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- EMDB-43729: Structure of Selenomonas sp. Cascade (SsCascade) -

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Basic information

Entry
Database: EMDB / ID: EMD-43729
TitleStructure of Selenomonas sp. Cascade (SsCascade)
Map data
Sample
  • Complex: Target DNA-bound SsCascade
    • Protein or peptide: Cas7
    • Protein or peptide: Cas8
    • Protein or peptide: Cas5
    • Protein or peptide: Cas6
    • DNA: Non-target strand DNA
    • RNA: crRNA
    • DNA: Target strand DNA
KeywordsCRISPR-Cas / Genome engineering / RNA-guided DNA endonuclease / Non-coding RNA / HNH nuclease / IMMUNE SYSTEM
Biological speciesSelenomonas sp. (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsHirano S / Zhang F
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)5R01HG009761-07 United States
CitationJournal: To Be Published
Title: Structural determinants of precise DNA cleavage by a CRISPR HNH-Cascade system
Authors: Hirano S / Altae-Tran H / Kannan S / Macrae RK / Zhang F
History
DepositionFeb 16, 2024-
Header (metadata) releaseAug 7, 2024-
Map releaseAug 7, 2024-
UpdateAug 7, 2024-
Current statusAug 7, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43729.png

Downloads & links

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Map

FileDownload / File: emd_43729.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.2 Å/pix.
x 264 pix.
= 317.328 Å		1.2 Å/pix.
x 264 pix.
= 317.328 Å		1.2 Å/pix.
x 264 pix.
= 317.328 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.202 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.32
Minimum - Maximum-1.3123261 - 2.2865336
Average (Standard dev.)0.0017494666 (±0.06191979)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 317.328 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43729_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43729_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Target DNA-bound SsCascade

EntireName: Target DNA-bound SsCascade
Components
  • Complex: Target DNA-bound SsCascade
    • Protein or peptide: Cas7
    • Protein or peptide: Cas8
    • Protein or peptide: Cas5
    • Protein or peptide: Cas6
    • DNA: Non-target strand DNA
    • RNA: crRNA
    • DNA: Target strand DNA

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Supramolecule #1: Target DNA-bound SsCascade

SupramoleculeName: Target DNA-bound SsCascade / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Selenomonas sp. (bacteria)

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Macromolecule #1: Cas7

MacromoleculeName: Cas7 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 38.700172 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAANKKATNV TLKSRPENLS FARCLNTTEA KFWQTDFLKR HTFKLPLLIT DKAVLASKGH EMPPDKLEKE IMDPNPQKSQ SCTLSTECD TLRIDFGIKV LPVKESMYSC SDYNYRTAIY QKIDEYIAED GFLTLAKRYV NNIANARFLW RNRKGAEIIE T IVTIEDKE ...String:
MAANKKATNV TLKSRPENLS FARCLNTTEA KFWQTDFLKR HTFKLPLLIT DKAVLASKGH EMPPDKLEKE IMDPNPQKSQ SCTLSTECD TLRIDFGIKV LPVKESMYSC SDYNYRTAIY QKIDEYIAED GFLTLAKRYV NNIANARFLW RNRKGAEIIE T IVTIEDKE YPSFNSKSFN LDTFVEDNAT INEIAQQIAD TFAGKREYLN IYVTCFVKIG CAMEVYPSQE MTFDDDDKGK KL FKFEGSA GMHSQKINNA LRTIDTWYPD YTTYEFPIPV ENYGAARSIG IPFRPDTKSF YKLIDRMILK NEDLPIEDKH YVM AILIRG GMFSKKQEK

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Macromolecule #2: Cas8

MacromoleculeName: Cas8 / type: protein_or_peptide / ID: 2
Details: This protein is histidine tagged at N-terminal region from residues 1 to 41. Residues 42 to 385 are derived from natural Cas8 protein sequences.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 43.706289 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MLRSKHHHHS GHHHTGHHHH SGSHHHTGGS GENLYFQGSG GLRNKILAAI SQKIPEEQKI NKYIEGLFQS IDKNHLATHV AKFTETNSP GNIGAYDILS SDMNCGYLDT ANAGWKEPDI VTNDAKYKRP QGFVAMEMSD GRTVMEHLQE DSAELRHEME E LTDKYDEI ...String:
MLRSKHHHHS GHHHTGHHHH SGSHHHTGGS GENLYFQGSG GLRNKILAAI SQKIPEEQKI NKYIEGLFQS IDKNHLATHV AKFTETNSP GNIGAYDILS SDMNCGYLDT ANAGWKEPDI VTNDAKYKRP QGFVAMEMSD GRTVMEHLQE DSAELRHEME E LTDKYDEI RDGILNMPSM QPYRTNQFIK QVFFPVGGSY HLLSILPSTV LNYEVSDRLY RSKIPKIRLR LLSSNAASTT GS RLVSKNK WPLVFQALPP KFLEKNLAKA LDKEYLLPDI NIDELEGVDN GCLIDEALLP LIIDEGKRKG EGNYRPRHLR DER KEETVQ AFLDKYGYCN IPVGYEVHHI VPLSQGGADS IKNMIMLSIE HHERVTEAHA SYFKWRNT

