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- EMDB-34809: Streptococcus thermophilus Cas1-Cas2- prespacer ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-34809
TitleStreptococcus thermophilus Cas1-Cas2- prespacer ternary complex
Map data
Sample
  • Complex: Streptococcus thermophilus Cas1-Cas2- prespacer ternary complex
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Protein or peptide: Type I-E CRISPR-associated endoribonuclease Cas2
    • DNA: DNA (26-MER)
    • DNA: DNA (31-MER)
KeywordsCRISPR / cas / adaptation / IMMUNE SYSTEM
Biological speciesStreptococcus thermophilus DGCC 7710 (bacteria) / Phage #D (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsChen Q / Luo Y
Funding support China, 8 items
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, 2022-
Header (metadata) releaseSep 13, 2023-
Map releaseSep 13, 2023-
UpdateSep 20, 2023-
Current statusSep 20, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34809.png

Downloads & links

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Map

FileDownload / File: emd_34809.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 256 pix.
= 217.6 Å		0.85 Å/pix.
x 256 pix.
= 217.6 Å		0.85 Å/pix.
x 256 pix.
= 217.6 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.282
Minimum - Maximum-3.214951 - 4.064062
Average (Standard dev.)0.00068607647 (±0.101702675)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 217.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_34809_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_34809_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Streptococcus thermophilus Cas1-Cas2- prespacer ternary complex

EntireName: Streptococcus thermophilus Cas1-Cas2- prespacer ternary complex
Components
  • Complex: Streptococcus thermophilus Cas1-Cas2- prespacer ternary complex
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Protein or peptide: Type I-E CRISPR-associated endoribonuclease Cas2
    • DNA: DNA (26-MER)
    • DNA: DNA (31-MER)

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Supramolecule #1: Streptococcus thermophilus Cas1-Cas2- prespacer ternary complex

SupramoleculeName: Streptococcus thermophilus Cas1-Cas2- prespacer ternary complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)

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Macromolecule #1: CRISPR-associated endonuclease Cas1

MacromoleculeName: CRISPR-associated endonuclease Cas1 / type: protein_or_peptide / ID: 1 / Details: WP_024704118.1 / Number of copies: 4 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)
Molecular weightTheoretical: 35.54957 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GASGSMVEKN GAKKTSLREL PKISDRVSFI YVEHAKINRV DSAITVLDSR GTVRIPAAMI GVLLLGPGTD ISHRAVELIG DTGTSMVWV GERGVRQYAH GRSLAHSTKF LEKQAKLVSN SRLRLAVARK MYQMRFPDED VSAMTMQQLR GREGARVRRV Y RLQSEKYQ ...String:
GASGSMVEKN GAKKTSLREL PKISDRVSFI YVEHAKINRV DSAITVLDSR GTVRIPAAMI GVLLLGPGTD ISHRAVELIG DTGTSMVWV GERGVRQYAH GRSLAHSTKF LEKQAKLVSN SRLRLAVARK MYQMRFPDED VSAMTMQQLR GREGARVRRV Y RLQSEKYQ VSWTKREYNP DDFEGGDIVN QALSAANVAL YGLVHSIVIA LGASPGLGFV HTGHDLSFIY DIADLYKAEL TI PLAFEIA ANFTEIDDIG KIARQKVRDS FVDGKLIVRI VQDIQYLFDL DDDEELLVDT LSLWDDKDML VKHGVSYKEE L

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Macromolecule #2: Type I-E CRISPR-associated endoribonuclease Cas2

MacromoleculeName: Type I-E CRISPR-associated endoribonuclease Cas2 / type: protein_or_peptide / ID: 2 / Details: WP_024704117.1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus DGCC 7710 (bacteria)
Molecular weightTheoretical: 13.94086 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MPFTVVTLKS VPPSLRGDLT KWMQEIAIGV YVGNFNSRIR EKLWNRIQAN VGEGEATISY YYRNEIGYQF DMINSQKSVV DFDGIPLVL IPNSKTSSEN YPKLGYSNAA KSRKIKRYSS YRG

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Macromolecule #3: DNA (26-MER)

MacromoleculeName: DNA (26-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Phage #D (virus)
Molecular weightTheoretical: 11.741589 KDa
SequenceString:
(DA)(DA)(DA)(DC)(DA)(DC)(DC)(DA)(DG)(DA) (DA)(DC)(DG)(DA)(DG)(DT)(DA)(DG)(DT)(DA) (DA)(DA)(DT)(DT)(DG)(DA)(DT)(DG)(DT) (DT)(DG)(DT)(DC)(DT)(DT)(DG)(DC)(DT)

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Macromolecule #4: DNA (31-MER)

MacromoleculeName: DNA (31-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Phage #D (virus)
Molecular weightTheoretical: 11.708511 KDa
SequenceString:
(DA)(DT)(DT)(DT)(DA)(DC)(DT)(DA)(DC)(DT) (DC)(DG)(DT)(DT)(DC)(DT)(DG)(DG)(DT)(DG) (DT)(DT)(DT)(DT)(DT)(DG)(DT)(DG)(DT) (DT)(DT)(DA)(DA)(DT)(DG)(DA)(DT)(DG)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.741 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 49.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.465 µm / Nominal defocus min: 1.213 µm

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 188628
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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