+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27180 | ||||||||||||
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Title | Structure of Cas12a2 ternary complex | ||||||||||||
Map data | Unsharpened map | ||||||||||||
Sample |
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Keywords | Cas12a2 / CRISPR / Nuclease / DNA Binding Protein-RNA complex | ||||||||||||
Function / homology | Transposase IS605, OrfB, C-terminal / Putative transposase DNA-binding domain / DNA binding / Cas12f1-like TNB domain-containing protein Function and homology information | ||||||||||||
Biological species | Sulfuricurvum sp. PC08-66 (bacteria) / synthetic construct (others) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.92 Å | ||||||||||||
Authors | Bravo JPK / Taylor DW | ||||||||||||
Funding support | United States, 3 items
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Citation | Journal: Nature / Year: 2023 Title: RNA targeting unleashes indiscriminate nuclease activity of CRISPR-Cas12a2. Authors: Jack P K Bravo / Thomson Hallmark / Bronson Naegle / Chase L Beisel / Ryan N Jackson / David W Taylor / Abstract: Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA following recognition of a ...Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA following recognition of a complementary RNA target, culminating in abortive infection. Here we report structures of Cas12a2 in binary, ternary and quaternary complexes to reveal a complete activation pathway. Our structures reveal that Cas12a2 is autoinhibited until binding a cognate RNA target, which exposes the RuvC active site within a large, positively charged cleft. Double-stranded DNA substrates are captured through duplex distortion and local melting, stabilized by pairs of 'aromatic clamp' residues that are crucial for double-stranded DNA degradation and in vivo immune system function. Our work provides a structural basis for this mechanism of abortive infection to achieve population-level immunity, which can be leveraged to create rational mutants that degrade a spectrum of collateral substrates. | ||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_27180.map.gz | 62.5 MB | EMDB map data format | |
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Header (meta data) | emd-27180-v30.xml emd-27180.xml | 18.2 KB 18.2 KB | Display Display | EMDB header |
Images | emd_27180.png | 57.2 KB | ||
Filedesc metadata | emd-27180.cif.gz | 6.3 KB | ||
Others | emd_27180_additional_1.map.gz emd_27180_half_map_1.map.gz emd_27180_half_map_2.map.gz | 118.1 MB 116.1 MB 116.1 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27180 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27180 | HTTPS FTP |
-Validation report
Summary document | emd_27180_validation.pdf.gz | 903.2 KB | Display | EMDB validaton report |
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Full document | emd_27180_full_validation.pdf.gz | 902.8 KB | Display | |
Data in XML | emd_27180_validation.xml.gz | 14 KB | Display | |
Data in CIF | emd_27180_validation.cif.gz | 16.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27180 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-27180 | HTTPS FTP |
-Related structure data
Related structure data | 8d4bMC 8d49C 8d4aC 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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-Map
File | Download / File: emd_27180.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Unsharpened map | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.81 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: Sharpened map
