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- EMDB-34810: Streptococcus thermophilus Cas1-Cas2-DnaQ-prespacer complex -

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Basic information

Entry
Database: EMDB / ID: EMD-34810
TitleStreptococcus thermophilus Cas1-Cas2-DnaQ-prespacer complex
Map dataStreptococcus thermophilus Cas1-Cas2-DnaQ-prespacer complex, conformation1
Sample
  • Complex: Cas1-Cas2-DnaQ-prespacer complex
    • Protein or peptide: Cas1
    • Protein or peptide: Cas2-DnaQ
KeywordsCRISPR-Cas / adaptation / IMMUNE SYSTEM
Biological speciesStreptococcus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.92 Å
AuthorsChen Q / Yu Y / Luo Y
Funding support1 items
OrganizationGrant numberCountry
Not funded
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_34810.png

Downloads & links

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Map

FileDownload / File: emd_34810.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStreptococcus thermophilus Cas1-Cas2-DnaQ-prespacer complex, conformation1
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.109
Minimum - Maximum-0.45913878 - 0.7147611
Average (Standard dev.)0.004653816 (±0.03460467)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 188.15999 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: the first half map of conformation1

Fileemd_34810_half_map_1.map
Annotationthe first half map of conformation1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: the second half map of conformation1

Fileemd_34810_half_map_2.map
Annotationthe second half map of conformation1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cas1-Cas2-DnaQ-prespacer complex

EntireName: Cas1-Cas2-DnaQ-prespacer complex
Components
  • Complex: Cas1-Cas2-DnaQ-prespacer complex
    • Protein or peptide: Cas1
    • Protein or peptide: Cas2-DnaQ

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Supramolecule #1: Cas1-Cas2-DnaQ-prespacer complex

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

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Macromolecule #1: Cas1

MacromoleculeName: Cas1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GASGSMVEKN GAKKTSLREL PKISDRVSFI YVEHAKINRV DSAITVLDSR GTVRIPAAMI GVLLLGPGTD ISHRAVELIG DTGTSMVWVG ERGVRQYAHG RSLAHSTKFL EKQAKLVSNS RLRLAVARKM YQMRFPDEDV SAMTMQQLRG REGARVRRVY RLQSEKYQVS ...String:
GASGSMVEKN GAKKTSLREL PKISDRVSFI YVEHAKINRV DSAITVLDSR GTVRIPAAMI GVLLLGPGTD ISHRAVELIG DTGTSMVWVG ERGVRQYAHG RSLAHSTKFL EKQAKLVSNS RLRLAVARKM YQMRFPDEDV SAMTMQQLRG REGARVRRVY RLQSEKYQVS WTKREYNPDD FEGGDIVNQA LSAANVALYG LVHSIVIALG ASPGLGFVHT GHDLSFIYDI ADLYKAELTI PLAFEIAANF TEIDDIGKIA RQKVRDSFVD GKLIVRIVQD IQYLFDLDDD EELLVDTLSL WDDKDMLVKH GVSYKEEL

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Macromolecule #2: Cas2-DnaQ

MacromoleculeName: Cas2-DnaQ / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPFTVVTLKS VPPSLRGDLT KWMQEIAIGV YVGNFNSRIR EKLWNRIQAN VGEGEATISY YYRNEIGYQF DMINSQKSVV DFDGIPLVLI PNSKTSSENY PKLGYSNAAK SRKIKRYSSY RGPQANSLKP YVVIDIETDG LDEKKNTIIE IGAVKFNGQQ VEEFNALIKY ...String:
MPFTVVTLKS VPPSLRGDLT KWMQEIAIGV YVGNFNSRIR EKLWNRIQAN VGEGEATISY YYRNEIGYQF DMINSQKSVV DFDGIPLVLI PNSKTSSENY PKLGYSNAAK SRKIKRYSSY RGPQANSLKP YVVIDIETDG LDEKKNTIIE IGAVKFNGQQ VEEFNALIKY EEKLPPTIFK LTGISKSLLD QEGRDLKEVL SEFLLFIGDL TLVGYNIHFD IQFINNKLNK FGLPLLINKT HDIMRYVKDE KLFLDNYQLQ TALKSYGIED SVPHRALKDA RLIYHLSTKV NKFLARMKEK S

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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

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GRAPHENE OXIDE / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.1500 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 65.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.465 µm / Nominal defocus min: 1.213 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.92 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 30496
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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