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- EMDB-24249: Oxidized PheRS G318W from Salmonella enterica serovar Typhimurium -

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Basic information

Entry
Database: EMDB / ID: EMD-24249
TitleOxidized PheRS G318W from Salmonella enterica serovar Typhimurium
Map dataoxidized PheRS from Salmonella enterica serovar Typhimurium
Sample
  • Complex: apo-PheRS tetramer structure
    • Protein or peptide: Phenylalanine--tRNA ligase alpha subunit
    • Protein or peptide: Phenylalanine--tRNA ligase beta subunit
Keywordssynthetase / tRNA-binding protein / tetrameric / oxidized / RNA BINDING PROTEIN / LIGASE
Function / homology
Function and homology information


phenylalanine-tRNA ligase / phenylalanine-tRNA ligase activity / phenylalanyl-tRNA aminoacylation / tRNA binding / magnesium ion binding / ATP binding / cytoplasm
Similarity search - Function
Phenylalanine-tRNA ligase, class II, N-terminal / Phenylalanine-tRNA ligase alpha chain 1, bacterial / Aminoacyl tRNA synthetase class II, N-terminal domain / Phenylalanine-tRNA ligase, class IIc, beta subunit, bacterial type / Phenylalanyl-tRNA synthetase, class IIc, alpha subunit / Phenylalanly tRNA synthetase, tRNA-binding-domain / tRNA synthetase, B5-domain / tRNA synthetase B5 domain / B5 domain profile. / tRNA synthetase B5 domain ...Phenylalanine-tRNA ligase, class II, N-terminal / Phenylalanine-tRNA ligase alpha chain 1, bacterial / Aminoacyl tRNA synthetase class II, N-terminal domain / Phenylalanine-tRNA ligase, class IIc, beta subunit, bacterial type / Phenylalanyl-tRNA synthetase, class IIc, alpha subunit / Phenylalanly tRNA synthetase, tRNA-binding-domain / tRNA synthetase, B5-domain / tRNA synthetase B5 domain / B5 domain profile. / tRNA synthetase B5 domain / B3/4 domain / Ferrodoxin-fold anticodon-binding domain / Ferrodoxin-fold anticodon-binding domain superfamily / Ferredoxin-fold anticodon binding domain / Ferredoxin-fold anticodon binding (FDX-ACB) domain profile. / Ferredoxin-fold anticodon binding domain / B3/B4 tRNA-binding domain / Phenylalanyl-tRNA synthetase-like, B3/B4 / Phenylalanyl tRNA synthetase beta chain, core domain / Phenylalanine-tRNA ligase, class IIc, beta subunit / Phenylalanyl tRNA synthetase beta chain CLM domain / B3/4 domain / Class I and II aminoacyl-tRNA synthetase, tRNA-binding arm / Phenylalanyl-tRNA synthetase / tRNA synthetases class II core domain (F) / tRNA-binding domain / Putative tRNA binding domain / tRNA-binding domain profile. / Putative DNA-binding domain superfamily / Aminoacyl-tRNA synthetase, class II / Aminoacyl-transfer RNA synthetases class-II family profile. / Class II Aminoacyl-tRNA synthetase/Biotinyl protein ligase (BPL) and lipoyl protein ligase (LPL) / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
Phenylalanine--tRNA ligase beta subunit / Phenylalanine--tRNA ligase alpha subunit
Similarity search - Component
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsSrinivas P / Dunham CM
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM065183 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)T32AI106699 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM008602 United States
CitationJournal: Nucleic Acids Res / Year: 2021
Title: Oxidation alters the architecture of the phenylalanyl-tRNA synthetase editing domain to confer hyperaccuracy.
Authors: Pooja Srinivas / Rebecca E Steiner / Ian J Pavelich / Ricardo Guerrero-Ferreira / Puneet Juneja / Michael Ibba / Christine M Dunham /
Abstract: High fidelity during protein synthesis is accomplished by aminoacyl-tRNA synthetases (aaRSs). These enzymes ligate an amino acid to a cognate tRNA and have proofreading and editing capabilities that ...High fidelity during protein synthesis is accomplished by aminoacyl-tRNA synthetases (aaRSs). These enzymes ligate an amino acid to a cognate tRNA and have proofreading and editing capabilities that ensure high fidelity. Phenylalanyl-tRNA synthetase (PheRS) preferentially ligates a phenylalanine to a tRNAPhe over the chemically similar tyrosine, which differs from phenylalanine by a single hydroxyl group. In bacteria that undergo exposure to oxidative stress such as Salmonella enterica serovar Typhimurium, tyrosine isomer levels increase due to phenylalanine oxidation. Several residues are oxidized in PheRS and contribute to hyperactive editing, including against mischarged Tyr-tRNAPhe, despite these oxidized residues not being directly implicated in PheRS activity. Here, we solve a 3.6 Å cryo-electron microscopy structure of oxidized S. Typhimurium PheRS. We find that oxidation results in widespread structural rearrangements in the β-subunit editing domain and enlargement of its editing domain. Oxidization also enlarges the phenylalanyl-adenylate binding pocket but to a lesser extent. Together, these changes likely explain why oxidation leads to hyperaccurate editing and decreased misincorporation of tyrosine. Taken together, these results help increase our understanding of the survival of S. Typhimurium during human infection.
History
DepositionJun 16, 2021-
Header (metadata) releaseOct 6, 2021-
Map releaseOct 6, 2021-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7n8y
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24249.png

