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- PDB-9no4: Cryo-EM structure of Csm/AcrIIIA2/enolase 3:2 complex -

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Basic information

Entry
Database: PDB / ID: 9no4
TitleCryo-EM structure of Csm/AcrIIIA2/enolase 3:2 complex
Components
  • (CRISPR system Cms endoribonuclease ...) x 2
  • (CRISPR system Cms protein ...) x 3
  • AcrIIIA2
  • CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
  • Enolase
  • crRNA RNA (35-MER)
KeywordsRNA BINDING PROTEIN/RNA / ANTI-CRISPR / TYPE III CRISPR / CRYO-EM / STRUCTURAL BIOLOY / ANTI-BACTERIAL / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


phosphopyruvate hydratase / phosphopyruvate hydratase complex / phosphopyruvate hydratase activity / exonuclease activity / peptidoglycan-based cell wall / glycolytic process / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / transferase activity / endonuclease activity / defense response to virus ...phosphopyruvate hydratase / phosphopyruvate hydratase complex / phosphopyruvate hydratase activity / exonuclease activity / peptidoglycan-based cell wall / glycolytic process / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / transferase activity / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / hydrolase activity / magnesium ion binding / cell surface / RNA binding / extracellular region / ATP binding
Similarity search - Function
: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal domain / CRISPR-associated protein, Csm2 Type III-A / CRISPR-associated protein Csm5 / Csm2 Type III-A / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 ...: / CRISPR RNA silencing complex Cmr2 subunit, second helical domain / Csm4, C-terminal / CRISPR Csm4 C-terminal domain / CRISPR-associated protein, Csm2 Type III-A / CRISPR-associated protein Csm5 / Csm2 Type III-A / CRISPR-associated RAMP Csm3 / CRISPR type III-associated RAMP protein Csm4 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / Csm1, subunit domain B / Csm1 subunit domain B / : / Enolase / Enolase, conserved site / Enolase, C-terminal TIM barrel domain / Enolase, N-terminal / Enolase, C-terminal TIM barrel domain / Enolase, N-terminal domain / Enolase signature. / Enolase, C-terminal TIM barrel domain / Enolase, N-terminal domain / : / Cas10/Cmr2, second palm domain / : / CRISPR type III-associated protein / RAMP superfamily / HD domain / GGDEF domain profile. / GGDEF domain / HD domain / Enolase-like, N-terminal / Enolase-like, C-terminal domain superfamily / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
RNA / RNA (> 10) / CRISPR system Cms protein Csm5 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / Uncharacterized protein / CRISPR system Cms protein Csm4 / CRISPR system Cms protein Csm2 / CRISPR system Cms endoribonuclease Csm3 / Enolase
Similarity search - Component
Biological speciesStreptococcus thermophilus (bacteria)
Streptococcus phage 2972 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.67 Å
AuthorsGoswami, H.N. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM152081 United States
CitationJournal: Nat Microbiol / Year: 2025
Title: A phage-encoded anti-CRISPR protein co-opts host enolase to prevent type III CRISPR immunity.
Authors: Katie A Johnson / Hemant N Goswami / Ryan J Catchpole / Fozieh Ahmadizadeh / Peng Zhao / Lance Wells / Hong Li / Michael P Terns /
Abstract: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems provide powerful adaptive immunity against phage infection. In response, phages use anti-CRISPR (Acr) proteins to evade ...CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems provide powerful adaptive immunity against phage infection. In response, phages use anti-CRISPR (Acr) proteins to evade CRISPR immunity. The few type III Acrs identified so far show conditional effectiveness in countering type III immunity or rely on unknown or poorly understood inhibitory mechanisms. Here we report the discovery of AcrIIIA2, a type III-A Acr encoded by Streptococcus thermophilus phages. Biochemical and structural analyses reveal that phage AcrIIIA2 co-opts host enolase, a highly abundant glycolysis enzyme, to form a ternary complex with the S. thermophilus type III-A (Csm) CRISPR ribonucleoprotein complex, obstructing its immune responses. The enolase-chaperoned AcrIIIA2 blocks the initial step of phage RNA binding, thereby preventing downstream type III anti-phage immune responses. Enolase participates in the anti-immune response by serving as an essential structural scaffold, stabilizing Acr-CRISPR interactions. These findings uncover a new anti-defence strategy that exploits a well-conserved host factor to block CRISPR immunity.
History
DepositionMar 7, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 12, 2025Provider: repository / Type: Initial release
Revision 1.0Nov 12, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Nov 12, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Nov 12, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 29, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.1Jul 29, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: CRISPR system Cms protein Csm4
E: CRISPR system Cms endoribonuclease Csm3
F: CRISPR system Cms endoribonuclease Csm3
G: CRISPR system Cms endoribonuclease Csm3
N: crRNA RNA (35-MER)
C: CRISPR system Cms protein Csm2
D: CRISPR system Cms protein Csm2
H: CRISPR system Cms protein Csm5
I: Enolase
J: Enolase
R: AcrIIIA2
K: Enolase
L: Enolase
M: Enolase
O: Enolase
P: Enolase
Q: Enolase
A: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)665,56526
Polymers665,37018
Non-polymers1948
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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CRISPR system Cms protein ... , 3 types, 4 molecules BCDH

