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- PDB-7xex: Crystal strucutre of apoCasDinG -

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Basic information

Entry
Database: PDB / ID: 7xex
TitleCrystal strucutre of apoCasDinG
ComponentsCasDinG
KeywordsSTRUCTURAL PROTEIN / Nuclease
Function / homologyP-loop containing nucleotide triphosphate hydrolases / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Function and homology information
Biological speciesPseudomonas aeruginosa (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3 Å
AuthorsZhang, J.T. / Cui, N. / Liu, Y.R. / Huang, H.D. / Jia, N.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Mol Cell / Year: 2023
Title: Type IV-A CRISPR-Csf complex: Assembly, dsDNA targeting, and CasDinG recruitment.
Authors: Ning Cui / Jun-Tao Zhang / Yongrui Liu / Yanhong Liu / Xiao-Yu Liu / Chongyuan Wang / Hongda Huang / Ning Jia /
Abstract: Type IV CRISPR-Cas systems, which are primarily found on plasmids and exhibit a strong plasmid-targeting preference, are the only one of the six known CRISPR-Cas types for which the mechanistic ...Type IV CRISPR-Cas systems, which are primarily found on plasmids and exhibit a strong plasmid-targeting preference, are the only one of the six known CRISPR-Cas types for which the mechanistic details of their function remain unknown. Here, we provide high-resolution functional snapshots of type IV-A Csf complexes before and after target dsDNA binding, either in the absence or presence of CasDinG, revealing the mechanisms underlying Csf complex assembly, "DWN" PAM-dependent dsDNA targeting, R-loop formation, and CasDinG recruitment. Furthermore, we establish that CasDinG, a signature DinG family helicase, harbors ssDNA-stimulated ATPase activity and ATP-dependent 5'-3' DNA helicase activity. In addition, we show that CasDinG unwinds the non-target strand (NTS) and target strand (TS) of target dsDNA from the Csf complex. These molecular details advance our mechanistic understanding of type IV-A CRISPR-Csf function and should enable Csf complexes to be harnessed as genome-engineering tools for biotechnological applications.
History
DepositionMar 31, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 26, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CasDinG
B: CasDinG
C: CasDinG


Theoretical massNumber of molelcules
Total (without water)203,8203
Polymers203,8203
Non-polymers00
Water00
1
A: CasDinG


Theoretical massNumber of molelcules
Total (without water)67,9401
Polymers67,9401
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: CasDinG


Theoretical massNumber of molelcules
Total (without water)67,9401
Polymers67,9401
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: CasDinG


Theoretical massNumber of molelcules
Total (without water)67,9401
Polymers67,9401
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)264.828, 72.232, 139.428
Angle α, β, γ (deg.)90.000, 121.220, 90.000
Int Tables number5
Space group name H-MC121

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Components

#1: Protein CasDinG


Mass: 67939.883 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: Sequence based on database WP_088922490.1 / Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli)

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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: 2.8 Å3/Da / Density % sol: 56.03 %
Crystal growTemperature: 289.15 K / Method: vapor diffusion, hanging drop / Details: 8% PEG 6000, 0.1M HEPES 7.4

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL18U1 / Wavelength: 0.97915 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Jul 19, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97915 Å / Relative weight: 1
ReflectionResolution: 3→40 Å / Num. obs: 44576 / % possible obs: 98.1 % / Redundancy: 6.7 % / Biso Wilson estimate: 54.72 Å2 / CC1/2: 0.845 / Net I/σ(I): 12
Reflection shellResolution: 3→3.11 Å / Num. unique obs: 4125 / CC1/2: 0.845

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Processing

Software
NameVersionClassification
PHENIX1.20.1-4487refinement
HKL-3000data scaling
PDB_EXTRACT3.27data extraction
HKL-3000data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6FWR

6fwr


Resolution: 3→38.37 Å / SU ML: 0.37 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 28.09 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2602 1875 4.96 %
Rwork0.2132 35892 -
obs0.2156 37767 83.04 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 135.13 Å2 / Biso mean: 53.8184 Å2 / Biso min: 10.75 Å2
Refinement stepCycle: final / Resolution: 3→38.37 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13577 0 0 0 13577
Num. residues----1777
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 13

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3-3.080.4417690.33771169123836
3.08-3.170.3799740.3091542161647
3.17-3.280.3251010.30871937203858
3.28-3.390.34531180.29382373249172
3.39-3.530.31581410.27052727286883
3.53-3.690.31161620.25263158332096
3.69-3.880.28221420.22533302344499
3.88-4.130.25841870.20963263345099
4.13-4.440.26511740.18313271344598
4.45-4.890.22771620.16783283344598
4.89-5.60.24161840.19243297348199
5.6-7.040.22951610.21933278343998
7.05-38.370.19982000.16973292349296
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.9870.86771.42342.21970.58931.60690.0710.5066-0.2490.16390.1059-0.07370.1850.19030.00930.1347-0.01030.0060.1160.10790.1427-62.97518.79887.1009
22.49130.13990.16871.36680.1031.391-0.0101-0.3646-0.11960.179-0.00140.33460.00660.02-0.01150.26880.10470.08640.24760.00290.1831-94.151645.759834.0485
32.3087-0.30550.38041.2916-0.17141.606-0.0298-0.3062-0.32260.02390.10360.14-0.2018-0.1051-0.01020.1360.0496-0.01530.112-0.03530.0523-51.545628.902121.6725
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1(chain 'A' and resid 11 through 624)A11 - 624
2X-RAY DIFFRACTION2(chain 'B' and resid 7 through 624)B7 - 624
3X-RAY DIFFRACTION3(chain 'C' and resid 5 through 624)C5 - 624

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