Downloads & links

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Map

FileDownload / File: emd_24249.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationoxidized PheRS from Salmonella enterica serovar Typhimurium
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 256 pix.
= 266.24 Å		1.04 Å/pix.
x 256 pix.
= 266.24 Å		1.04 Å/pix.
x 256 pix.
= 266.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0088 / Movie #1: 0.013
Minimum - Maximum-0.09327989 - 0.14374422
Average (Standard dev.)-0.0000033943036 (±0.0038418653)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 266.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z266.240266.240266.240
α/β/γ90.00090.00090.000
start NX/NY/NZ484647
NX/NY/NZ97101117
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0930.144-0.000

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Supplemental data

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Sample components

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Entire : apo-PheRS tetramer structure

EntireName: apo-PheRS tetramer structure
Components
  • Complex: apo-PheRS tetramer structure
    • Protein or peptide: Phenylalanine--tRNA ligase alpha subunit
    • Protein or peptide: Phenylalanine--tRNA ligase beta subunit

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Supramolecule #1: apo-PheRS tetramer structure

SupramoleculeName: apo-PheRS tetramer structure / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)

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Macromolecule #1: Phenylalanine--tRNA ligase alpha subunit

MacromoleculeName: Phenylalanine--tRNA ligase alpha subunit / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: phenylalanine-tRNA ligase
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Molecular weightTheoretical: 36.799645 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSHLAELVAN AAAAINQASD VAALDNVRVE YLGKKGHLTL QMTTLRDLPP EERPAAGAVI NAAKEQVQQA LNARKAELES AALNARLAA ETIDISLPGR RIENGGLHPV TRTIDRIESF FGELGFTVAT GPEIEDDYHN FDALNIPGHH PARADHDTFW F DATRLLRT ...String:
MSHLAELVAN AAAAINQASD VAALDNVRVE YLGKKGHLTL QMTTLRDLPP EERPAAGAVI NAAKEQVQQA LNARKAELES AALNARLAA ETIDISLPGR RIENGGLHPV TRTIDRIESF FGELGFTVAT GPEIEDDYHN FDALNIPGHH PARADHDTFW F DATRLLRT QTSGVQIRTM KAQQPPIRII APGRVYRNDY DQTHTPMFHQ MEGLIVDTNI SFTNLKGTLH DFLRNFFEED LQ IRFRPSY FPFTEPSAEV DVMGKNGKWL EVLGCGMVHP NVLRNVGIDP EIYSGFAFGM GMERLTMLRY GVTDLRSFFE NDL RFLKQF K

UniProtKB: Phenylalanine--tRNA ligase alpha subunit

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Macromolecule #2: Phenylalanine--tRNA ligase beta subunit

MacromoleculeName: Phenylalanine--tRNA ligase beta subunit / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: phenylalanine-tRNA ligase
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Molecular weightTheoretical: 87.484883 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKFSELWLRE WVNPAIDSDA LANQITMAGL EVDGVEPVAG SFNGVVVGEV VECAQHPNAD KLRVTKVNVG GERLLDIVCG APNCRQGLK VAVATIGAIL PGDFKIKAAK LRGEPSEGML CSFSELGISD DHSGIIELPA DAPLGTDIRE YLKLDDNTIE I SVTPNRAD ...String:
MKFSELWLRE WVNPAIDSDA LANQITMAGL EVDGVEPVAG SFNGVVVGEV VECAQHPNAD KLRVTKVNVG GERLLDIVCG APNCRQGLK VAVATIGAIL PGDFKIKAAK LRGEPSEGML CSFSELGISD DHSGIIELPA DAPLGTDIRE YLKLDDNTIE I SVTPNRAD CLGIIGVARD VAVLNKAPLQ EPEMAPVTAT ISDTLPITVE AADACPRYLG RVVKGINVNA PTPLWMKEKL RR CGIRSID AVVDVTNYVL LELGQPMHAF DKDRIDGGIV VRMAKEGETV VLLDGSEATL NADTLVIADH HKALGIAGIF WGE HSGVNG ETQNVLLECA YFNPLSITGR ARRHGLHTDA SHRYERGVDP ALQYKAIERA TRLLLDICGG DAGPIIDVSN EATL PKRAT ITLRRSKLDR LIGHHIADEQ VSDILRRLGC EVTEGQDEWK AVAPTWRFDM EIEEDLVEEV ARVYGYNNIP DEPIQ AGLI MGTHREADLS LKRVKTMLND KGYQEVITYS FVDPKVQQLI HPGAEALLLP NPISVEMSAM RLSLWSGLLA TVVYNQ NRQ QNRVRIFETG LRFVPDTQAN LGIRQDLMLA GVICGNRYDE HWNLAKETVD FYDLKGDLEA VLDLTGKLGD IQFKAEM NP ALHPGQSAAI YLKDERIGFI GVVHPELERK LDLNGRTLVF ELEWNKLADR IVPQAREISR FPANRRDIAV VVAENVPA A DILSECKKVG VNQVVGVNLF DVYRGKGVAE GYKSLAISLI LQDTNRTLEE EEIAATVAKC VEALKERFQA SLRD

UniProtKB: Phenylalanine--tRNA ligase beta subunit

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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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average exposure time: 12.0 sec. / Average electron dose: 64.67 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

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

3pco

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 204902
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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