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- EMDB-34919: CryoEM structure of HpaCas9-sgRNA-dsDNA in the presence of AcrIIC4 -

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Basic information

Entry
Database: EMDB / ID: EMD-34919
TitleCryoEM structure of HpaCas9-sgRNA-dsDNA in the presence of AcrIIC4
Map data
Sample
  • Complex: Complex of HpaCas9-sgRNA-DNA in the presence of AcrIIC4
    • Complex: HpaCas9/AcrIIC4
      • Protein or peptide: CRISPR-associated endonuclease Cas9
      • Protein or peptide: anti-CRISPR protein AcrIIC4
    • Complex: RNA/DNA
      • RNA: sgRNASubgenomic mRNA
      • DNA: target strand
      • DNA: non-target strand
KeywordsCas9 / 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
RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesHaemophilus parainfluenzae (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSun W / Cheng Z / Wang J / Yang X / Wang Y
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: mBio / Year: 2018
Title: Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and AcrIIC5 Anti-CRISPR Proteins.
Authors: Jooyoung Lee / Aamir Mir / Alireza Edraki / Bianca Garcia / Nadia Amrani / Hannah E Lou / Ildar Gainetdinov / April Pawluk / Raed Ibraheim / Xin D Gao / Pengpeng Liu / Alan R Davidson / ...Authors: Jooyoung Lee / Aamir Mir / Alireza Edraki / Bianca Garcia / Nadia Amrani / Hannah E Lou / Ildar Gainetdinov / April Pawluk / Raed Ibraheim / Xin D Gao / Pengpeng Liu / Alan R Davidson / Karen L Maxwell / Erik J Sontheimer /
Abstract: In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used ...In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017). We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature (K. L. Maxwell, Mol Cell 68:8-14, 2017, https://doi.org/10.1016/j.molcel.2017.09.002; A. Pawluk, A. R. Davidson, and K. L. Maxwell, Nat Rev Microbiol 16:12-17, 2018, https://doi.org/10.1038/nrmicro.2017.120; E. J. Sontheimer and A. R. Davidson, Curr Opin Microbiol 37:120-127, 2017, https://doi.org/10.1016/j.mib.2017.06.003). We have identified new anti-CRISPRs using the same bioinformatic approach that successfully identified previous Acr proteins (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017) against Cas9 (NmeCas9). In this work, we report two novel anti-CRISPR families in strains of and , both of which harbor type II-C CRISPR-Cas systems (A. Mir, A. Edraki, J. Lee, and E. J. Sontheimer, ACS Chem Biol 13:357-365, 2018, https://doi.org/10.1021/acschembio.7b00855). We characterize the type II-C Cas9 orthologs from and , show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from and reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect coevolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications. As one of their countermeasures against CRISPR-Cas immunity, bacteriophages have evolved natural inhibitors known as anti-CRISPR (Acr) proteins. Despite the existence of such examples for type II CRISPR-Cas systems, we currently know relatively little about the breadth of Cas9 inhibitors, and most of their direct Cas9 targets are uncharacterized. In this work we identify two new type II-C anti-CRISPRs and their cognate Cas9 orthologs, validate their functionality and in bacteria, define their inhibitory spectrum against a panel of Cas9 orthologs, demonstrate that they act before Cas9 DNA binding, and document their utility as off-switches for Cas9-based tools in mammalian applications. The discovery of diverse anti-CRISPRs, the mechanistic analysis of their cognate Cas9s, and the definition of Acr inhibitory mechanisms afford deeper insight into the interplay between Cas9 orthologs and their inhibitors and provide greater scope for exploiting Acrs for CRISPR-based genome engineering.
History
DepositionDec 8, 2022-
Header (metadata) releaseJul 19, 2023-
Map releaseJul 19, 2023-
UpdateAug 2, 2023-
Current statusAug 2, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34919.png

Downloads & links

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Map

FileDownload / File: emd_34919.map.gz / Format: CCP4 / Size: 23.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.17136587 - 0.24574105
Average (Standard dev.)0.00037615726 (±0.009194293)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions184184184
Spacing184184184
CellA=B=C: 191.35999 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_34919_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34919_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of HpaCas9-sgRNA-DNA in the presence of AcrIIC4

EntireName: Complex of HpaCas9-sgRNA-DNA in the presence of AcrIIC4
Components
  • Complex: Complex of HpaCas9-sgRNA-DNA in the presence of AcrIIC4
    • Complex: HpaCas9/AcrIIC4
      • Protein or peptide: CRISPR-associated endonuclease Cas9
      • Protein or peptide: anti-CRISPR protein AcrIIC4
    • Complex: RNA/DNA
      • RNA: sgRNASubgenomic mRNA
      • DNA: target strand
      • DNA: non-target strand

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Supramolecule #1: Complex of HpaCas9-sgRNA-DNA in the presence of AcrIIC4

SupramoleculeName: Complex of HpaCas9-sgRNA-DNA in the presence of AcrIIC4
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Haemophilus parainfluenzae (bacteria)
Molecular weightTheoretical: 194 KDa

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Supramolecule #2: HpaCas9/AcrIIC4

SupramoleculeName: HpaCas9/AcrIIC4 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #5

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Supramolecule #3: RNA/DNA

SupramoleculeName: RNA/DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#4

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Macromolecule #1: CRISPR-associated endonuclease Cas9

