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- EMDB-15952: Map from local refinement with a mask around T3 SAM lyase from th... -

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Basic information

Entry
Database: EMDB / ID: EMD-15952
TitleMap from local refinement with a mask around T3 SAM lyase from the complex of T3 SAM lyase with MetK.
Map data
Sample
  • Complex: T3 SAM lyase in complex with E. coli S-adenosylmethionine synthase.
    • Complex: S-adenosylmethionine synthase in complex with T3 SAM lyase.
      • Protein or peptide: S-adenosylmethionine synthase
      • Protein or peptide: T3 SAM lyase
    • Complex: T3 SAM lyase in complex with S-adenosylmethionine synthase.
KeywordsSAM lyase / complex / LYASE
Function / homology
Function and homology information


methionine adenosyltransferase / methionine adenosyltransferase activity / S-adenosylmethionine cycle / S-adenosylmethionine biosynthetic process / potassium ion binding / one-carbon metabolic process / magnesium ion binding / ATP binding / identical protein binding / cytosol
Similarity search - Function
S-adenosylmethionine synthetase / S-adenosylmethionine synthetase, N-terminal / S-adenosylmethionine synthetase, central domain / S-adenosylmethionine synthetase, C-terminal / S-adenosylmethionine synthetase, conserved site / S-adenosylmethionine synthetase superfamily / S-adenosylmethionine synthetase, N-terminal domain / S-adenosylmethionine synthetase, central domain / S-adenosylmethionine synthetase, C-terminal domain / S-adenosylmethionine synthase signature 1. / S-adenosylmethionine synthase signature 2.
Similarity search - Domain/homology
S-adenosylmethionine synthase
Similarity search - Component
Biological speciesEnterobacteria phage T3 (virus) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsTriguis S / Selmer M
Funding support Sweden, 2 items
OrganizationGrant numberCountry
Swedish Research Council2017-03827 Sweden
Knut and Alice Wallenberg FoundationEvolution of new genes and proteins Sweden
CitationJournal: Cell Rep / Year: 2023
Title: Phage T3 overcomes the BREX defense through SAM cleavage and inhibition of SAM synthesis by SAM lyase.
Authors: Aleksandr Andriianov / Silvia Trigüis / Alena Drobiazko / Nicolas Sierro / Nikolai V Ivanov / Maria Selmer / Konstantin Severinov / Artem Isaev /
Abstract: Bacteriophage T3 encodes a SAMase that, through cleavage of S-adenosyl methionine (SAM), circumvents the SAM-dependent type I restriction-modification (R-M) defense. We show that SAMase also allows ...Bacteriophage T3 encodes a SAMase that, through cleavage of S-adenosyl methionine (SAM), circumvents the SAM-dependent type I restriction-modification (R-M) defense. We show that SAMase also allows T3 to evade the BREX defense. Although SAM depletion weakly affects BREX methylation, it completely inhibits the defensive function of BREX, suggesting that SAM could be a co-factor for BREX-mediated exclusion of phage DNA, similar to its anti-defense role in type I R-M. The anti-BREX activity of T3 SAMase is mediated not just by enzymatic degradation of SAM but also by direct inhibition of MetK, the host SAM synthase. We present a 2.8 Å cryoelectron microscopy (cryo-EM) structure of the eight-subunit T3 SAMase-MetK complex. Structure-guided mutagenesis reveals that this interaction stabilizes T3 SAMase in vivo, further stimulating its anti-BREX activity. This work provides insights in the versatility of bacteriophage counterdefense mechanisms and highlights the role of SAM as a co-factor of diverse bacterial immunity systems.
History
DepositionOct 12, 2022-
Header (metadata) releaseOct 18, 2023-
Map releaseOct 18, 2023-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15952.png

Downloads & links

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Map

FileDownload / File: emd_15952.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1125 Å
Density
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-0.5993054 - 1.2701172
Average (Standard dev.)-0.0002488576 (±0.0090163415)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 534.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_15952_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15952_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 : T3 SAM lyase in complex with E. coli S-adenosylmethionine synthase.

EntireName: T3 SAM lyase in complex with E. coli S-adenosylmethionine synthase.
Components
  • Complex: T3 SAM lyase in complex with E. coli S-adenosylmethionine synthase.
    • Complex: S-adenosylmethionine synthase in complex with T3 SAM lyase.
      • Protein or peptide: S-adenosylmethionine synthase
      • Protein or peptide: T3 SAM lyase
    • Complex: T3 SAM lyase in complex with S-adenosylmethionine synthase.

