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- EMDB-0456: The cryo-EM structure of hexameric ArnA -

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Basic information

Entry
Database: EMDB / ID: EMD-0456
TitleThe cryo-EM structure of hexameric ArnA
Map dataLocal sharpened map
Sample
  • Complex: ArnA
    • Protein or peptide: A recombined hexameric ArnA molecule based on the cryo-EM map
Function / homology
Function and homology information


: / UDP-4-deoxy-4-formamido-beta-L-arabinopyranose biosynthetic process / UDP-glucuronic acid dehydrogenase activity / UDP-4-amino-4-deoxy-L-arabinose formyltransferase activity / UDP-glucuronic acid dehydrogenase (UDP-4-keto-hexauronic acid decarboxylating) / UDP-4-amino-4-deoxy-L-arabinose formyltransferase / UDP-D-xylose biosynthetic process / UDP-glucuronate decarboxylase activity / lipopolysaccharide biosynthetic process / lipid A biosynthetic process ...: / UDP-4-deoxy-4-formamido-beta-L-arabinopyranose biosynthetic process / UDP-glucuronic acid dehydrogenase activity / UDP-4-amino-4-deoxy-L-arabinose formyltransferase activity / UDP-glucuronic acid dehydrogenase (UDP-4-keto-hexauronic acid decarboxylating) / UDP-4-amino-4-deoxy-L-arabinose formyltransferase / UDP-D-xylose biosynthetic process / UDP-glucuronate decarboxylase activity / lipopolysaccharide biosynthetic process / lipid A biosynthetic process / NAD+ binding / response to antibiotic / protein-containing complex / identical protein binding
Similarity search - Function
Bifunctional polymyxin resistance protein, ArnA / Bifunctional polymyxin resistance protein ArnA-like, extended (e) SDRs / Formyl transferase, C-terminal / Formyl transferase, C-terminal domain / Formyl transferase-like, C-terminal domain superfamily / Formyl transferase, N-terminal / Formyl transferase / Formyl transferase, N-terminal domain superfamily / NAD-dependent epimerase/dehydratase / NAD dependent epimerase/dehydratase family / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Bifunctional polymyxin resistance protein ArnA / Bifunctional polymyxin resistance protein ArnA / Bifunctional polymyxin resistance protein ArnA
Similarity search - Component
Biological speciesEscherichia coli DH5[alpha] (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsYang M / Gehring K
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research152569 Canada
CitationJournal: J Struct Biol / Year: 2019
Title: Cryo-electron microscopy structures of ArnA, a key enzyme for polymyxin resistance, revealed unexpected oligomerizations and domain movements.
Authors: Meng Yang / Yu Seby Chen / Muneyoshi Ichikawa / Daniel Calles-Garcia / Kaustuv Basu / Rayan Fakih / Khanh Huy Bui / Kalle Gehring /
Abstract: Gram-negative bacteria evade the attack of cationic antimicrobial peptides through modifying their lipid A structure in their outer membranes with 4-amino-4-deoxy-L-arabinose (Ara4N). ArnA is a ...Gram-negative bacteria evade the attack of cationic antimicrobial peptides through modifying their lipid A structure in their outer membranes with 4-amino-4-deoxy-L-arabinose (Ara4N). ArnA is a crucial enzyme in the lipid A modification pathway and its deletion abolishes the polymyxin resistance of gram-negative bacteria. Previous studies by X-ray crystallography have shown that full-length ArnA forms a three-bladed propeller-shaped hexamer. Here, the structures of ArnA determined by cryo-electron microscopy (cryo-EM) reveal that ArnA exists in two 3D architectures, hexamer and tetramer. This is the first observation of a tetrameric ArnA. The hexameric cryo-EM structure is similar to previous crystal structures but shows differences in domain movements and conformational changes. We propose that ArnA oligomeric states are in a dynamic equilibrium, where the hexamer state is energetically more favorable, and its domain movements are important for cooperating with downstream enzymes in the lipid A-Ara4N modification pathway. The results provide us with new possibilities to explore inhibitors targeting ArnA.
History
DepositionJan 14, 2019-
Header (metadata) releaseJan 23, 2019-
Map releaseAug 7, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 11
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 11
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6pih
  • Surface level: 11
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0456.png