File | emd_27180_additional_1.map | ||||||||||||
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Annotation | Sharpened map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half Map 1
File | emd_27180_half_map_1.map | ||||||||||||
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Annotation | Half Map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half Map 2
File | emd_27180_half_map_2.map | ||||||||||||
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Annotation | Half Map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Cas12a2 ternary complex
Entire | Name: Cas12a2 ternary complex |
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Components |
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-Supramolecule #1: Cas12a2 ternary complex
Supramolecule | Name: Cas12a2 ternary complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Sulfuricurvum sp. PC08-66 (bacteria) |
-Macromolecule #1: OrfB_Zn_ribbon domain-containing protein
Macromolecule | Name: OrfB_Zn_ribbon domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Sulfuricurvum sp. PC08-66 (bacteria) |
Molecular weight | Theoretical: 143.172797 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MLHAFTNQYQ LSKTLRFGAT LKEDEKKCKS HEELKGFVDI SYENMKSSAT IAESLNENEL VKKCERCYSE IVKFHNAWEK IYYRTDQIA VYKDFYRQLS RKARFDAGKQ NSQLITLASL CGMYQGAKLS RYITNYWKDN ITRQKSFLKD FSQQLHQYTR A LEKSDKAH ...String: MLHAFTNQYQ LSKTLRFGAT LKEDEKKCKS HEELKGFVDI SYENMKSSAT IAESLNENEL VKKCERCYSE IVKFHNAWEK IYYRTDQIA VYKDFYRQLS RKARFDAGKQ NSQLITLASL CGMYQGAKLS RYITNYWKDN ITRQKSFLKD FSQQLHQYTR A LEKSDKAH TKPNLINFNK TFMVLANLVN EIVIPLSNGA ISFPNISKLE DGEESHLIEF ALNDYSQLSE LIGELKDAIA TN GGYTPFA KVTLNHYTAE QKPHVFKNDI DAKIRELKLI GLVETLKGKS SEQIEEYFSN LDKFSTYNDR NQSVIVRTQC FKY KPIPFL VKHQLAKYIS EPNGWDEDAV AKVLDAVGAI RSPAHDYANN QEGFDLNHYP IKVAFDYAWE QLANSLYTTV TFPQ EMCEK YLNSIYGCEV SKEPVFKFYA DLLYIRKNLA VLEHKNNLPS NQEEFICKIN NTFENIVLPY KISQFETYKK DILAW INDG HDHKKYTDAK QQLGFIRGGL KGRIKAEEVS QKDKYGKIKS YYENPYTKLT NEFKQISSTY GKTFAELRDK FKEKNE ITK ITHFGIIIED KNRDRYLLAS ELKHEQINHV STILNKLDKS SEFITYQVKS LTSKTLIKLI KNHTTKKGAI SPYADFH TS KTGFNKNEIE KNWDNYKREQ VLVEYVKDCL TDSTMAKNQN WAEFGWNFEK CNSYEDIEHE IDQKSYLLQS DTISKQSI A SLVEGGCLLL PIINQDITSK ERKDKNQFSK DWNHIFEGSK EFRLHPEFAV SYRTPIEGYP VQKRYGRLQF VCAFNAHIV PQNGEFINLK KQIENFNDED VQKRNVTEFN KKVNHALSDK EYVVIGIDRG LKQLATLCVL DKRGKILGDF EIYKKEFVRA EKRSESHWE HTQAETRHIL DLSNLRVETT IEGKKVLVDQ SLTLVKKNRD TPDEEATEEN KQKIKLKQLS YIRKLQHKMQ T NEQDVLDL INNEPSDEEF KKRIEGLISS FGEGQKYADL PINTMREMIS DLQGVIARGN NQTEKNKIIE LDAADNLKQG IV ANMIGIV NYIFAKYSYK AYISLEDLSR AYGGAKSGYD GRYLPSTSQD EDVDFKEQQN QMLAGLGTYQ FFEMQLLKKL QKI QSDNTV LRFVPAFRSA DNYRNILRLE ETKYKSKPFG VVHFIDPKFT SKKCPVCSKT NVYRDKDDIL VCKECGFRSD SQLK ERENN IHYIHNGDDN GAYHIALKSV ENLIQMK UniProtKB: Cas12f1-like TNB domain-containing protein |
-Macromolecule #2: RNA (41-MER)
Macromolecule | Name: RNA (41-MER) / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 13.151831 KDa |
Sequence | String: AUUUCUACUA UUGUAGAUUG GAGCAACACC UGAAGAAGGC U |
-Macromolecule #3: RNA (28-MER)
Macromolecule | Name: RNA (28-MER) / type: rna / ID: 3 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 8.934285 KDa |
Sequence | String: AGCCUUCUUC AGGUGUUGCU UUAGAAAG |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | TFS KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 70.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: NONE |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 192639 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |