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- EMDB-34832: CryoEM structure of an anti-CRISPR protein AcrIIC5 bound to Nme1C... -

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Basic information

Entry
Database: EMDB / ID: EMD-34832
TitleCryoEM structure of an anti-CRISPR protein AcrIIC5 bound to Nme1Cas9-sgRNA complex
Map data
Sample
  • Complex: Complex of AcrIIC5 with sgRNA-bound Nme1Cas9
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: sgRNA
    • Protein or peptide: Phage protein
KeywordsCas9 / anti-CRISPR proteins / cleavage inhibition / ANTIMICROBIAL PROTEIN / HYDROLASE-RNA-ANTIMICROBIAL PROTEIN 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 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9 / Phage protein
Similarity search - Component
Biological speciesNeisseria meningitidis 8013 (bacteria) / Neisseria meningitidis serogroup C (strain 8013) (bacteria) / Simonsiella muelleri ATCC 29453 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsWang Y / Sun W
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Anti-CRISPR AcrIIC5 is a dsDNA mimic that inhibits type II-C Cas9 effectors by blocking PAM recognition.
Authors: Wei Sun / Xiaolong Zhao / Jinlong Wang / Xiaoqi Yang / Zhi Cheng / Shuo Liu / Jiuyu Wang / Gang Sheng / Yanli Wang /
Abstract: Anti-CRISPR proteins are encoded by phages to inhibit the CRISPR-Cas systems of the hosts. AcrIIC5 inhibits several naturally high-fidelity type II-C Cas9 enzymes, including orthologs from Neisseria ...Anti-CRISPR proteins are encoded by phages to inhibit the CRISPR-Cas systems of the hosts. AcrIIC5 inhibits several naturally high-fidelity type II-C Cas9 enzymes, including orthologs from Neisseria meningitidis (Nme1Cas9) and Simonsiella muelleri (SmuCas9). Here, we solve the structure of AcrIIC5 in complex with Nme1Cas9 and sgRNA. We show that AcrIIC5 adopts a novel fold to mimic the size and charge distribution of double-stranded DNA, and uses its negatively charged grooves to bind and occlude the protospacer adjacent motif (PAM) binding site in the target DNA cleft of Cas9. AcrIIC5 is positioned into the crevice between the WED and PI domains of Cas9, and one end of the anti-CRISPR interacts with the phosphate lock loop and a linker between the RuvC and BH domains. We employ biochemical and mutational analyses to build a model for AcrIIC5's mechanism of action, and identify residues on both the anti-CRISPR and Cas9 that are important for their interaction and inhibition. Together, the structure and mechanism of AcrIIC5 reveal convergent evolution among disparate anti-CRISPR proteins that use a DNA-mimic strategy to inhibit diverse CRISPR-Cas surveillance complexes, and provide new insights into a tool for potent inhibition of type II-C Cas9 orthologs.
History
DepositionNov 22, 2022-
Header (metadata) releaseJan 25, 2023-
Map releaseJan 25, 2023-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34832.png

Downloads & links

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Map

FileDownload / File: emd_34832.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.7229153 - 1.196273
Average (Standard dev.)-0.00012589883 (±0.023361202)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 270.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_34832_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34832_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 : Complex of AcrIIC5 with sgRNA-bound Nme1Cas9

EntireName: Complex of AcrIIC5 with sgRNA-bound Nme1Cas9
Components
  • Complex: Complex of AcrIIC5 with sgRNA-bound Nme1Cas9
    • Protein or peptide: CRISPR-associated endonuclease Cas9
    • RNA: sgRNA
    • Protein or peptide: Phage protein

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Supramolecule #1: Complex of AcrIIC5 with sgRNA-bound Nme1Cas9

SupramoleculeName: Complex of AcrIIC5 with sgRNA-bound Nme1Cas9 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Neisseria meningitidis 8013 (bacteria)
Molecular weightTheoretical: 183.04 kDa/nm

