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- EMDB-43762: Aca2 from Pectobacterium phage ZF40 bound to RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-43762
TitleAca2 from Pectobacterium phage ZF40 bound to RNA
Map dataSharpened map of Aca2 bound to RNA
Sample
  • Complex: Anti-CRISPR associated (Aca) protein, Aca2 bound to its 5'UTR RNA (IR2 and IR-RBS)
    • Protein or peptide: Anti-CRISPR associated (Aca) protein, Aca2
    • RNA: IR2 and IR-RBS RNA
KeywordsHTH protein / CRISPR / RNA-binding protein / 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 / DUF1870 family protein
Function and homology information
Biological speciesPectobacterium phage ZF40 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsWilkinson ME / Birkholz N / Kimanius D / Fineran PC
Funding support United States, New Zealand, Germany, 3 items
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, 2024-
Header (metadata) releaseJul 24, 2024-
Map releaseJul 24, 2024-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43762.png

Downloads & links

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Map

FileDownload / File: emd_43762.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of Aca2 bound to RNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 128 pix.
= 135.91 Å		1.06 Å/pix.
x 128 pix.
= 135.91 Å		1.06 Å/pix.
x 128 pix.
= 135.91 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0618 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.18737018 - 0.2828002
Average (Standard dev.)-0.00014782272 (±0.008583539)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 135.9104 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43762_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Aca2-RNA refinement, half-map 1

Fileemd_43762_half_map_1.map
AnnotationAca2-RNA refinement, half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Aca2-RNA refinement, half-map 2

Fileemd_43762_half_map_2.map
AnnotationAca2-RNA refinement, half-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 : Anti-CRISPR associated (Aca) protein, Aca2 bound to its 5'UTR RNA...

EntireName: Anti-CRISPR associated (Aca) protein, Aca2 bound to its 5'UTR RNA (IR2 and IR-RBS)
Components
  • Complex: Anti-CRISPR associated (Aca) protein, Aca2 bound to its 5'UTR RNA (IR2 and IR-RBS)
    • Protein or peptide: Anti-CRISPR associated (Aca) protein, Aca2
    • RNA: IR2 and IR-RBS RNA

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Supramolecule #1: Anti-CRISPR associated (Aca) protein, Aca2 bound to its 5'UTR RNA...

SupramoleculeName: Anti-CRISPR associated (Aca) protein, Aca2 bound to its 5'UTR RNA (IR2 and IR-RBS)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pectobacterium phage ZF40 (virus)
Molecular weightTheoretical: 39.5 KDa

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

MacromoleculeName: Anti-CRISPR associated (Aca) protein, Aca2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pectobacterium phage ZF40 (virus)
Molecular weightTheoretical: 13.710401 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
SHGSMTNKEL QAIRKLLMLD VSEAAEHIGR VSARSWQYWE SGRSAVPDDV EQEMLDLASV RIEMMSAIDK RLADGERPKL RFYNKLDEY LADNPDHNVI GWRLSQSVAA LYYTEGHADL I

UniProtKB: DUF1870 family protein

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Macromolecule #2: IR2 and IR-RBS RNA

MacromoleculeName: IR2 and IR-RBS RNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Pectobacterium phage ZF40 (virus)
Molecular weightTheoretical: 13.463021 KDa
SequenceString:
AUCGGUUCGA GAUGGCUCGA AUCGCUCCUA ACGAGGAUUC CA

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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

Concentration5.1 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
19.0 mMTris-HCl pH 7.4
86.0 mMsodium chlorideNaCl
5.0 mMmagnesium chlorideMgCl2
GridModel: Quantifoil / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: 25 mA plasma
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 285 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 11232 / Average exposure time: 1.33 sec. / Average electron dose: 72.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6449886
Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5.0) / Details: BLUSH regularisation used during refinement / Number images used: 301832
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final 3D classificationNumber classes: 4 / Avg.num./class: 254000 / Software - Name: RELION (ver. 5.0) / Details: BLUSH regularisation used
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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