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- PDB-8w35: Aca2 from Pectobacterium phage ZF40 bound to RNA -

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Basic information

Entry
Database: PDB / ID: 8w35
TitleAca2 from Pectobacterium phage ZF40 bound to RNA
Components
  • Anti-CRISPR associated (Aca) protein, Aca2
  • IR2 and IR-RBS RNA
KeywordsRNA BINDING PROTEIN / HTH protein / CRISPR / RNA-binding protein
Function / homologyProtein of unknown function DUF1870 / Domain of unknown function (DUF1870) / YdiL domain superfamily / Lambda repressor-like, DNA-binding domain superfamily / DNA binding / metal ion binding / RNA / RNA (> 10) / DUF1870 family protein
Function and homology information
Biological speciesPectobacterium phage ZF40 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsWilkinson, M.E. / Birkholz, N. / Kimanius, D. / Fineran, P.C.
Funding support United States, New Zealand, Germany, 3items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Other government New Zealand
Alexander von Humboldt Foundation Germany
CitationJournal: Nature / Year: 2024
Title: Phage anti-CRISPR control by an RNA- and DNA-binding helix-turn-helix protein.
Authors: Nils Birkholz / Kotaro Kamata / Maximilian Feussner / Max E Wilkinson / Christian Cuba Samaniego / Angela Migur / Dari Kimanius / Marijn Ceelen / Sam C Went / Ben Usher / Tim R Blower / ...Authors: Nils Birkholz / Kotaro Kamata / Maximilian Feussner / Max E Wilkinson / Christian Cuba Samaniego / Angela Migur / Dari Kimanius / Marijn Ceelen / Sam C Went / Ben Usher / Tim R Blower / Chris M Brown / Chase L Beisel / Zasha Weinberg / Robert D Fagerlund / Simon A Jackson / Peter C Fineran /
Abstract: In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins. For instance, in the arms race between ...In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.
History
DepositionFeb 21, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 24, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 14, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Anti-CRISPR associated (Aca) protein, Aca2
B: Anti-CRISPR associated (Aca) protein, Aca2
C: IR2 and IR-RBS RNA


Theoretical massNumber of molelcules
Total (without water)40,8843
Polymers40,8843
Non-polymers00
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

#1: Protein Anti-CRISPR associated (Aca) protein, Aca2


Mass: 13710.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pectobacterium phage ZF40 (virus) / Gene: ZF40_0030 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: H9C180
#2: RNA chain IR2 and IR-RBS RNA


Mass: 13463.021 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pectobacterium phage ZF40 (virus)

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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: Anti-CRISPR associated (Aca) protein, Aca2 bound to its 5'UTR RNA (IR2 and IR-RBS)
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.0395 MDa / Experimental value: NO
Source (natural)Organism: Pectobacterium phage ZF40 (virus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
119 mMTris-HCl pH 7.41
286 mMsodium chlorideNaCl1
35 mMmagnesium chlorideMgCl21
SpecimenConc.: 5.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 25 mA plasma / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 285 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.33 sec. / Electron dose: 72.2 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11232
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7ISOLDEmodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
12RELION4final Euler assignment
13RELION5classification
14RELION53D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6449886
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 301832 / Details: BLUSH regularisation used during refinement / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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