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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 serogroup C (strain 8013) (bacteria)
Molecular weightTheoretical: 124.700961 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SMAAFKPNSI NYILGLDIGI ASVGWAMVEI DEEENPIRLI DLGVRVFERA EVPKTGDSLA MARRLARSVR RLTRRRAHRL LRTRRLLKR EGVLQAANFD ENGLIKSLPN TPWQLRAAAL DRKLTPLEWS AVLLHLIKHR GYLSQRKNEG ETADKELGAL L KGVAGNAH ...String:
SMAAFKPNSI NYILGLDIGI ASVGWAMVEI DEEENPIRLI DLGVRVFERA EVPKTGDSLA MARRLARSVR RLTRRRAHRL LRTRRLLKR EGVLQAANFD ENGLIKSLPN TPWQLRAAAL DRKLTPLEWS AVLLHLIKHR GYLSQRKNEG ETADKELGAL L KGVAGNAH ALQTGDFRTP AELALNKFEK ESGHIRNQRS DYSHTFSRKD LQAELILLFE KQKEFGNPHV SGGLKEGIET LL MTQRPAL SGDAVQKMLG HCTFEPAEPK AAKNTYTAER FIWLTKLNNL RILEQGSERP LTDTERATLM DEPYRKSKLT YAQ ARKLLG LEDTAFFKGL RYGKDNAEAS TLMEMKAYHA ISRALEKEGL KDKKSPLNLS PELQDEIGTA FSLFKTDEDI TGRL KDRIQ PEILEALLKH ISFDKFVQIS LKALRRIVPL MEQGKRYDEA CAEIYGDHYG KKNTEEKIYL PPIPADEIRN PVVLR ALSQ ARKVINGVVR RYGSPARIHI ETAREVGKSF KDRKEIEKRQ EENRKDREKA AAKFREYFPN FVGEPKSKDI LKLRLY EQQ HGKCLYSGKE INLGRLNEKG YVEIDHALPF SRTWDDSFNN KVLVLGSENQ NKGNQTPYEY FNGKDNSREW QEFKARV ET SRFPRSKKQR ILLQKFDEDG FKERNLNDTR YVNRFLCQFV ADRMRLTGKG KKRVFASNGQ ITNLLRGFWG LRKVRAEN D RHHALDAVVV ACSTVAMQQK ITRFVRYKEM NAFDGKTIDK ETGEVLHQKT HFPQPWEFFA QEVMIRVFGK PDGKPEFEE ADTLEKLRTL LAEKLSSRPE AVHEYVTPLF VSRAPNRKMS GQGHMETVKS AKRLDEGVSV LRVPLTQLKL KDLEKMVNRE REPKLYEAL KARLEAHKDD PAKAFAEPFY KYDKAGNRTQ QVKAVRVEQV QKTGVWVRNH NGIADNATMV RVDVFEKGDK Y YLVPIYSW QVAKGILPDR AVVQGKDEED WQLIDDSFNF KFSLHPNDLV EVITKKARMF GYFASCHRGT GNINIRIHDL DH KIGKNGI LEGIGVKTAL SFQKYQIDEL GKEIRPCRLK KRPPVR

UniProtKB: CRISPR-associated endonuclease Cas9

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Macromolecule #3: Phage protein

MacromoleculeName: Phage protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Simonsiella muelleri ATCC 29453 (bacteria)
Molecular weightTheoretical: 15.399833 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
SMNNSIKFHV SYDGTARALF NTKEQAEKYC LVEEINDEMN GYKRKSWEEK LREENCASVQ DWVEKNYTSS YSDLFNICEI EVSSAGQLV KIDNTEVDDF VENCYGFTLE DDLEEFNKAK QYLQKFYAEC EN

UniProtKB: Phage protein

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Neisseria meningitidis 8013 (bacteria)
Molecular weightTheoretical: 43.059352 KDa
SequenceString:
GGUCACUCUG CUAUUUAACU UUACGUUGUA GCUCCCUUUC UCGAAAGAGA ACCGUUGCUA CAAUAAGGCC GUCUGAAAAG AUGUGCCGC AACGCUCUGC CCCUUAAAGC UCCUGCUUUA AGGGGCAUCG UUUAUC

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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
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMTristris(hydroxymethyl)aminomethane
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6jdq

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 291265
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: ANGULAR RECONSTITUTION

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