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- EMDB-27178: Structure of Cas12a2 binary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-27178
TitleStructure of Cas12a2 binary complex
Map dataStructure of Cas12a2 binary complex
Sample
  • Complex: Cas12a2 nuclease
    • Protein or peptide: OrfB_Zn_ribbon domain-containing protein
    • RNA: RNA (26-MER)
KeywordsCas12a2 / CRISPR / Nuclease / DNA Binding Protein-RNA complex
Function / homologyTransposase IS605, OrfB, C-terminal / Cas12f1-like, TNB domain / DNA binding / Cas12f1-like TNB domain-containing protein
Function and homology information
Biological speciesSulfuricurvum sp. PC08-66 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBravo JPK / Taylor DW
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
Welch FoundationF-1938 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR160088 United States
CitationJournal: 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
DepositionJun 1, 2022-
Header (metadata) releaseJan 18, 2023-
Map releaseJan 18, 2023-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27178.png

Downloads & links

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Map

FileDownload / File: emd_27178.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of Cas12a2 binary complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.94 Å/pix.
x 320 pix.
= 300.8 Å		0.94 Å/pix.
x 320 pix.
= 300.8 Å		0.94 Å/pix.
x 320 pix.
= 300.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.94 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.24
Minimum - Maximum-0.32462883 - 0.8220373
Average (Standard dev.)0.00031703355 (±0.018072067)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 300.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Additional Map

Fileemd_27178_additional_1.map
AnnotationAdditional Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 1

Fileemd_27178_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_27178_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cas12a2 nuclease

EntireName: Cas12a2 nuclease
Components
  • Complex: Cas12a2 nuclease
    • Protein or peptide: OrfB_Zn_ribbon domain-containing protein
    • RNA: RNA (26-MER)

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Supramolecule #1: Cas12a2 nuclease

SupramoleculeName: Cas12a2 nuclease / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Sulfuricurvum sp. PC08-66 (bacteria)

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Macromolecule #1: OrfB_Zn_ribbon domain-containing protein

MacromoleculeName: OrfB_Zn_ribbon domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sulfuricurvum sp. PC08-66 (bacteria)
Molecular weightTheoretical: 143.172797 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #2: RNA (26-MER)

MacromoleculeName: RNA (26-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.105683 KDa
SequenceString:
AUUUCUACUA UUGUAGAUCU UUUUUU

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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
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 400 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 97470
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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