Downloads & links

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Map

FileDownload / File: emd_0456.map.gz / Format: CCP4 / Size: 35.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal sharpened map
Voxel sizeX=Y=Z: 1.073 Å
Density
Contour LevelBy AUTHOR: 11 / Movie #1: 11
Minimum - Maximum-16.00808 - 25.454664
Average (Standard dev.)0.1909696 (±2.3596401)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions210210210
Spacing210210210
CellA=B=C: 225.32999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0731.0731.073
M x/y/z210210210
origin x/y/z0.0000.0000.000
length x/y/z225.330225.330225.330
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS210210210
D min/max/mean-16.00825.4550.191

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Supplemental data

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Half map: ArnA EM consensus half map

Fileemd_0456_half_map_1.map
AnnotationArnA EM consensus half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: ArnA EM consensus half map

Fileemd_0456_half_map_2.map
AnnotationArnA EM consensus half 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 : ArnA

EntireName: ArnA
Components
  • Complex: ArnA
    • Protein or peptide: A recombined hexameric ArnA molecule based on the cryo-EM map

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Supramolecule #1: ArnA

SupramoleculeName: ArnA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Full length ArnA from E.coli
Source (natural)Organism: Escherichia coli DH5[alpha] (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightExperimental: 440 KDa

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Macromolecule #1: A recombined hexameric ArnA molecule based on the cryo-EM map

MacromoleculeName: A recombined hexameric ArnA molecule based on the cryo-EM map
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli DH5[alpha] (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKTVVFAYHD MGCLGIEALL AAGYEISAIF THTDNPGEKA FYGSVARLAA ERGIPVYAPD NVNHPLWVE RIAQLSPDVI FSFYYRHLIY DEILQLAPAG AFNLHGSLLP KYRGRAPLNW V LVNGETET GVTLHRMVKR ADAGAIVAQL RIAIAPDDIA ITLHHKLCHA ...String:
MKTVVFAYHD MGCLGIEALL AAGYEISAIF THTDNPGEKA FYGSVARLAA ERGIPVYAPD NVNHPLWVE RIAQLSPDVI FSFYYRHLIY DEILQLAPAG AFNLHGSLLP KYRGRAPLNW V LVNGETET GVTLHRMVKR ADAGAIVAQL RIAIAPDDIA ITLHHKLCHA ARQLLEQTLP AI KHGNILE IAQRENEATC FGRRTPDDSF LEWHKPASVL HNMVRAVADP WPGAFSYVGN QKF TVWSSR VHPHASKAQP GSVISVAPLL IACGDGALEI VTGQAGDGIT MQGSQLAQTL GLVQ GSRLN SQPACTARRR TRVLILGVNG FIGNHLTERL LREDHYEVYG LDIGSDAISR FLNHP HFHF VEGDISIHSE WIEYHVKKCD VVLPLVAIAT PIEYTRNPLR VFELDFEENL RIIRYC VKY RKRIIFPSTS EVYGMCSDKY FDEDHSNLIV GPVNKPRWIY SVSKQLLDRV IWAYGEK EG LQFTLFRPFN WMGPRLDNLN AARIGSSRAI TQLILNLVEG SPIKLIDGGK QKRCFTDI R DGIEALYRII ENAGNRCDGE IINIGNPENE ASIEELGEML LASFEKHPLR HHFPPFAGF RVVESSSYYG KGYQDVEHRK PSIRNAHRCL DWEPKIDMQE TIDETLDFFL RTVDLTDKPS

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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

Concentration1.2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
20.0 mMC4H11NO3Tris
1.0 mMC9H15O6PHClTCEP-HCl
GridModel: C-flat-2/2 4C / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 35.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

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

4wkg

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 70822
FSC plot (resolution estimation)

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