#1: Protein CRISPR system Cms protein Csm4


Mass: 33828.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria)
Gene: csm, csm4, STCNRZ302_04685, STHERMO_1022, STHERMO_1028, STHERMO_1031
Production host: Escherichia coli (E. coli) / References: UniProt: A0A811IGE5
#5: Protein CRISPR system Cms protein Csm2 / CRISPR type III A-associated protein Csm2


Mass: 15361.617 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: csm, STHERMO_1026 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8D6U6W4
#6: Protein CRISPR system Cms protein Csm5 / CRISPR type III A-associated protein Csm5


Mass: 41226.324 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: csm5 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0A7HF79

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CRISPR system Cms endoribonuclease ... , 2 types, 3 molecules EFG

#2: Protein CRISPR system Cms endoribonuclease Csm3 / CRISPR type III A-associated RAMP protein Csm3


Mass: 24585.840 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: csm, STHERMO_1021 / Production host: Escherichia coli (E. coli) / References: UniProt: A0AAN1ZZJ5
#3: Protein CRISPR system Cms endoribonuclease Csm3 / CRISPR type III A-associated RAMP protein Csm3


Mass: 24555.857 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: csm, STHERMO_1021 / Production host: Escherichia coli (E. coli) / References: UniProt: A0AAN1ZZJ5

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Protein , 3 types, 10 molecules IJKLMOPQRA

#7: Protein
Enolase / 2-phospho-D-glycerate hydro-lyase / 2-phosphoglycerate dehydratase


Mass: 46948.484 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: eno, STER_0684 / Production host: Escherichia coli (E. coli) / References: UniProt: Q03LI0, phosphopyruvate hydratase
#8: Protein AcrIIIA2


Mass: 12226.886 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus phage 2972 (virus) / Gene: CHPC1156_0026 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A3G8FB66
#9: Protein CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / ssDNase Cas10 / Cyclic oligoadenylate synthase / StCas10


Mass: 86930.672 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Cas10 / Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: cas10, csm1 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A0A7HFE1, Hydrolases; Acting on ester bonds, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases

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RNA chain / Non-polymers , 2 types, 9 molecules N

#10: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: RNA chain crRNA RNA (35-MER)


Mass: 11118.652 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Production host: Escherichia coli (E. coli)

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Csm/AcrIIIA2/enolase complex / Type: COMPLEX
Details: AcrIIIA2 forms a stable ternary complex with the type III-A (Csm) crRNP (CRISPR RNA-Protein complex) and host enolase
Entity ID: #9, #1-#8 / Source: RECOMBINANT
Molecular weightValue: .680 MDa / Experimental value: NO
Source (natural)Organism: Streptococcus thermophilus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: 250mM NaCl, 30mM HEPES, 10mM Beta-mercaptoethanol
Buffer componentConc.: 250 mM / Name: Sodium Chloride / Formula: NaCl
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: OTHER / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2PHENIX1.21.2_5419model refinement
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 311708 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 63.06 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.014847417
ELECTRON MICROSCOPYf_angle_d1.254164226
ELECTRON MICROSCOPYf_chiral_restr0.07377130
ELECTRON MICROSCOPYf_plane_restr0.01448289
ELECTRON MICROSCOPYf_dihedral_angle_d7.93496887

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