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- EMDB-27179: Cas12a2 quaternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-27179
TitleCas12a2 quaternary complex
Map dataUnsharpened map
Sample
  • Complex: Cas12a2 ternary complex
    • Protein or peptide: OrfB_Zn_ribbon domain-containing protein
    • RNA: RNA (41-MER)
    • RNA: RNA (28-MER)
  • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • DNA: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: water
Function / homologyTransposase IS605, OrfB, C-terminal / Putative transposase DNA-binding 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: 2.74 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-
UpdateFeb 1, 2023-
Current statusFeb 1, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27179.png

Downloads & links

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Map

FileDownload / File: emd_27179.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map
Voxel sizeX=Y=Z: 0.94 Å
Density
Contour LevelBy AUTHOR: 0.142
Minimum - Maximum-0.3698893 - 1.1136614
Average (Standard dev.)0.00013527651 (±0.018820459)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 360.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Additional Map

Fileemd_27179_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_27179_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_27179_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 ternary complex

EntireName: Cas12a2 ternary complex
Components
  • Complex: Cas12a2 ternary complex
    • Protein or peptide: OrfB_Zn_ribbon domain-containing protein
    • RNA: RNA (41-MER)
    • RNA: RNA (28-MER)
  • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • DNA: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Cas12a2 ternary complex

SupramoleculeName: Cas12a2 ternary complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3
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

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Macromolecule #2: RNA (41-MER)

MacromoleculeName: RNA (41-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.151831 KDa
SequenceString:
AUUUCUACUA UUGUAGAUUG GAGCAACACC UGAAGAAGGC U

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Macromolecule #3: RNA (28-MER)

MacromoleculeName: RNA (28-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.934285 KDa
SequenceString:
AGCCUUCUUC AGGUGUUGCU UUAGAAAG

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Macromolecule #4: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')

MacromoleculeName: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3') / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.301163 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)

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Macromolecule #5: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')

MacromoleculeName: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3') / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.400317 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 1 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 104857

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