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- EMDB-35154: Cryo-EM structure of Cas12g-sgRNA binary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-35154
TitleCryo-EM structure of Cas12g-sgRNA binary complex
Map data
Sample
  • Complex: Cas12g-sgRNA binary complex
    • Complex: Cas12g
      • Protein or peptide: Cas12g
    • Complex: RNA
      • RNA: RNA (139-MER)
  • Ligand: ZINC ION
KeywordsCRISPR-Cas / RNA BINDING PROTEIN
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsOuyang SY / Liu MX / Li ZK
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)82172287 China
CitationJournal: PLoS Genet / Year: 2023
Title: Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage.
Authors: Mengxi Liu / Zekai Li / Jing Chen / Jinying Lin / Qiuhua Lu / Yangmiao Ye / Hongmin Zhang / Bo Zhang / Songying Ouyang /
Abstract: Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR-Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC ...Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR-Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In this study, we determined the crystal structure of apo-Cas12g, the cryo-EM structure of the Cas12g-sgRNA binary complex and investigated conformational changes that occur during the transition from the apo state to the Cas12g-sgRNA binary complex. The conserved zinc finger motifs in Cas12g undergo an ordered-to-disordered transition from the apo to the sgRNA-bound state and their mutations negatively impact on target RNA cleavage. Moreover, we identified a lid motif in the RuvC domain that undergoes transformation from a helix to loop to regulate the access to the RuvC active site and subsequent cleavage of the RNA substrate. Overall, our study provides valuable insights into the mechanisms by which Cas12g recognizes sgRNA and the conformational changes it undergoes from sgRNA binding to the activation of the RNase active site, thereby laying a foundation for the potential repurposing of Cas12g as a tool for RNA-editing.
History
DepositionJan 17, 2023-
Header (metadata) releaseSep 6, 2023-
Map releaseSep 6, 2023-
UpdateOct 4, 2023-
Current statusOct 4, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35154.png

Downloads & links

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Map

FileDownload / File: emd_35154.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.0084
Minimum - Maximum-0.035974536 - 0.06703414
Average (Standard dev.)0.00018351615 (±0.0018147479)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 201.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_35154_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35154_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35154_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Cas12g-sgRNA binary complex

EntireName: Cas12g-sgRNA binary complex
Components
  • Complex: Cas12g-sgRNA binary complex
    • Complex: Cas12g
      • Protein or peptide: Cas12g
    • Complex: RNA
      • RNA: RNA (139-MER)
  • Ligand: ZINC ION

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Supramolecule #1: Cas12g-sgRNA binary complex

SupramoleculeName: Cas12g-sgRNA binary complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #2: Cas12g

SupramoleculeName: Cas12g / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2

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Supramolecule #3: RNA

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

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Macromolecule #1: RNA (139-MER)

MacromoleculeName: RNA (139-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 44.771703 KDa
SequenceString:
GGGAUGCUUA CUUAGUCAUC UGGUUGGCAA ACCUCCGCGG ACCUUCGGGA CCAAUGGAGA GGAACCCAGC CGAGAAGCAU CGAGCCGGU AAAUGAAUUU ACCGGCUCUG ACACCAAUUC GAAAUUAACA CAAACAAGCU

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Macromolecule #2: Cas12g

MacromoleculeName: Cas12g / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 89.847672 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAQASSTPAV SPRPRPRYRE ERTLVRKLLP RPGQSKQEFR ENVKKLRKAF LQFNADVSGV CQWAIQFRPR YGKPAEPTET FWKFFLEPE TSLPPNDSRS PEFRRLQAFE AAAGINGAAA LDDPAFTNEL RDSILAVASR PKTKEAQRLF SRLKDYQPAH R MILAKVAA ...String:
MAQASSTPAV SPRPRPRYRE ERTLVRKLLP RPGQSKQEFR ENVKKLRKAF LQFNADVSGV CQWAIQFRPR YGKPAEPTET FWKFFLEPE TSLPPNDSRS PEFRRLQAFE AAAGINGAAA LDDPAFTNEL RDSILAVASR PKTKEAQRLF SRLKDYQPAH R MILAKVAA EWIESRYRRA HQNWERNYEE WKKEKQEWEQ NHPELTPEIR EAFNQIFQQL EVKEKRVRIC PAARLLQNKD NC QYAGKNK HSVLCNQFNE FKKNHLQGKA IKFFYKDAEK YLRCGLQSLK PNVQGPFRED WNKYLRYMNL KEETLRGKNG GRL PHCKNL GQECEFNPHT ALCKQYQQQL SSRPDLVQHD ELYRKWRREY WREPRKPVFR YPSVKRHSIA KIFGENYFQA DFKN SVVGL RLDSMPAGQY LEFAFAPWPR NYRPQPGETE ISSVHLHFVG TRPRIGFRFR VPHKRSRFDC TQEELDELRS RTFPR KAQD QKFLEAARKR LLETFPGNAE QELRLLAVAL GTDSARAAFF IGKTFQQAFP LKIVKIEKLY EQWPNQKQAG DRRDAS SKQ PRPGLSRDHV GRHLQKMRAQ ASEIAQKRQE LTGTPAPETT TDQAAKKATL QPFDLRGLTV HTARMIRDWA RLNARQI IQ LAEENQVDLI VLESLRGFRP PGYENLDQEK KRRVAFFAHG RIRRKVTEKA VERGMRVVTV PYLASSKVCA ECRKKQKD N KQWEKNKKRG LFKCEGCGSQ AQVDENAARV LGRVFWGEIE LPTAIP

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / 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

Concentration3 mg/mL
BufferpH: 8.5
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: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (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: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 88696

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