_peptide / ID: 4 ; Details: CRISPR-associated protein Cas7 from the type I-F PseCAST (Tn7016) Cascade complex of Pseudoalteromonas sp. S983.; Number of copies: 6 / Enantiomer: LEVO
• Source (natural): Organism: Pseudoalteromonas sp. S983 (bacteria)
• Molecular weight: Theoretical: 39.845145 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

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

UniProtKB: Cas7.1

Macromolecule #5: Cas8

• Macromolecule: Name: Cas8 / type: protein_or_peptide / ID: 5 ; Details: CRISPR-associated protein Cas8 from the type I-F PseCAST (Tn7016) Cascade complex of Pseudoalteromonas sp. S983.; Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Pseudoalteromonas sp. S983 (bacteria)
• Molecular weight: Theoretical: 79.290211 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

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

UniProtKB: Cas8

Macromolecule #6: Cas6

• Macromolecule: Name: Cas6 / type: protein_or_peptide / ID: 6 ; Details: CRISPR-associated protein Cas6 from the type I-F PseCAST (Tn7016) Cascade complex of Pseudoalteromonas sp. S983, bearing a C-terminal Twin-Strep tag.; Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Pseudoalteromonas sp. S983 (bacteria)
• Molecular weight: Theoretical: 26.631074 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

MQRYYFTVHF LPKQANLALL TGRCISIMHG FILKHNIEGM GVTFPAWSDS SIGNEIAFVY TDKEILNTLK DQAYFVDMQD CGFFKVSQV LAVPDSCEEV RFIRNQAVAK IFTGESRRRL KRLQKRALAR GEDFNPKKIE APREIDIFHR VAMTSKSSQE D YILHIQKQ DVDCQAEPYF SNYGLASNEK FKGTVPDLSP SIDRNSAWSH PQFEKGGGSG GGSGGSAWSH PQFEKSGGG

UniProtKB: Cas6

Macromolecule #7: TniQ.1

• Macromolecule: Name: TniQ.1 / type: protein_or_peptide / ID: 7 ; Details: CRISPR-associated protein TniQ from the type I-F PseCAST (Tn7016) system of Pseudoalteromonas sp. S983, bearing an N-terminal His10 tag followed by a TEV protease cleavage site.; Number of copies: 2 / Enantiomer: LEVO
• Source (natural): Organism: Pseudoalteromonas sp. S983 (bacteria)
• Molecular weight: Theoretical: 49.832406 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

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

UniProtKB: TniQ.1

Macromolecule #8: AAA+ ATPase domain-containing protein

• Macromolecule: Name: AAA+ ATPase domain-containing protein / type: protein_or_peptide / ID: 8 ; Details: AAA+ ATPase TnsC from the type I-F PseCAST (Tn7016) system of Pseudoalteromonas sp. S983.; Number of copies: 7 / Enantiomer: LEVO
• Source (natural): Organism: Pseudoalteromonas sp. S983 (bacteria)
• Molecular weight: Theoretical: 38.609902 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

MNALTEIQIE KLRNFSDCIV MHPQIKTIFN DFDELRLNRK FQSDQQCMLL IGDTGVGKSH TINHYKKRVL ATQNYSRNTM PVLVSRISR GKGLDATLVQ MLADLELFGS SQIKKRGYKT DLTKKLVESL IKAQVELLII NEFQELIEFK SVQERQQIAN G LKFISEEA KVPIVLVGMP WAAKIAEEPQ WASRLVRKRK LEYFSLKNDS KYFRQYLMGL AKKMPFDVPP KLESKNTTIA LF AACRGEN RALKHLLLEA LKLALSCNEY LENKHFITAY DKFDFFNDKE KLKSKNPFKQ DIKDIEIYEV IKNSSYNPNA LDP EDMLTD RVFAIVK

UniProtKB: UNIPROTKB: A0A290S0Z4

Macromolecule #9: Maltose/maltodextrin-binding periplasmic protein,TnsA endonucleas...

