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- PDB-7xf0: Crystal strucutre of CasDinG in complex with ATP -

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Basic information

Entry
Database: PDB / ID: 7xf0
TitleCrystal strucutre of CasDinG in complex with ATP
ComponentsCasDinG
KeywordsSTRUCTURAL PROTEIN / Nuclease
Function / homologyP-loop containing nucleotide triphosphate hydrolases / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta / ADENOSINE-5'-TRIPHOSPHATE
Function and homology information
Biological speciesPseudomonas aeruginosa (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.1 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
C: CasDinG
A: CasDinG
B: CasDinG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)205,3416
Polymers203,8203
Non-polymers1,5223
Water00
1
C: CasDinG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)68,4472
Polymers67,9401
Non-polymers5071
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area730 Å2
ΔGint-2 kcal/mol
Surface area24790 Å2
MethodPISA
2
A: CasDinG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)68,4472
Polymers67,9401
Non-polymers5071
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area760 Å2
ΔGint-1 kcal/mol
Surface area23730 Å2
MethodPISA
3
B: CasDinG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)68,4472
Polymers67,9401
Non-polymers5071
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area720 Å2
ΔGint-2 kcal/mol
Surface area25100 Å2
MethodPISA
Unit cell
Length a, b, c (Å)135.546, 72.095, 224.230
Angle α, β, γ (deg.)90.000, 90.840, 90.000
Int Tables number5
Space group name H-MI121

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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)
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
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: 2.69 Å3/Da / Density % sol: 54.23 %
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: BL02U1 / Wavelength: 0.97918 Å
DetectorType: DECTRIS EIGER2 S 9M / Detector: PIXEL / Date: Sep 21, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 3.1→38.91 Å / Num. obs: 39721 / % possible obs: 99.9 % / Redundancy: 6.7 % / Biso Wilson estimate: 67.01 Å2 / CC1/2: 0.992 / Rmerge(I) obs: 0.166 / Rpim(I) all: 0.069 / Rrim(I) all: 0.18 / Net I/σ(I): 9 / Num. measured all: 267256 / Scaling rejects: 1205
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
3.1-3.237.113136244320.9140.4031.0793100
11.18-38.916.40.08456978890.9880.0360.0921797.7

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Processing

Software
NameVersionClassification
DIALS0.7.1data scaling
PHENIX1.20.1-4487refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6FWR

6fwr


Resolution: 3.1→38.91 Å / SU ML: 0.42 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 28.55 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2605 2015 5.08 %
Rwork0.2235 37614 -
obs0.2253 39629 99.77 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 169.66 Å2 / Biso mean: 74.3619 Å2 / Biso min: 29.14 Å2
Refinement stepCycle: final / Resolution: 3.1→38.91 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13604 0 93 0 13697
Biso mean--94.81 --
Num. residues----1782
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.1-3.180.361350.328326632798100
3.18-3.260.3231530.295326602813100
3.26-3.360.3541510.279626102761100
3.36-3.470.30411460.26127002846100
3.47-3.590.29831460.264726582804100
3.59-3.740.3261400.278326482788100
3.74-3.910.25731670.230626542821100
3.91-4.110.23881420.225326932835100
4.11-4.370.26751520.206326822834100
4.37-4.70.22771380.192726802818100
4.7-5.180.21671360.195127192855100
5.18-5.920.27711330.215127232856100
5.92-7.460.2441330.225727332866100
7.46-38.910.20921430.17752791293499
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)
11.744-0.1409-0.00261.0668-0.24092.0580.14420.0732-0.10330.0598-0.00490.0705-0.1706-0.2663-0.14160.3554-0.0374-0.02040.32650.00390.311222.6049-31.851132.3567
21.55310.1542-0.56632.694-0.62813.64170.02230.07870.1903-0.16720.1328-0.1252-0.2243-0.2408-0.13730.487-0.09990.0690.3907-0.02790.387236.1741-10.9121-8.6372
32.04990.28820.11311.9386-0.02913.26060.0599-0.14160.0417-0.2479-0.0072-0.4525-0.33590.4457-0.04190.3097-0.0390.08410.4471-0.0880.544476.2689-12.113249.8684
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1(chain 'C' and resid 5 through 625)C5 - 625
2X-RAY DIFFRACTION2(chain 'A' and resid 8 through 625)A8 - 625
3X-RAY DIFFRACTION3(chain 'B' and resid 6 through 624)B6 - 624

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