MacromoleculeName: CRISPR-associated endonuclease Cas9 / type: protein_or_peptide / ID: 1
Details: Two mutations D13A and H581A were introduced to inactivate the catalytic sites of HpaCas9. The first residue 'Ser' of the sample sequence is the one expressed from the vector left after tag cleavage.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Haemophilus parainfluenzae (bacteria)
Molecular weightTheoretical: 121.605695 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SMENKNLNYI LGLALGIASV GWAVVEIDEK ENPLRLIDVG VRTFERAEVP KTGESLALSR RLARSARRLT QRRVARLKKA KRLLKSENI LLSTDERLPH QVWQLRVEGL DHKLERQEWA AVLLHLIKHR GYLSQRKNES KSENKELGAL LSGVDNNHKL L QQATYRSP ...String:
SMENKNLNYI LGLALGIASV GWAVVEIDEK ENPLRLIDVG VRTFERAEVP KTGESLALSR RLARSARRLT QRRVARLKKA KRLLKSENI LLSTDERLPH QVWQLRVEGL DHKLERQEWA AVLLHLIKHR GYLSQRKNES KSENKELGAL LSGVDNNHKL L QQATYRSP AELAVKKFEV EEGHIRNQQG AYTHTFSRLD LLAEMELLFS RQQHFGNPFA SEKLLENLTA LLMWQKPALS GE AILKMLG KCTFEDEYKA AKNTYSAERF VWITKLNNLR IQENGLERAL NDNERLALME QPYDKNRLFY SQVRSILKLS DEA IFKGLR YSGEDKKAIE TKAVLMEMKA YHQIRKVLEG NNLKAEWAEL KANPTLLDEI GTAFSLYKTD EDISAYLAGK LSQP VLNAL LENLSFDKFI QLSLKALYKL LPLMQQGLRY DEACREIYGD HYGKKTEENH HFLPQIPADE IRNPVVLRTL TQARK VING VVRLYGSPAR IHIETGREVG KSYKDRRELE KRQEENRKQR ENAIKEFKEY FPHFAGEPKA KDILKMRLYK QQNAKC LYS GKPIELHRLL EKGYVEVDAA LPFSRTWDDS FNNKVLVLAN ENQNKGNLTP FEWLDGKHNS ERWRAFKALV ETSAFPY AK KQRILSQKLD EKGFIERNLN DTRYVARFLC NFIADNMHLT GEGKRKVFAS NGQITALLRS RWGLAKSRED NDRHHALD A VVVACSTVAM QQKITRFVRF EAGDVFTGER IDRETGEIIP LHFPTPWQFF KQEVEIRIFS DNPKLELENR LPDRPQANH EFVQPLFVSR MPTRKMTGQG HMETVKSAKR LNEGISVIKM PLTKLKLKDL ELMVNREREK DLYDTLKARL EAFNDDPAKA FAEPFIKKG GAIVKSVRVE QIQKSGVLVR EGNGVADNAS MVRVDVFTKG GKYFLVPIYT WQVAKGILPN KAATQYKDEE D WEVMDNSA TFKFSLHPND LVKLVTKKKT ILGYFNGLNR ATGNIDIKEH DLDKSKGKQG IFEGVGIKLA LSFEKYQVDE LG KNIRLCK PSKRQPVR

UniProtKB: CRISPR-associated endonuclease Cas9

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Macromolecule #5: anti-CRISPR protein AcrIIC4

MacromoleculeName: anti-CRISPR protein AcrIIC4 / type: protein_or_peptide / ID: 5
Details: WP_049372635.1;The first residue 'Ser' of the sample sequence is the one expressed from the vector left after tag cleavage.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Haemophilus parainfluenzae (bacteria)
Molecular weightTheoretical: 10.072395 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
SMKITSSNFA TIATSENFAK LSVLPKNHRE PIKGLFKSAV EQFSSARDFF KNENYSKELA EKFNKEAVNE AVEKLQKAID LAEKQGIQF

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2
Details: RNA is originally derived from Haemophilus parainfluenzae and modified by author.
Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 40.860125 KDa
SequenceString:
GGUCACUCUA ACAUUUAAUC ACACGUUGUA GCUCCCUUUU UCGAAAGAAA AACGUUGUUA CAAUAAGAGA AAAGAUUUCU CGCAAAGCU CUGUCCCUUG AAAUGUAAGU UUCAAGGGAC AUCUUUUUC

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Macromolecule #3: target strand

MacromoleculeName: target strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Haemophilus parainfluenzae (bacteria)
Molecular weightTheoretical: 10.865029 KDa
SequenceString:
(DT)(DG)(DA)(DA)(DA)(DT)(DC)(DA)(DT)(DA) (DT)(DG)(DT)(DG)(DT)(DG)(DA)(DT)(DT)(DA) (DA)(DA)(DT)(DG)(DT)(DT)(DA)(DG)(DA) (DG)(DT)(DG)(DA)(DC)(DC)

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Macromolecule #4: non-target strand

MacromoleculeName: non-target strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Haemophilus parainfluenzae (bacteria)
Molecular weightTheoretical: 10.766954 KDa
SequenceString:
(DG)(DG)(DT)(DC)(DA)(DC)(DT)(DC)(DT)(DA) (DA)(DC)(DA)(DT)(DT)(DT)(DA)(DA)(DT)(DG) (DT)(DG)(DT)(DG)(DA)(DT)(DA)(DT)(DG) (DA)(DT)(DT)(DT)(DC)(DA)

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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
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chloridesodium chloride
20.0 mMTristris(hydroxymethyl)aminomethane
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.6 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6jdq

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 212049

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-8hnv:
CryoEM structure of HpaCas9-sgRNA-dsDNA in the presence of AcrIIC4

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