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- EMDB-36123: Cryo-EM structure of the NmeCas9-sgRNA-AcrIIC4 ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-36123
TitleCryo-EM structure of the NmeCas9-sgRNA-AcrIIC4 ternary complex
Map data
Sample
  • Complex: a protein complex
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • Protein or peptide: Uncharacterized protein
    • RNA: RNA (117-MER)
Keywordsa protein complex / VIRAL PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
Uncharacterized protein / CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesNeisseria meningitidis (bacteria) / Haemophilus parainfluenzae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsYin H / Li Z / Yu GM / Li XZ
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Inhibitory mechanism of CRISPR-Cas9 by AcrIIC4.
Authors: Xuzichao Li / Fumeng Liao / Jiaqi Gao / Guangyong Song / Chendi Zhang / Nan Ji / Xiaoshen Wang / Jing Wen / Jia He / Yong Wei / Heng Zhang / Zhuang Li / Guimei Yu / Hang Yin /
Abstract: CRISPR-Cas systems act as the adaptive immune systems of bacteria and archaea, targeting and destroying invading foreign mobile genetic elements (MGEs) such as phages. MGEs have also evolved anti- ...CRISPR-Cas systems act as the adaptive immune systems of bacteria and archaea, targeting and destroying invading foreign mobile genetic elements (MGEs) such as phages. MGEs have also evolved anti-CRISPR (Acr) proteins to inactivate the CRISPR-Cas systems. Recently, AcrIIC4, identified from Haemophilus parainfluenzae phage, has been reported to inhibit the endonuclease activity of Cas9 from Neisseria meningitidis (NmeCas9), but the inhibition mechanism is not clear. Here, we biochemically and structurally investigated the anti-CRISPR activity of AcrIIC4. AcrIIC4 folds into a helix bundle composed of three helices, which associates with the REC lobe of NmeCas9 and sgRNA. The REC2 domain of NmeCas9 is locked by AcrIIC4, perturbing the conformational dynamics required for the target DNA binding and cleavage. Furthermore, mutation of the key residues in the AcrIIC4-NmeCas9 and AcrIIC4-sgRNA interfaces largely abolishes the inhibitory effects of AcrIIC4. Our study offers new insights into the mechanism of AcrIIC4-mediated suppression of NmeCas9 and provides guidelines for the design of regulatory tools for Cas9-based gene editing applications.
History
DepositionMay 5, 2023-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateFeb 26, 2025-
Current statusFeb 26, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36123.png

Downloads & links

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Map

FileDownload / File: emd_36123.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 280 pix.
= 238. Å		0.85 Å/pix.
x 280 pix.
= 238. Å		0.85 Å/pix.
x 280 pix.
= 238. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.63
Minimum - Maximum-3.2231185 - 5.2748337
Average (Standard dev.)-0.0014016938 (±0.17069)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 238.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_36123_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_36123_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 : a protein complex

EntireName: a protein complex
Components
  • Complex: a protein complex
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • Protein or peptide: Uncharacterized protein
    • RNA: RNA (117-MER)

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Supramolecule #1: a protein complex

SupramoleculeName: a protein complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Neisseria meningitidis (bacteria)

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Macromolecule #1: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Neisseria meningitidis (bacteria)
Molecular weightTheoretical: 124.613883 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAAFKPNSIN YILGLDIGIA SVGWAMVEID EEENPIRLID LGVRVFERAE VPKTGDSLAM ARRLARSVRR LTRRRAHRLL RTRRLLKRE GVLQAANFDE NGLIKSLPNT PWQLRAAALD RKLTPLEWSA VLLHLIKHRG YLSQRKNEGE TADKELGALL K GVAGNAHA ...String:
MAAFKPNSIN YILGLDIGIA SVGWAMVEID EEENPIRLID LGVRVFERAE VPKTGDSLAM ARRLARSVRR LTRRRAHRLL RTRRLLKRE GVLQAANFDE NGLIKSLPNT PWQLRAAALD RKLTPLEWSA VLLHLIKHRG YLSQRKNEGE TADKELGALL K GVAGNAHA LQTGDFRTPA ELALNKFEKE SGHIRNQRSD YSHTFSRKDL QAELILLFEK QKEFGNPHVS GGLKEGIETL LM TQRPALS GDAVQKMLGH CTFEPAEPKA AKNTYTAERF IWLTKLNNLR ILEQGSERPL TDTERATLMD EPYRKSKLTY AQA RKLLGL EDTAFFKGLR YGKDNAEAST LMEMKAYHAI SRALEKEGLK DKKSPLNLSP ELQDEIGTAF SLFKTDEDIT GRLK DRIQP EILEALLKHI SFDKFVQISL KALRRIVPLM EQGKRYDEAC AEIYGDHYGK KNTEEKIYLP PIPADEIRNP VVLRA LSQA RKVINGVVRR YGSPARIHIE TAREVGKSFK DRKEIEKRQE ENRKDREKAA AKFREYFPNF VGEPKSKDIL KLRLYE QQH GKCLYSGKEI NLGRLNEKGY VEIDHALPFS RTWDDSFNNK VLVLGSENQN KGNQTPYEYF NGKDNSREWQ EFKARVE TS RFPRSKKQRI LLQKFDEDGF KERNLNDTRY VNRFLCQFVA DRMRLTGKGK KRVFASNGQI TNLLRGFWGL RKVRAEND R HHALDAVVVA CSTVAMQQKI TRFVRYKEMN AFDGKTIDKE TGEVLHQKTH FPQPWEFFAQ EVMIRVFGKP DGKPEFEEA DTLEKLRTLL AEKLSSRPEA VHEYVTPLFV SRAPNRKMSG QGHMETVKSA KRLDEGVSVL RVPLTQLKLK DLEKMVNRER EPKLYEALK ARLEAHKDDP AKAFAEPFYK YDKAGNRTQQ VKAVRVEQVQ KTGVWVRNHN GIADNATMVR VDVFEKGDKY Y LVPIYSWQ VAKGILPDRA VVQGKDEEDW QLIDDSFNFK FSLHPNDLVE VITKKARMFG YFASCHRGTG NINIRIHDLD HK IGKNGIL EGIGVKTALS FQKYQIDELG KEIRPCRLKK RPPVR

UniProtKB: CRISPR-associated endonuclease Cas9

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Macromolecule #2: Uncharacterized protein

MacromoleculeName: Uncharacterized protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Haemophilus parainfluenzae (bacteria)
Molecular weightTheoretical: 9.985316 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MKITSSNFAT IATSENFAKL SVLPKNHREP IKGLFKSAVE QFSSARDFFK NENYSKELAE KFNKEAVNEA VEKLQKAIDL AEKQGIQF

UniProtKB: Uncharacterized protein

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Macromolecule #3: RNA (117-MER)

MacromoleculeName: RNA (117-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Neisseria meningitidis (bacteria)
Molecular weightTheoretical: 37.367059 KDa
SequenceString:
UAACUUUACG UUGUAGCUCC CUUUCUCGAA AGAGAACCGU UGCUACAAUA AGGCCGUCUG AAAAGAUGUG CCGCAACGCU CUGCCCCUU AAAGCUCCUG CUUUAAGGGG CAUCGUUU

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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
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: 40.0 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: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 251996
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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