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- PDB-9asc: Cryo-EM Structure of the Glycosyltransferase ArnC from Salmonella... -

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Basic information

Entry
Database: PDB / ID: 9asc
TitleCryo-EM Structure of the Glycosyltransferase ArnC from Salmonella enterica in the UDP-bound State Determined on Talos Arctica microscope
ComponentsUndecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase
KeywordsTRANSFERASE / Glycosyltransferase / undecaprenyl phosphate / aminoarabinose / polymyxin resistance / GT-A
Function / homology
Function and homology information


undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase / undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase activity / 4-amino-4-deoxy-alpha-L-arabinopyranosyl undecaprenyl phosphate biosynthetic process / phosphotransferase activity, for other substituted phosphate groups / lipopolysaccharide biosynthetic process / lipid A biosynthetic process / response to antibiotic / plasma membrane
Similarity search - Function
Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase / : / Glycosyltransferase 2-like / Glycosyl transferase family 2 / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
: / URIDINE-5'-DIPHOSPHATE / Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase
Similarity search - Component
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsAshraf, K.U. / Punetha, A. / Petrou, V.I.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM123228 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM150831 United States
CitationJournal: bioRxiv / Year: 2025
Title: Structural basis of undecaprenyl phosphate glycosylation leading to polymyxin resistance in Gram-negative bacteria.
Authors: Khuram U Ashraf / Mariana Bunoro-Batista / T Bertie Ansell / Ankita Punetha / Stephannie Rosario-Garrido / Emre Firlar / Jason T Kaelber / Phillip J Stansfeld / Vasileios I Petrou /
Abstract: In Gram-negative bacteria, the enzymatic modification of Lipid A with aminoarabinose (L-Ara4N) leads to resistance against polymyxin antibiotics and cationic antimicrobial peptides. ArnC, an integral ...In Gram-negative bacteria, the enzymatic modification of Lipid A with aminoarabinose (L-Ara4N) leads to resistance against polymyxin antibiotics and cationic antimicrobial peptides. ArnC, an integral membrane glycosyltransferase, attaches a formylated form of aminoarabinose to the lipid undecaprenyl phosphate, enabling its association with the bacterial inner membrane. Here, we present cryo-electron microscopy structures of ArnC from in and nucleotide-bound conformations. These structures reveal a conformational transition that takes place upon binding of the partial donor substrate. Using coarse-grained and atomistic simulations, we provide insights into substrate coordination before and during catalysis, and we propose a catalytic mechanism that may operate on all similar metal-dependent polyprenyl phosphate glycosyltransferases. The reported structures provide a new target for drug design aiming to combat polymyxin resistance.
History
DepositionFeb 24, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 5, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase
B: Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase
C: Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase
D: Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)164,73912
Polymers162,9024
Non-polymers1,8368
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
Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase / Polymyxin resistance protein PmrF / Undecaprenyl-phosphate Ara4FN transferase / Ara4FN transferase


Mass: 40725.539 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Gene: arnC, pbgP2, pmrF, STM2298 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: O52324, undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase
#2: Chemical
ChemComp-UDP / URIDINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 404.161 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C9H14N2O12P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: UDP*YM
#3: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
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: Salmonella enterica ArnC in MSP1E3D1 nanodisc bound to UDP and Mn2+
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.1627 MDa / Experimental value: NO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMSodium chlorideNaCl1
31 mMManganese chlorideMnCl21
42 mMUridine 5-diphosphate disodiumC9H12N2Na2O12P21
SpecimenConc.: 1.5 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: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: 3 microliters of ArnC incorporated into nanodiscs was applied to a glow-discharged UltraAuFoil (1.2/1.3) 300 mesh grids (Quantifoil), blotted with filter paper for 3.5 s, and flash-frozen by ...Details: 3 microliters of ArnC incorporated into nanodiscs was applied to a glow-discharged UltraAuFoil (1.2/1.3) 300 mesh grids (Quantifoil), blotted with filter paper for 3.5 s, and flash-frozen by plunging in liquid ethane cooled with liquid nitrogen. Grids were stored in liquid nitrogen.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 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: 5.3 sec. / Electron dose: 39.88 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7925
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 53 / Used frames/image: 1-53

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.32particle selection
2EPUimage acquisition
4cryoSPARC3.32CTF correction
7Coot0.9.8.1model fitting
9PHENIX1.20.1-4487model refinement
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARC3.3.2final Euler assignment
12cryoSPARC3.3.2classification
13cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2382682
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 261104 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00310256
ELECTRON MICROSCOPYf_angle_d0.44213912
ELECTRON MICROSCOPYf_dihedral_angle_d10.0973764
ELECTRON MICROSCOPYf_chiral_restr0.0391616
ELECTRON MICROSCOPYf_plane_restr0.0041760

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