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- PDB-8hi1: Streptococcus thermophilus Cas1-Cas2- prespacer ternary complex -

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Basic information

Entry
Database: PDB / ID: 8hi1
TitleStreptococcus thermophilus Cas1-Cas2- prespacer ternary complex
Components
  • CRISPR-associated endonuclease Cas1
  • DNA (26-MER)
  • DNA (31-MER)
  • Type I-E CRISPR-associated endoribonuclease Cas2
KeywordsIMMUNE SYSTEM / CRISPR / cas / adaptation
Function / homologyDNA / DNA (> 10)
Function and homology information
Biological speciesStreptococcus thermophilus DGCC 7710 (bacteria)
Phage #D (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsChen, Q. / Luo, Y.
Funding support China, 8items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32270761 China
National Natural Science Foundation of China (NSFC)31741027 China
National Natural Science Foundation of China (NSFC)81672722 China
Ministry of Science and Technology (MoST, China)2022YFC2303700 China
Ministry of Science and Technology (MoST, China)2021YFA301900 China
National Natural Science Foundation of China (NSFC)32222040 China
National Natural Science Foundation of China (NSFC)32070049 China
Other government2022M712272 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
DepositionNov 18, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 13, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2023Group: Database references / Category: citation
Item: _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas1
B: CRISPR-associated endonuclease Cas1
C: CRISPR-associated endonuclease Cas1
D: CRISPR-associated endonuclease Cas1
E: Type I-E CRISPR-associated endoribonuclease Cas2
F: Type I-E CRISPR-associated endoribonuclease Cas2
G: DNA (26-MER)
H: DNA (31-MER)


Theoretical massNumber of molelcules
Total (without water)193,5308
Polymers193,5308
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
CRISPR-associated endonuclease Cas1


Mass: 35549.570 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: WP_024704118.1
Source: (gene. exp.) Streptococcus thermophilus DGCC 7710 (bacteria)
Gene: cas1e, cas1, DXC09_04770 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: Hydrolases; Acting on ester bonds
#2: Protein Type I-E CRISPR-associated endoribonuclease Cas2


Mass: 13940.860 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: WP_024704117.1
Source: (gene. exp.) Streptococcus thermophilus DGCC 7710 (bacteria)
Gene: cas2e, DXC09_04765 / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: DNA chain DNA (26-MER)


Mass: 11741.589 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Phage #D (virus)
#4: DNA chain DNA (31-MER)


Mass: 11708.511 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Phage #D (virus)

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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: Streptococcus thermophilus Cas1-Cas2- prespacer ternary complex
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 0.741 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2465 nm / Nominal defocus min: 1213 nm
Image recordingElectron dose: 49 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)
Image scansMovie frames/image: 5

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 188628 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00421306
ELECTRON MICROSCOPYf_angle_d0.71938378
ELECTRON MICROSCOPYf_dihedral_angle_d16.6298394
ELECTRON MICROSCOPYf_chiral_restr0.0431751
ELECTRON MICROSCOPYf_plane_restr0.0032942

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