• Macromolecule: Name: Maltose/maltodextrin-binding periplasmic protein,TnsA endonuclease N-terminal domain-containing protein,TnsB transposase; type: protein_or_peptide / ID: 9 ; Details: C-terminal hook motif of TnsB, part of the TnsA-TnsB fusion protein. The fusion protein is composed of TnsA (res 1-209, NCBI Reference Sequence WP_249364174.1) and TnsB (res 239-613, NCBI Reference Sequence TMP85090.1) from the type I-F PseCAST (Tn7016) system of Pseudoalteromonas sp. S983, connected by a synthetic NLS linker (res 210-238) and bearing an N-terminal His6-MBP tag followed by a TEV protease cleavage site. The hook comprises residues 605-613 of TnsB (843-851 of the TnsA-TnsB fusion).,C-terminal hook motif of TnsB, part of the TnsA-TnsB fusion protein. The fusion protein is composed of TnsA (res 1-209, NCBI Reference Sequence WP_249364174.1) and TnsB (res 239-613, NCBI Reference Sequence TMP85090.1) from the type I-F PseCAST (Tn7016) system of Pseudoalteromonas sp. S983, connected by a synthetic NLS linker (res 210-238) and bearing an N-terminal His6-MBP tag followed by a TEV protease cleavage site. The hook comprises residues 605-613 of TnsB (843-851 of the TnsA-TnsB fusion).,C-terminal hook motif of TnsB, part of the TnsA-TnsB fusion protein. The fusion protein is composed of TnsA (res 1-209, NCBI Reference Sequence WP_249364174.1) and TnsB (res 239-613, NCBI Reference Sequence TMP85090.1) from the type I-F PseCAST (Tn7016) system of Pseudoalteromonas sp. S983, connected by a synthetic NLS linker (res 210-238) and bearing an N-terminal His6-MBP tag followed by a TEV protease cleavage site. The hook comprises residues 605-613 of TnsB (843-851 of the TnsA-TnsB fusion).; Number of copies: 7 / Enantiomer: LEVO
• Source (natural): Organism: Pseudoalteromonas sp. S983 (bacteria)
• Molecular weight: Theoretical: 142.301844 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

MGSSHHHHHH GSSMKIEEGK LVIWINGDKG YNGLAEVGKK FEKDTGIKVT VEHPDKLEEK FPQVAATGDG PDIIFWAHDR FGGYAQSGL LAEITPDKAF QDKLYPFTWD AVRYNGKLIA YPIAVEALSL IYNKDLLPNP PKTWEEIPAL DKELKAKGKS A LMFNLQEP YFTWPLIAAD GGYAFKYENG KYDIKDVGVD NAGAKAGLTF LVDLIKNKHM NADTDYSIAE AAFNKGETAM TI NGPWAWS NIDTSKVNYG VTVLPTFKGQ PSKPFVGVLS AGINAASPNK ELAKEFLENY LLTDEGLEAV NKDKPLGAVA LKS YEEELA KDPRIAATME NAQKGEIMPN IPQMSAFWYA VRTAVINAAS GRQTVDEALK DAQTNSSSNN NNNNNNNNLG IEEN LYFQS NAMYIRNLRK PSPNKNVFKF ASTKVSSVVM CESSLEFDAC FHHEYNDLIE SFGSQPEGFK YEFMGKSLPY TPDAL ISYT DKTQKYHEYK PYSKIASPLF RAEFAAKRAA SLKLGIDLVL VTDRQIRVNP ILNNLKLLHR YSGVYGISGI QKELLS FIH KSGVIKLNDI SSQVGIPIGE TRSFLFGLMH KGLVKADLGC DDLTNNPTLW ATPGSGSGKR TADGSEFESP KKKRKVG SG SGGMTDFFNE FDESLVPLKP QTPTQYVKLD DANLIQRDLD TFSDTFKNQA LQRYKLISTI DKKLSRGWTQ RNLDPILD E LFKGGDVVRP NWRTVARWRK KYIESNGDIA SLADKNHKMG NRTNRIKGDD KFFDKALERF LDAKRPTIAT AYQYYKDLI VIENESIVEG KIPIISYNAF NKRIKAIPPY AVAVARHGKF KADQWFAYCA AHVPPTRILE RVEIDHTPLD LILLDDELLI PIGRPYLTL LIDVFSGCVL GFHLSYKSPS YVSAAKAITH AIKPKSLDAL NIELQNDWPC FGKFENLVVD NGAEFWSKNL E HACQSAGI NIQYNPVRKP WLKPFIERFF GVMNEYFLPE LPGKTFSNIL EKEEYKPEKD AIMRFSTFVE EFHRWIADVY HQ DSNSRET RIPIKRWQQG FDAYPPLTMN EEEETRFSML MRISDSRTLT RNGFKYQELM YDSTALADYR KHYPQTKETV KKL IKVDPD DISKIYVYLE ELESYLEVPC TDPTGYTDGL SIYEHKTIKK INREVIRESK DSLGLAKARM AIHERVKQEQ EVFI ESKTK AKITAVKKQA QIADVSNTGT STIKVSEESA APVQKHISND NSDDWDDDLE AFE

UniProtKB: Maltose/maltodextrin-binding periplasmic protein

Macromolecule #10: ADENOSINE-5'-TRIPHOSPHATE

• Macromolecule: Name: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 10 / Number of copies: 7 / Formula: ATP
• Molecular weight: Theoretical: 507.181 Da

Chemical component information

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

Macromolecule #11: MAGNESIUM ION

• Macromolecule: Name: MAGNESIUM ION / type: ligand / ID: 11 / Number of copies: 7 / Formula: MG
• Molecular weight: Theoretical: 24.305 Da

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 2.6 mg/mL

Buffer

pH: 7.5
Component:

Concentration	Formula	Name
20.0 mM	C8H17KN2O4S	HEPES potassium salt
10.0 mM	MgCl2	magnesium chloride
300.0 mM	KCl	potassium chloride
1.0 mM	C4H10O2S2	dithiothreitol
0.025 %	C58H114O26	polysorbate 20 (Tween 20)
1.0 mM	C10H14N5Na2O13P3	adenosin-5'-triphosphate disodium salt
10.0 mM	C12H22O11	maltose

• Grid: Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
• Vitrification: Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
• Details: The sample was prepared by co-precipitation, using His-MBP-tagged TnsAB bound to a double-stranded right-end substrate as bait to capture Cascade-TniQ and TnsC.

Electron microscopy

• Microscope: TFS KRIOS
• Image recording: Film or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 11374 / Average exposure time: 1.25 sec. / Average electron dose: 60.95 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 329059
• CTF correction: Software - Name: cryoSPARC (ver. 4.7); Software - details: Patch CTF Estimation was used for CTF correction; Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
• Startup model: Type of model: INSILICO MODEL; In silico model: AlphaFold 3 models of the individual chains were rigid-body fitted into the cryo-EM density map.
• Final reconstruction: Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7) / Number images used: 138834
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7); Software - details: Ab-Initio Reconstruction was used for initial angular assignment
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7); Software - details: Non-Uniform Refinement was used fo final angular assignment
• Final 3D classification: Number classes: 2 / Software - Name: cryoSPARC (ver. 4.7); Software - details: Ab-Initio Reconstruction was used for final classification

Atomic model buiding 1

• Initial model: Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
• Refinement: Space: REAL / Protocol: FLEXIBLE FIT; Target criteria: Real-space map-model correlation and geometry restraints

Output model

PDB-30gb:
Cryo-EM structure of the PseCascade-TniQ-TnsC complex bound to PseTnsB-hook motifs