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Yorodumi- EMDB-34832: CryoEM structure of an anti-CRISPR protein AcrIIC5 bound to Nme1C... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-34832 | |||||||||
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Title | CryoEM structure of an anti-CRISPR protein AcrIIC5 bound to Nme1Cas9-sgRNA complex | |||||||||
Map data | ||||||||||
Sample |
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Keywords | Cas9 / 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 | |||||||||
Biological species | Neisseria meningitidis 8013 (bacteria) / Neisseria meningitidis serogroup C (strain 8013) (bacteria) / Simonsiella muelleri ATCC 29453 (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||
Authors | Wang Y / Sun W | |||||||||
Funding support | China, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_34832.map.gz | 63.3 MB | EMDB map data format | |
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Header (meta data) | emd-34832-v30.xml emd-34832.xml | 17.3 KB 17.3 KB | Display Display | EMDB header |
Images | emd_34832.png | 104.2 KB | ||
Filedesc metadata | emd-34832.cif.gz | 6.5 KB | ||
Others | emd_34832_half_map_1.map.gz emd_34832_half_map_2.map.gz | 62.1 MB 62.1 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-34832 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-34832 | HTTPS FTP |
-Validation report
Summary document | emd_34832_validation.pdf.gz | 972.5 KB | Display | EMDB validaton report |
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Full document | emd_34832_full_validation.pdf.gz | 972 KB | Display | |
Data in XML | emd_34832_validation.xml.gz | 12.3 KB | Display | |
Data in CIF | emd_34832_validation.cif.gz | 14.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-34832 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-34832 | HTTPS FTP |
-Related structure data
Related structure data | 8hj4MC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_34832.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.04 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_34832_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_34832_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Complex of AcrIIC5 with sgRNA-bound Nme1Cas9
Entire | Name: Complex of AcrIIC5 with sgRNA-bound Nme1Cas9 |
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Components |
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-Supramolecule #1: Complex of AcrIIC5 with sgRNA-bound Nme1Cas9
Supramolecule | Name: Complex of AcrIIC5 with sgRNA-bound Nme1Cas9 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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Source (natural) | Organism: Neisseria meningitidis 8013 (bacteria) |
Molecular weight | Theoretical: 183.04 kDa/nm |
-Macromolecule #1: CRISPR-associated endonuclease Cas9
Macromolecule | Name: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds |
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Source (natural) | Organism: Neisseria meningitidis serogroup C (strain 8013) (bacteria) |
Molecular weight | Theoretical: 124.700961 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: 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 |
-Macromolecule #3: Phage protein
Macromolecule | Name: Phage protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Simonsiella muelleri ATCC 29453 (bacteria) |
Molecular weight | Theoretical: 15.399833 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: SMNNSIKFHV SYDGTARALF NTKEQAEKYC LVEEINDEMN GYKRKSWEEK LREENCASVQ DWVEKNYTSS YSDLFNICEI EVSSAGQLV KIDNTEVDDF VENCYGFTLE DDLEEFNKAK QYLQKFYAEC EN UniProtKB: Phage protein |
-Macromolecule #2: sgRNA
Macromolecule | Name: sgRNA / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: Neisseria meningitidis 8013 (bacteria) |
Molecular weight | Theoretical: 43.059352 KDa |
Sequence | String: GGUCACUCUG CUAUUUAACU UUACGUUGUA GCUCCCUUUC UCGAAAGAGA ACCGUUGCUA CAAUAAGGCC GUCUGAAAAG AUGUGCCGC AACGCUCUGC CCCUUAAAGC UCCUGCUUUA AGGGGCAUCG UUUAUC |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 Component:
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Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 60.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination 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 |
-Image processing
Startup model | Type of model: PDB ENTRY PDB model - PDB ID: |
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Final reconstruction | Applied 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 assignment | Type: PROJECTION MATCHING |
Final angle assignment | Type: ANGULAR RECONSTITUTION |