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- EMDB-33183: CryoEM structure of type IV-A NTS-nicked dsDNA bound Csf-crRNA te... -

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Basic information

Entry
Database: EMDB / ID: EMD-33183
TitleCryoEM structure of type IV-A NTS-nicked dsDNA bound Csf-crRNA ternary complex
Map data
Sample
  • Complex: Type IV-A NTS-nicked dsDNA bound Csf-crRNA ternary complex
    • Complex: Csf
      • Protein or peptide: Csf1
      • Protein or peptide: Csf3
      • Protein or peptide: Csf2
      • Protein or peptide: Csf5
    • Complex: crRNA
      • RNA: crRNA
    • Complex: dsDNA
      • DNA: NTS
      • DNA: TS
  • Ligand: ZINC ION
KeywordsNuclease / STRUCTURAL PROTEIN-RNA-DNA COMPLEX
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsZhang JT / Cui N / Huang HD / Jia N
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Mol Cell / Year: 2023
Title: Type IV-A CRISPR-Csf complex: Assembly, dsDNA targeting, and CasDinG recruitment.
Authors: Ning Cui / Jun-Tao Zhang / Yongrui Liu / Yanhong Liu / Xiao-Yu Liu / Chongyuan Wang / Hongda Huang / Ning Jia /
Abstract: Type IV CRISPR-Cas systems, which are primarily found on plasmids and exhibit a strong plasmid-targeting preference, are the only one of the six known CRISPR-Cas types for which the mechanistic ...Type IV CRISPR-Cas systems, which are primarily found on plasmids and exhibit a strong plasmid-targeting preference, are the only one of the six known CRISPR-Cas types for which the mechanistic details of their function remain unknown. Here, we provide high-resolution functional snapshots of type IV-A Csf complexes before and after target dsDNA binding, either in the absence or presence of CasDinG, revealing the mechanisms underlying Csf complex assembly, "DWN" PAM-dependent dsDNA targeting, R-loop formation, and CasDinG recruitment. Furthermore, we establish that CasDinG, a signature DinG family helicase, harbors ssDNA-stimulated ATPase activity and ATP-dependent 5'-3' DNA helicase activity. In addition, we show that CasDinG unwinds the non-target strand (NTS) and target strand (TS) of target dsDNA from the Csf complex. These molecular details advance our mechanistic understanding of type IV-A CRISPR-Csf function and should enable Csf complexes to be harnessed as genome-engineering tools for biotechnological applications.
History
DepositionApr 2, 2022-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33183.png

Downloads & links

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Map

FileDownload / File: emd_33183.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.)
1.1 Å/pix.
x 256 pix.
= 281.6 Å		1.1 Å/pix.
x 256 pix.
= 281.6 Å		1.1 Å/pix.
x 256 pix.
= 281.6 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.08917657 - 0.17000891
Average (Standard dev.)0.00018791165 (±0.00341385)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_33183_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

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Half map: #2

Fileemd_33183_half_map_1.map
Projections & Slices
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Histogram (log scale)

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Half map: #1

Fileemd_33183_half_map_2.map
Projections & Slices
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Sample components

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Entire : Type IV-A NTS-nicked dsDNA bound Csf-crRNA ternary complex

EntireName: Type IV-A NTS-nicked dsDNA bound Csf-crRNA ternary complex
Components
  • Complex: Type IV-A NTS-nicked dsDNA bound Csf-crRNA ternary complex
    • Complex: Csf
      • Protein or peptide: Csf1
      • Protein or peptide: Csf3
      • Protein or peptide: Csf2
      • Protein or peptide: Csf5
    • Complex: crRNA
      • RNA: crRNA
    • Complex: dsDNA
      • DNA: NTS
      • DNA: TS
  • Ligand: ZINC ION

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Supramolecule #1: Type IV-A NTS-nicked dsDNA bound Csf-crRNA ternary complex

SupramoleculeName: Type IV-A NTS-nicked dsDNA bound Csf-crRNA ternary complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)

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Supramolecule #2: Csf

SupramoleculeName: Csf / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)

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Supramolecule #3: crRNA

SupramoleculeName: crRNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5

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Supramolecule #4: dsDNA

SupramoleculeName: dsDNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #6-#7

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Macromolecule #1: Csf1

MacromoleculeName: Csf1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 28.675768 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: HHHHHHHHHH MRYPSDIVDQ VLKAGPDKGL LTWEGVDAAC SHCSRPIQNG DLYSPSSVGA FFSDTRDLAS TSRSICWRCV VLRKKPMLY GLSAAVVTQD GIYSISKDVN KAWLFTTPPP APFLVVHSSS TMQHLSWRTP VTLDNRRIHV RYGPNLFIVR P EVVRKALS ...String:
HHHHHHHHHH MRYPSDIVDQ VLKAGPDKGL LTWEGVDAAC SHCSRPIQNG DLYSPSSVGA FFSDTRDLAS TSRSICWRCV VLRKKPMLY GLSAAVVTQD GIYSISKDVN KAWLFTTPPP APFLVVHSSS TMQHLSWRTP VTLDNRRIHV RYGPNLFIVR P EVVRKALS IADRVNEGQK KWVTPVYFDR KAAAMGHGLI TRAGAEMLTQ EEQEFFQSVT PGERWALSYL MHSKRPEPEV GE CITEKVM TSLNKE

