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- PDB-9gs9: Tn7016 PseCAST QCascade -

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Basic information

Entry
Database: PDB / ID: 9gs9
TitleTn7016 PseCAST QCascade
Components
  • Cas6
  • Cas7.1
  • Cas8
  • NT-DNA
  • T-DNA
  • TniQ.1
  • crRNA
KeywordsRNA / CRISPR / transposon / genome
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesPseudoalteromonas (bacteria)
Pseudoalteromonas agarivorans S816 (bacteria)
RNA satellites (virus)
DNA molecule (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsLampe, G.D. / Liang, A.R. / Zhang, D.J. / Fernandez, I.S. / Sternberg, S.H.
Funding support United States, 1items
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, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 16, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: crRNA
2: T-DNA
3: NT-DNA
A: Cas8
B: Cas7.1
C: Cas7.1
D: Cas7.1
E: Cas7.1
F: Cas7.1
G: Cas7.1
H: Cas6
I: TniQ.1
J: TniQ.1


Theoretical massNumber of molelcules
Total (without water)486,96713
Polymers486,96713
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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RNA chain , 1 types, 1 molecules 1

#1: RNA chain crRNA


Mass: 19501.658 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) RNA satellites (virus)

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DNA chain , 2 types, 2 molecules 23

#2: DNA chain T-DNA


Mass: 22801.545 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#3: DNA chain NT-DNA


Mass: 3296.191 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)

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Protein , 4 types, 10 molecules ABCDEFGHIJ

#4: Protein Cas8


Mass: 79290.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudoalteromonas (bacteria) / Production host: Escherichia coli (E. coli)
#5: Protein
Cas7.1


Mass: 39845.145 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudoalteromonas (bacteria) / Production host: Escherichia coli (E. coli)
#6: Protein Cas6


Mass: 23341.666 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudoalteromonas (bacteria) / Production host: Escherichia coli (E. coli)
#7: Protein TniQ.1


Mass: 49832.406 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudoalteromonas agarivorans S816 (bacteria)
Production host: Escherichia coli (E. coli)

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Details

Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Tn7016 PseCAST QCascade / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Pseudoalteromonas aestuariivivens (bacteria)
Source (recombinant)Organism: Escherichia coli B (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 150000 / Symmetry type: POINT
RefinementResolution: 2.6→2.6 Å / Cor.coef. Fo:Fc: 0.91 / SU B: 9.718 / SU ML: 0.18 / ESU R: 0.347
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.26136 --
obs0.26136 188454 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 94.995 Å2
Baniso -1Baniso -2Baniso -3
1-0.16 Å2-0.1 Å20.39 Å2
2--0.35 Å2-1.17 Å2
3----0.51 Å2
Refinement stepCycle: 1 / Total: 20705
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0030.01321362
ELECTRON MICROSCOPYr_bond_other_d0.0010.01718703
ELECTRON MICROSCOPYr_angle_refined_deg1.2741.58829347
ELECTRON MICROSCOPYr_angle_other_deg1.0961.64943515
ELECTRON MICROSCOPYr_dihedral_angle_1_deg12.1455.762865
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.0523.34943
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.156153202
ELECTRON MICROSCOPYr_dihedral_angle_4_deg15.2781584
ELECTRON MICROSCOPYr_chiral_restr0.0580.2012874
ELECTRON MICROSCOPYr_gen_planes_refined0.0030.0222685
ELECTRON MICROSCOPYr_gen_planes_other0.0020.024515
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it1.81310.3479650
ELECTRON MICROSCOPYr_mcbond_other1.81310.3479649
ELECTRON MICROSCOPYr_mcangle_it3.39315.50512038
ELECTRON MICROSCOPYr_mcangle_other3.39315.50512039
ELECTRON MICROSCOPYr_scbond_it1.24110.20711712
ELECTRON MICROSCOPYr_scbond_other1.24110.20711713
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other2.38415.33617310
ELECTRON MICROSCOPYr_long_range_B_refined7.9226411
ELECTRON MICROSCOPYr_long_range_B_other7.9226412
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3→3.078 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.335 14042 -
obs--100 %

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