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- PDB-7oxd: Crystal structure of Scytonema hofmanni transposition protein TniQ -

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Basic information

Entry
Database: PDB / ID: 7oxd
TitleCrystal structure of Scytonema hofmanni transposition protein TniQ
ComponentsShTniQ
KeywordsDNA BINDING PROTEIN / transposition protein / zinc-finger protein
Biological speciesScytonema hofmannii (Cyanobacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.3 Å
AuthorsQuerques, I. / Jinek, M.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_182567 Switzerland
European Research Council (ERC)ERC-CoG-820152
CitationJournal: Nature / Year: 2021
Title: Target site selection and remodelling by type V CRISPR-transposon systems.
Authors: Irma Querques / Michael Schmitz / Seraina Oberli / Christelle Chanez / Martin Jinek /
Abstract: Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided ...Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ, but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools.
History
DepositionJun 22, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 1, 2021Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ShTniQ
hetero molecules


Theoretical massNumber of molelcules
Total (without water)17,7613
Polymers17,6311
Non-polymers1312
Water4,252236
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area8360 Å2
MethodPISA
Unit cell
γ
α
β
Length a, b, c (Å)41.636, 49.875, 86.800
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein ShTniQ


Mass: 17630.566 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (Cyanobacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 236 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.56 Å3/Da / Density % sol: 51.88 %
Crystal growTemperature: 280 K / Method: vapor diffusion, hanging drop
Details: 100 mM HEPES pH 7.5 20% PEG 6000 250 mM sodium chloride

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Aug 16, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.3→43.24 Å / Num. obs: 85839 / % possible obs: 99.75 % / Redundancy: 6.7 % / CC1/2: 1 / CC star: 1 / Rmerge(I) obs: 0.04553 / Rpim(I) all: 0.01314 / Rrim(I) all: 0.04743 / Net I/σ(I): 32.11
Reflection shellResolution: 1.3→1.346 Å / Redundancy: 12.1 % / Rmerge(I) obs: 0.9636 / Num. unique obs: 53615 / CC1/2: 0.889 / Rpim(I) all: 0.2847 / % possible all: 99.8

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Processing

Software
NameVersionClassification
PHENIX1.19.1_4122refinement
PDB_EXTRACT3.27data extraction
XDSdata reduction
autoPROCdata scaling
AutoSolphasing
RefinementMethod to determine structure: SAD / Resolution: 1.3→43.24 Å / SU ML: 0.13 / Cross valid method: THROUGHOUT / σ(F): 1.29 / Phase error: 21.24 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1952 4319 5.04 %
Rwork0.1881 81333 -
obs0.1889 45126 99.74 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 50 Å2 / Biso mean: 20.7887 Å2 / Biso min: 10.96 Å2
Refinement stepCycle: final / Resolution: 1.3→43.24 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1232 0 2 236 1470
Biso mean--17.18 30.31 -
Num. residues----155
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.3-1.310.35061420.35452641278399
1.31-1.330.2611420.269826952837100
1.33-1.350.28081440.259527632907100
1.35-1.360.27761410.237626732814100
1.36-1.380.29121500.247927642914100
1.38-1.40.24341460.236326912837100
1.4-1.420.23911460.232527262872100
1.42-1.440.27431430.227227372880100
1.44-1.460.20781460.218226842830100
1.46-1.490.23991360.220527232859100
1.49-1.510.24091470.201327452892100
1.51-1.540.22311420.198427362878100
1.54-1.570.23221410.189726892830100
1.57-1.60.2381440.181427302874100
1.6-1.640.20271460.173926832829100
1.64-1.680.20681430.181127362879100
1.68-1.720.16961460.190327422888100
1.72-1.760.25261470.179726992846100
1.76-1.820.20061480.192727312879100
1.82-1.870.1771450.190126942839100
1.87-1.940.18341380.21562607274596
1.94-2.020.1941480.179827172865100
2.02-2.110.16291450.180627352880100
2.11-2.220.18191430.18062701284499
2.22-2.360.22671360.19412692282899
2.36-2.540.15521440.180927052849100
2.54-2.80.20861410.188827462887100
2.8-3.210.19561500.183527162866100
3.21-4.040.16851440.167227122856100
4.04-43.40.17591450.178627202865100

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