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Macromolecule #2: Csf3

MacromoleculeName: Csf3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 24.421953 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVNLKVTIDL SNPMMEPGDL LHLDALLGAL RVSRARAEHG DAINPRDYHY DLPLERYQAP SGDWVFKASA FKLKRQLPNQ MWMQTGRLS IVEAARHRQS GYLQLRAGKP NPAGGPFKTS IYHRPIVQAE LTAFCVGDQQ GIEALLSECR QIGGKRGVGF G QVAGFKVE ...String:
MVNLKVTIDL SNPMMEPGDL LHLDALLGAL RVSRARAEHG DAINPRDYHY DLPLERYQAP SGDWVFKASA FKLKRQLPNQ MWMQTGRLS IVEAARHRQS GYLQLRAGKP NPAGGPFKTS IYHRPIVQAE LTAFCVGDQQ GIEALLSECR QIGGKRGVGF G QVAGFKVE PVAETDCPWS WRALPADADP RLVTSEHARC IAAIRGPYWD RTLHVEALAP TP

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Macromolecule #3: Csf2

MacromoleculeName: Csf2 / type: protein_or_peptide / ID: 3 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 37.322234 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQIEVTVRNI TPIFSAAPGS NYITIDGTIN PPPGVSRFPL VRTRMMYVAA DVGDGVIKSV PLQIVPGNTM RSLLRRTMLK HVIEPALVE KGNKLSIGAY ATAYSGNATG NPDGVPSSFD EIATMRAHPF IGLFGGGPRM LEGRLMVDSL YPIHTNAERI L GAGYENEM ...String:
MQIEVTVRNI TPIFSAAPGS NYITIDGTIN PPPGVSRFPL VRTRMMYVAA DVGDGVIKSV PLQIVPGNTM RSLLRRTMLK HVIEPALVE KGNKLSIGAY ATAYSGNATG NPDGVPSSFD EIATMRAHPF IGLFGGGPRM LEGRLMVDSL YPIHTNAERI L GAGYENEM MSGPITQVVW ARRMDPILNL GSSEDVEVIN GGAVAANGWI QDLLANSKAA ASKKKKAAAD EDESDGAAEE NG RGLKAFN AHEVVIPGLK WVWRISLDRP TDAQVGLVLL ALNKMTNERI AGGHSKDYGR FVIDGVSLNG EQVWSQSGIT GGE QYFDAV AEAIDGLSSK EFEQFAQSAK EA

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Macromolecule #4: Csf5

MacromoleculeName: Csf5 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 29.152414 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFVKQVFFDL GARIHADEAR ALVAKLLDDT QPGLVSALMN YMPASKTSKT EFPLVQFSNF NQGFALLGFG EVGAQILSDA TPIIHDAMA KLFASRGQGV VVQVSSRDVP LSCEKRPYGL QYTVAKMVVQ KKHEHRERLA NPETGKVFLE GLFLRSLERQ A AAVGMVLP ...String:
MFVKQVFFDL GARIHADEAR ALVAKLLDDT QPGLVSALMN YMPASKTSKT EFPLVQFSNF NQGFALLGFG EVGAQILSDA TPIIHDAMA KLFASRGQGV VVQVSSRDVP LSCEKRPYGL QYTVAKMVVQ KKHEHRERLA NPETGKVFLE GLFLRSLERQ A AAVGMVLP RDLVVSFKGA ERVSSVKLRP DSTLAHGSLR HAVFEVNARL GGLWSVGFLL SKGFGHINTD LQLGQGGTRH AL SKWSHPQ FEKGGGSGGG SGGWSHPQFE K

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Macromolecule #5: crRNA

MacromoleculeName: crRNA / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 19.89982 KDa
SequenceString:
GUGAACGGUG GAGCAACACC UGAAGGAAGG CUUGAUGAGC GUGUUCCCCG CAUACGCGGG (23G)

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Macromolecule #6: NTS

MacromoleculeName: NTS / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 13.086473 KDa
SequenceString:
(DA)(DA)(DC)(DA)(DC)(DC)(DC)(DT)(DT)(DT) (DC)(DA)(DT)(DT)(DA)(DT)(DT)(DA)(DT)(DT) (DA)(DT)(DT)(DA)(DT)(DT)(DA)(DT)(DT) (DA)(DT)(DT)(DA)(DT)(DT)(DA)(DT)(DT)(DA) (DT) (DT)(DA)(DT)

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Macromolecule #7: TS

MacromoleculeName: TS / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 16.547568 KDa
SequenceString: (DC)(DT)(DG)(DC)(DC)(DG)(DC)(DA)(DC)(DT) (DT)(DG)(DC)(DT)(DC)(DA)(DT)(DC)(DA)(DA) (DG)(DC)(DC)(DT)(DT)(DC)(DC)(DT)(DT) (DC)(DA)(DG)(DG)(DT)(DG)(DT)(DT)(DG)(DC) (DT) (DC)(DC)(DA)(DG)(DA)(DA) ...String:
(DC)(DT)(DG)(DC)(DC)(DG)(DC)(DA)(DC)(DT) (DT)(DG)(DC)(DT)(DC)(DA)(DT)(DC)(DA)(DA) (DG)(DC)(DC)(DT)(DT)(DC)(DC)(DT)(DT) (DC)(DA)(DG)(DG)(DT)(DG)(DT)(DT)(DG)(DC) (DT) (DC)(DC)(DA)(DG)(DA)(DA)(DA)(DG) (DG)(DG)(DT)(DG)(DT)(DT)

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 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: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 136552
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT

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