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- EMDB-22258: Cryo-EM structure of Cas12g ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-22258
TitleCryo-EM structure of Cas12g ternary complex
Map dataunsharpened
Sample
  • Complex: CRISP-Cas complex
    • Protein or peptide: CRISPR-Cas
    • RNA: RNA (130-MER)
    • RNA: RNA (5'-R(P*UP*UP*AP*AP*UP*GP*CP*GP*GP*UP*AP*GP*UP*UP*UP*AP*UP*CP*AP*CP*AP*GP*UP*U)-3')
  • Ligand: ZINC ION
KeywordsCRISPR / HYDROLASE-RNA complex
Biological speciesmetagenome (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsChang L / Li Z / Zhang H
CitationJournal: Nat Chem Biol / Year: 2021
Title: Cryo-EM structure of the RNA-guided ribonuclease Cas12g.
Authors: Zhuang Li / Heng Zhang / Renjian Xiao / Ruijie Han / Leifu Chang /
Abstract: Cas12g, the type V-G CRISPR-Cas effector, is an RNA-guided ribonuclease that targets single-stranded RNA substrate. The CRISPR-Cas12g system offers a potential platform for transcriptome engineering ...Cas12g, the type V-G CRISPR-Cas effector, is an RNA-guided ribonuclease that targets single-stranded RNA substrate. The CRISPR-Cas12g system offers a potential platform for transcriptome engineering and diagnostic applications. We determined the structures of Cas12g-guide RNA complexes in the absence and presence of target RNA by cryo-EM to a resolution of 3.1 Å and 4.8 Å, respectively. Cas12g adopts a bilobed structure with miniature REC2 and Nuc domains, whereas the guide RNAs fold into a flipped 'F' shape, which is primarily recognized by the REC lobe. Target RNA and the CRISPR RNA (crRNA) guide form a duplex that inserts into the central cavity between the REC and NUC lobes, inducing conformational changes in both lobes to activate Cas12g. The structural insights would facilitate the development of Cas12g-based applications.
History
DepositionJun 30, 2020-
Header (metadata) releaseJan 13, 2021-
Map releaseJan 13, 2021-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0125
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0125
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xmg
  • Surface level: 0.0125
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22258.png

Downloads & links

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Map

FileDownload / File: emd_22258.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.0125 / Movie #1: 0.0125
Minimum - Maximum-0.0136332745 - 0.0397625
Average (Standard dev.)0.00022854292 (±0.0021772753)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 209.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z210.000210.000210.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ520520520
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0140.0400.000

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Supplemental data

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Sample components

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Entire : CRISP-Cas complex

EntireName: CRISP-Cas complex
Components
  • Complex: CRISP-Cas complex
    • Protein or peptide: CRISPR-Cas
    • RNA: RNA (130-MER)
    • RNA: RNA (5'-R(P*UP*UP*AP*AP*UP*GP*CP*GP*GP*UP*AP*GP*UP*UP*UP*AP*UP*CP*AP*CP*AP*GP*UP*U)-3')
  • Ligand: ZINC ION

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Supramolecule #1: CRISP-Cas complex

SupramoleculeName: CRISP-Cas complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: metagenome (others)

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Macromolecule #1: CRISPR-Cas

MacromoleculeName: CRISPR-Cas / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 89.89168 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 QELRLLAVDL 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 #2: RNA (130-MER)

MacromoleculeName: RNA (130-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 43.777043 KDa
SequenceString:
GGGAUGCUUA CUUAGUCAUC UGGUUGGCAA ACCUCCGCGG ACCUUCGGGA CCAAUGGAGA GGAACCCAGC CGAGAAGCAU CGAGCCGGU AAAUGUUUAC CGGCUCUGAC ACCAACUGUG AUAAACUACC GCAUUAA

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Macromolecule #3: RNA (5'-R(P*UP*UP*AP*AP*UP*GP*CP*GP*GP*UP*AP*GP*UP*UP*UP*AP*UP*CP...

MacromoleculeName: RNA (5'-R(P*UP*UP*AP*AP*UP*GP*CP*GP*GP*UP*AP*GP*UP*UP*UP*AP*UP*CP*AP*CP*AP*GP*UP*U)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 7.633511 KDa
SequenceString:
UUAAUGCGGU AGUUUAUCAC AGUU

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / 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: 7.5
GridDetails: unspecified
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: 1.35 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 38238
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD

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