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- EMDB-44302: Cryo-EM Structure of the Glycosyltransferase ArnC from Salmonella... -

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Basic information

Entry
Database: EMDB / ID: EMD-44302
TitleCryo-EM Structure of the Glycosyltransferase ArnC from Salmonella enterica in the Apo State Determined on Krios microscope
Map dataArnC apo - Krios - sharpened map
Sample
  • Complex: Salmonella enterica ArnC in MSP1E3D1 nanodisc in the apo state
    • Protein or peptide: Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase
  • Ligand: water
KeywordsGlycosyltransferase / undecaprenyl phosphate / aminoarabinose / polymyxin resistance / GT-A / TRANSFERASE
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
Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase
Similarity search - Component
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsAshraf KU / Punetha A / Petrou VI
Funding support United States, 2 items
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
DepositionMar 27, 2024-
Header (metadata) releaseFeb 5, 2025-
Map releaseFeb 5, 2025-
UpdateMar 5, 2025-
Current statusMar 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44302.png

Downloads & links

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Map

FileDownload / File: emd_44302.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationArnC apo - Krios - sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.67 Å/pix.
x 480 pix.
= 321.6 Å		0.67 Å/pix.
x 480 pix.
= 321.6 Å		0.67 Å/pix.
x 480 pix.
= 321.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.67 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.072
Minimum - Maximum-0.55579793 - 0.7266368
Average (Standard dev.)0.00011900259 (±0.013758153)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 321.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: ArnC apo - Krios - half map A

Fileemd_44302_half_map_1.map
AnnotationArnC apo - Krios - half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: ArnC apo - Krios - half map B

Fileemd_44302_half_map_2.map
AnnotationArnC apo - Krios - half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Salmonella enterica ArnC in MSP1E3D1 nanodisc in the apo state

EntireName: Salmonella enterica ArnC in MSP1E3D1 nanodisc in the apo state
Components
  • Complex: Salmonella enterica ArnC in MSP1E3D1 nanodisc in the apo state
    • Protein or peptide: Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase
  • Ligand: water

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Supramolecule #1: Salmonella enterica ArnC in MSP1E3D1 nanodisc in the apo state

SupramoleculeName: Salmonella enterica ArnC in MSP1E3D1 nanodisc in the apo state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Molecular weightTheoretical: 162.7 KDa

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Macromolecule #1: Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase

MacromoleculeName: Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
EC number: undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Molecular weightTheoretical: 40.725539 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MDYKDDDDKH HHHHHHHHHE NLYFQSYVGG GSGGGSMFDA APIKKVSVVI PVYNEQESLP ELIRRTTTAC ESLGKAWEIL LIDDGSSDS SAELMVKASQ EADSHIISIL LNRNYGQHAA IMAGFSHVSG DLIITLDADL QNPPEEIPRL VAKADEGFDV V GTVRQNRQ ...String:
MDYKDDDDKH HHHHHHHHHE NLYFQSYVGG GSGGGSMFDA APIKKVSVVI PVYNEQESLP ELIRRTTTAC ESLGKAWEIL LIDDGSSDS SAELMVKASQ EADSHIISIL LNRNYGQHAA IMAGFSHVSG DLIITLDADL QNPPEEIPRL VAKADEGFDV V GTVRQNRQ DSLFRKSASK IINLLIQRTT GKAMGDYGCM LRAYRRPIID TMLRCHERST FIPILANIFA RRATEIPVHH AE REFGDSK YSFMRLINLM YDLVTCLTTT PLRLLSLLGS VIAIGGFSLS VLLIVLRLAL GPQWAAEGVF MLFAVLFTFI GAQ FIGMGL LGEYIGRIYN DVRARPRYFV QQVIYPESTP FTEESHQ

UniProtKB: Undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 130 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration1.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloride
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
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

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) / Detector mode: COUNTING / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Digitization - Frames/image: 1-35 / Number grids imaged: 1 / Number real images: 23259 / Average exposure time: 1.2 sec. / Average electron dose: 57.42 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: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
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: 4186210
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 490807
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: experimental model
Details: The initial model consisted of the complete biological assembly for PDB entry 8VXH
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-9b77:
Cryo-EM Structure of the Glycosyltransferase ArnC from Salmonella enterica in the Apo State Determined on Krios microscope

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