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- PDB-8h2f: Crystal structure of DnaQ domain in complex witn TMP of Streptoco... -

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Basic information

Entry
Database: PDB / ID: 8h2f
TitleCrystal structure of DnaQ domain in complex witn TMP of Streptococcus thermophilus strain DGCC 7710
ComponentsDnaQ
KeywordsLYASE / nuclease
Function / homologyTHYMIDINE-5'-PHOSPHATE
Function and homology information
Biological speciesStreptococcus thermophilus DGCC 7710 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.448 Å
AuthorsChen, Q. / Yu, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)111111 China
CitationJournal: Innovation (Camb) / Year: 2023
Title: DnaQ mediates directional spacer acquisition in the CRISPR-Cas system by a time-dependent mechanism.
Authors: Dongmei Tang / Tingting Jia / Yongbo Luo / Biqin Mou / Jie Cheng / Shiqian Qi / Shaohua Yao / Zhaoming Su / Yamei Yu / Qiang Chen /
Abstract: In the spacer acquisition stage of CRISPR-Cas immunity, spacer orientation and protospacer adjacent motif (PAM) removal are two prerequisites for functional spacer integration. Cas4 has been ...In the spacer acquisition stage of CRISPR-Cas immunity, spacer orientation and protospacer adjacent motif (PAM) removal are two prerequisites for functional spacer integration. Cas4 has been implicated in both processing the prespacer and determining the spacer orientation. In Cas4-lacking systems, host 3'-5' DnaQ family exonucleases were recently reported to play a Cas4-like role. However, the molecular details of DnaQ functions remain elusive. Here, we characterized the spacer acquisition of the adaptation module of the type I-E system, in which a DnaQ domain naturally fuses with Cas2. We presented X-ray crystal structures and cryo-electron microscopy structures of this adaptation module. Our biochemical data showed that DnaQ trimmed PAM-containing and PAM-deficient overhangs with different efficiencies. Based on these results, we proposed a time-dependent model for DnaQ-mediated spacer acquisition to elucidate PAM removal and spacer orientation determination in Cas4-lacking CRISPR-Cas systems.
History
DepositionOct 5, 2022Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 13, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2023Group: Database references / Structure summary / Category: citation / struct
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _struct.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DnaQ
hetero molecules


Theoretical massNumber of molelcules
Total (without water)20,9234
Polymers20,5521
Non-polymers3713
Water3,909217
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area780 Å2
ΔGint-25 kcal/mol
Surface area9210 Å2
MethodPISA
Unit cell
Length a, b, c (Å)96.139, 96.139, 61.419
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number146
Space group name H-MH3

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Components

#1: Protein DnaQ


Mass: 20551.707 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus DGCC 7710 (bacteria)
Production host: Escherichia coli (E. coli)
#2: Chemical ChemComp-TMP / THYMIDINE-5'-PHOSPHATE


Mass: 322.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N2O8P / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 217 / 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: 2.66 Å3/Da / Density % sol: 53.72 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 200mM ammonium sulfate, 36% (w/v) PEG8000, 100mM sodium cacodylate trihydrate pH 6.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.97855 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Dec 12, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97855 Å / Relative weight: 1
ReflectionResolution: 1.448→50 Å / Num. obs: 37677 / % possible obs: 100 % / Redundancy: 10.1 % / Rmerge(I) obs: 0.105 / Rpim(I) all: 0.034 / Rrim(I) all: 0.11 / Χ2: 0.997 / Net I/σ(I): 5.4
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.45-1.487.90.80118790.7630.2950.8560.93999.8
1.48-1.59.20.7118840.8530.2440.7520.95799.9
1.5-1.539.70.64519210.8720.2160.6810.963100
1.53-1.569.80.54818570.9040.1830.5790.96599.9
1.56-1.69.30.48118890.9080.1650.509199.8
1.6-1.639.10.40418750.9240.1410.4290.986100
1.63-1.6710.70.37618430.9520.120.3951.023100
1.67-1.7210.70.31919200.9680.1020.3351.023100
1.72-1.7710.70.27518880.9710.0880.2891.013100
1.77-1.8310.70.23218650.9770.0740.2441.028100
1.83-1.8910.60.19918830.9830.0640.2091.046100
1.89-1.9710.60.16219110.9870.0520.171.03999.9
1.97-2.0610.40.13718680.9910.0440.1441.04299.9
2.06-2.179.80.11918850.9920.040.1251.03899.9
2.17-2.39.20.10318990.9910.0360.1091.006100
2.3-2.4811.10.09918920.9940.0310.1031.028100
2.48-2.73110.0918640.9950.0280.0940.979100
2.73-3.1210.90.08418930.9950.0270.0880.992100
3.12-3.9410.20.07518780.9960.0250.0790.94899.9
3.94-5010.90.07218830.9950.0230.0760.91199.9

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Processing

Software
NameVersionClassification
PHENIX1.15.2_3472refinement
HKL-3000data scaling
PDB_EXTRACT3.27data extraction
HKL-3000data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: IJ53

Resolution: 1.448→24.713 Å / SU ML: 0.15 / Cross valid method: THROUGHOUT / σ(F): 1.98 / Phase error: 22.76 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1881 1090 2.92 %
Rwork0.1755 36270 -
obs0.1759 37360 99.01 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 85.85 Å2 / Biso mean: 23.8856 Å2 / Biso min: 6.08 Å2
Refinement stepCycle: final / Resolution: 1.448→24.713 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1405 0 70 217 1692
Biso mean--39.61 32.64 -
Num. residues----172
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
1.448-1.51340.30271040.2678427593
1.5134-1.59320.23941190.22414588100
1.5932-1.6930.2251890.20844618100
1.693-1.82360.22091350.20084563100
1.8236-2.00710.20591480.18764572100
2.0071-2.29730.16851660.16684567100
2.2973-2.89370.17141480.16354556100
2.8937-24.7130.17781810.15274531100

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