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- EMDB-33185: CryoEM structure of type IV-A CasDinG bound NTS-nicked Csf-crRNA-... -

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Entry
Database: EMDB / ID: EMD-33185
TitleCryoEM structure of type IV-A CasDinG bound NTS-nicked Csf-crRNA-dsDNA quaternary complex in a second state
Map data
Sample
  • Complex: Type IV-A CasDinG bound NTS-nicked Csf-crRNA-dsDNA quaternary complex in a second state
    • Complex: Csf
      • Protein or peptide: Csf1
      • Protein or peptide: Csf3
      • Protein or peptide: Csf2
      • Protein or peptide: Csf5Markerville/Safron Farms Aerodrome
      • Protein or peptide: Csf4
    • Complex: crRNA
      • RNA: crRNA
    • Complex: dsDNADNA
      • DNA: NTS
      • DNA: TS
  • Ligand: ZINC ION
KeywordsNuclease / STRUCTURAL PROTEIN-RNA-DNA COMPLEX
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 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-
UpdateAug 9, 2023-
Current statusAug 9, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33185.png

Downloads & links

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Map

FileDownload / File: emd_33185.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.030970782 - 0.07178651
Average (Standard dev.)0.00008013353 (±0.001271464)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 396.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_33185_msk_1.map
Projections & Slices
AxesZYX

Projections

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Histogram (log scale)

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Half map: #2

Fileemd_33185_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33185_half_map_2.map
Projections & Slices
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Sample components

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Entire : Type IV-A CasDinG bound NTS-nicked Csf-crRNA-dsDNA quaternary com...

EntireName: Type IV-A CasDinG bound NTS-nicked Csf-crRNA-dsDNA quaternary complex in a second state
Components
  • Complex: Type IV-A CasDinG bound NTS-nicked Csf-crRNA-dsDNA quaternary complex in a second state
    • Complex: Csf
      • Protein or peptide: Csf1
      • Protein or peptide: Csf3
      • Protein or peptide: Csf2
      • Protein or peptide: Csf5Markerville/Safron Farms Aerodrome
      • Protein or peptide: Csf4
    • Complex: crRNA
      • RNA: crRNA
    • Complex: dsDNADNA
      • DNA: NTS
      • DNA: TS
  • Ligand: ZINC ION

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Supramolecule #1: Type IV-A CasDinG bound NTS-nicked Csf-crRNA-dsDNA quaternary com...

SupramoleculeName: Type IV-A CasDinG bound NTS-nicked Csf-crRNA-dsDNA quaternary complex in a second state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
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, #8
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 #8: Csf4

MacromoleculeName: Csf4 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 67.939883 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MELPADALPG EGAVREVLRP LLKQAAEKTA AGKIVFAEAA TGTGKGRMIA SLAAAAAIKG DTVVVSAPLA VTWQLVNDMK DIPEVRRVG LTLSLGRPNF ISPQRTLEWA IDNERADLAA WIEGGGKPLS LRSMETSKVI SHELCWLLED ALLLAEDLPA D SLLLTSED ...String:
MELPADALPG EGAVREVLRP LLKQAAEKTA AGKIVFAEAA TGTGKGRMIA SLAAAAAIKG DTVVVSAPLA VTWQLVNDMK DIPEVRRVG LTLSLGRPNF ISPQRTLEWA IDNERADLAA WIEGGGKPLS LRSMETSKVI SHELCWLLED ALLLAEDLPA D SLLLTSED PADCPAQQLY VAMRSNYTEA GIILCSHFML AAHTRMMQMR GLGNDEELDD EAPTGLSLPH FIDTLIVDEA HL LEQAFAS VYTHTLRLRP LMRTIEGLGS RGRKPALDAL KELFTQMQVA SARSTNTSLN VPLSDVPELI PALKDTVKTL GAL PTKGMS RDARSVIRIA TRAANDALSG HSRLRIEVTP VHSYPMLLSG RSNLQRALLG LWNATGGATL VSATLFTTGD NGSL TRWKL EVPTERAAFL PPVHPAWTTA PVLLHKEFCA HEPDDSPEWA TECAQTIQGV ASTAQGGTLV LCTSYQNTEL LAGRL GAAL GDRLIVQSKT SSAATCLAQF KAKHKAGIRP VWLGLGAAWT GIDLSDHSLP DNPELDRLLS DLVITRIPVG QNRSLT HER RTAIGGFRII SQEAAWHFRQ GLGRLVRRPG VTHKNLWVLD ARIYGGAAWV APFRQILDRY KKA

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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: 11.52044 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) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(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 #9: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 9 / 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 8882

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