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Macromolecule #3: Cas5

MacromoleculeName: Cas5 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 28.703135 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MMKGYILLEK VNIENANAFN NIIVGIPAIT SFLGFARALE RKLNAKEIAI RINGVGLEFH EYELKGYKNK RGQYVTSCPL PGSIPGQNE KKLDAHIMNQ AYIDLNMSFL LEVEGPHVDM STCKSIKSTM ETLRIAGGII RNYKKIRLID TLADIPYGYF L TLRQDNLN ...String:
MMKGYILLEK VNIENANAFN NIIVGIPAIT SFLGFARALE RKLNAKEIAI RINGVGLEFH EYELKGYKNK RGQYVTSCPL PGSIPGQNE KKLDAHIMNQ AYIDLNMSFL LEVEGPHVDM STCKSIKSTM ETLRIAGGII RNYKKIRLID TLADIPYGYF L TLRQDNLN DAAGDDMLDK MIHALQQEDT LVPIAVGFKA LSEVGHVEGQ RDPEKDHCFV ESIFSLGGFE CSKILEDINS CL WRYKTEE GLYLCTII

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Macromolecule #4: Cas6

MacromoleculeName: Cas6 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Selenomonas sp. (bacteria)
Molecular weightTheoretical: 20.735873 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MFSQILIIKP GTGISPNIII SEDIFPVLHS LFVEHDKKFG ITFPAYSFDK KGHLGNIIEV LSEDKEALAS LCLEEHLAEV TDYVKVKKE ITFTDDYVLF KRIREENQYE TTARRMRKRG HTELGRPLEM HIKKKNQQIF CHAYIKVKSA STGQSYNIFL A PTDIKHGS FSAYGLLRGD THA

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Macromolecule #5: Non-target strand DNA

MacromoleculeName: Non-target strand DNA / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 20.484176 KDa
SequenceString: (DG)(DA)(DG)(DT)(DT)(DG)(DC)(DA)(DG)(DC) (DA)(DA)(DG)(DC)(DG)(DT)(DA)(DC)(DG)(DG) (DA)(DG)(DA)(DA)(DG)(DT)(DC)(DA)(DT) (DT)(DT)(DA)(DA)(DT)(DA)(DA)(DG)(DG)(DC) (DC) (DA)(DC)(DT)(DG)(DT)(DT) ...String:
(DG)(DA)(DG)(DT)(DT)(DG)(DC)(DA)(DG)(DC) (DA)(DA)(DG)(DC)(DG)(DT)(DA)(DC)(DG)(DG) (DA)(DG)(DA)(DA)(DG)(DT)(DC)(DA)(DT) (DT)(DT)(DA)(DA)(DT)(DA)(DA)(DG)(DG)(DC) (DC) (DA)(DC)(DT)(DG)(DT)(DT)(DA)(DA) (DA)(DA)(DA)(DG)(DC)(DA)(DA)(DC)(DA)(DG) (DC)(DT) (DG)(DA)(DT)(DT)(DG)(DC)

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Macromolecule #7: Target strand DNA

MacromoleculeName: Target strand DNA / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 20.20293 KDa
SequenceString: (DG)(DC)(DA)(DA)(DT)(DC)(DA)(DG)(DC)(DT) (DG)(DT)(DT)(DG)(DC)(DT)(DT)(DT)(DT)(DT) (DA)(DA)(DC)(DA)(DG)(DT)(DG)(DG)(DC) (DC)(DT)(DT)(DA)(DT)(DT)(DA)(DA)(DA)(DT) (DG) (DA)(DC)(DT)(DT)(DC)(DT) ...String:
(DG)(DC)(DA)(DA)(DT)(DC)(DA)(DG)(DC)(DT) (DG)(DT)(DT)(DG)(DC)(DT)(DT)(DT)(DT)(DT) (DA)(DA)(DC)(DA)(DG)(DT)(DG)(DG)(DC) (DC)(DT)(DT)(DA)(DT)(DT)(DA)(DA)(DA)(DT) (DG) (DA)(DC)(DT)(DT)(DC)(DT)(DC)(DC) (DG)(DT)(DA)(DC)(DG)(DC)(DT)(DT)(DG)(DC) (DT)(DG) (DC)(DA)(DA)(DC)(DT)(DC)

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Macromolecule #6: crRNA

MacromoleculeName: crRNA / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 19.746777 KDa
SequenceString:
UUUAGAAGGA GAAGUCAUUU AAUAAGGCCA CUGUUAAAAA GUGUACCGCC GGAUAGGCGG U

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 1.05 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 80908
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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