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Supramolecule #1: T3 SAM lyase in complex with E. coli S-adenosylmethionine synthase.

SupramoleculeName: T3 SAM lyase in complex with E. coli S-adenosylmethionine synthase.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: T3 SAM lyase was recombinantly expressed in E. coli Top10 and co-purified in complex with S-adenosylmethionine synthase from the host.
Source (natural)Organism: Enterobacteria phage T3 (virus)
Molecular weightTheoretical: 239 KDa

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Supramolecule #2: S-adenosylmethionine synthase in complex with T3 SAM lyase.

SupramoleculeName: S-adenosylmethionine synthase in complex with T3 SAM lyase.
type: complex / ID: 2 / Parent: 1 / Macromolecule list: all
Details: T3 SAM lyase was recombinantly expressed in E. coli Top10 and co-purified in complex with S-adenosylmethionine synthase from the host.
Source (natural)Organism: Escherichia coli (E. coli) / Strain: Top10

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Supramolecule #3: T3 SAM lyase in complex with S-adenosylmethionine synthase.

SupramoleculeName: T3 SAM lyase in complex with S-adenosylmethionine synthase.
type: complex / ID: 3 / Parent: 1 / Macromolecule list: all
Details: T3 SAM lyase was recombinantly expressed in E. coli Top10 and co-purified in complex with S-adenosylmethionine synthase from the host.
Source (natural)Organism: Enterobacteria phage T3 (virus)

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Macromolecule #1: S-adenosylmethionine synthase

MacromoleculeName: S-adenosylmethionine synthase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: methionine adenosyltransferase
Source (natural)Organism: Escherichia coli (E. coli) / Strain: Top10
SequenceString: MAKHLFTSES VSEGHPDKIA DQISDAVLDA ILEQDPKARV ACETYVKTGM VLVGGEITTS AWVDIEEITR NTVREIGYVH SDMGFDANS CAVLSAIGKQ SPDINQGVDR ADPLEQGAGD QGLMFGYATN ETDVLMPAPI TYAHRLVQRQ AEVRKNGTLP W LRPDAKSQ ...String:
MAKHLFTSES VSEGHPDKIA DQISDAVLDA ILEQDPKARV ACETYVKTGM VLVGGEITTS AWVDIEEITR NTVREIGYVH SDMGFDANS CAVLSAIGKQ SPDINQGVDR ADPLEQGAGD QGLMFGYATN ETDVLMPAPI TYAHRLVQRQ AEVRKNGTLP W LRPDAKSQ VTFQYDDGKI VGIDAVVLST QHSEEIDQKS LQEAVMEEII KPILPAEWLT SATKFFINPT GRFVIGGPMG DC GLTGRKI IVDTYGGMAR HGGGAFSGKD PSKVDRSAAY AARYVAKNIV AAGLADRCEI QVSYAIGVAE PTSIMVETFG TEK VPSEQL TLLVREFFDL RPYGLIQMLD LLHPIYKETA AYGHFGREHF PWEKTDKAQL LRDAAGLK

UniProtKB: S-adenosylmethionine synthase

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Macromolecule #2: T3 SAM lyase

MacromoleculeName: T3 SAM lyase / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO / EC number: ec: 3.3.1.2
Source (natural)Organism: Enterobacteria phage T3 (virus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MIFTKEPANV FYVLVSAFRS NLCDEVNMSR HRHMVSTLRA APGLYGSVES TDLTGCYREA ISSAPTEEKT VRVRCKDKAQ ALNVARLAC NEWEQDCVLV YKSQTHTAGL VYAKGIDGYK AERLPGSFQE VPKGAPLQGC FTIDEFGRRW QVQHHHHHH

GENBANK: GENBANK: NP_523296.1

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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

Concentration0.125 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
25.0 mMNH2C(CH2OH)3Tris(Hydroxymethyl)aminomethane
150.0 mMNaClSodium chloride
2.5 mMHSCH2CH2OH2-Mercaptoethanol
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting for 3 seconds before plunging..

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 5447 / Average exposure time: 3.0 sec. / Average electron dose: 47.75 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.1 µm / Calibrated defocus min: 0.35000000000000003 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
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: 1445186
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 979648
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3)
Details: 3D classification was used to remove particles with incomplete occupancy of T3 SAMase.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1p7l


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER

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