+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27178 | ||||||||||||
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Title | Structure of Cas12a2 binary complex | ||||||||||||
Map data | Structure of Cas12a2 binary complex | ||||||||||||
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: 3.2 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_27178.map.gz | 62.7 MB | EMDB map data format | |
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Header (meta data) | emd-27178-v30.xml emd-27178.xml | 18.1 KB 18.1 KB | Display Display | EMDB header |
Images | emd_27178.png | 55.8 KB | ||
Filedesc metadata | emd-27178.cif.gz | 6.4 KB | ||
Others | emd_27178_additional_1.map.gz emd_27178_half_map_1.map.gz emd_27178_half_map_2.map.gz | 118.1 MB 116 MB 116 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27178 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27178 | HTTPS FTP |
-Related structure data
Related structure data | 8d49MC 8d4aC 8d4bC 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_27178.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Structure of Cas12a2 binary complex | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.94 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: Additional Map
File | emd_27178_additional_1.map | ||||||||||||
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Annotation | Additional Map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half Map 1
File | emd_27178_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_27178_half_map_2.map | ||||||||||||
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Annotation | Half Map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Cas12a2 nuclease
Entire | Name: Cas12a2 nuclease |
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Components |
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-Supramolecule #1: Cas12a2 nuclease
Supramolecule | Name: Cas12a2 nuclease / 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 (26-MER)
Macromolecule | Name: RNA (26-MER) / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 8.105683 KDa |
Sequence | String: AUUUCUACUA UUGUAGAUCU UUUUUU |
-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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Grid | Model: C-flat-2/2 / Material: COPPER / Mesh: 400 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | TFS GLACIOS |
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Image recording | Film or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 200 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 |
-Image processing
Startup model | Type of model: NONE |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 97470 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |