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- EMDB-64789: cryo-EM structure of hexameric ArnA -

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Basic information

Entry
Database: EMDB / ID: EMD-64789
Titlecryo-EM structure of hexameric ArnA
Map data
Sample
  • Complex: Hexameric Bifunctional polymyxin resistance protein ArnA
    • Protein or peptide: Bifunctional polymyxin resistance protein ArnA
    • Protein or peptide: Bifunctional polymyxin resistance protein ArnA
KeywordsLipid A modification Polymyxin resistance Bifunctional enzyme UDP-glucuronic acid dehydrogenase UDP-4-amino-4-deoxy-L-arabinose / transferase Lipopolysaccharide biosynthesis Antibiotic resistance Aminotransferase Dehydrogenase LPS modification pathway L-Ara4N biosynthesis / TRANSFERASE
Function / homology
Function and homology information


UDP-4-deoxy-4-formamido-beta-L-arabinopyranose biosynthetic process / UDP-glucuronate 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 ...UDP-4-deoxy-4-formamido-beta-L-arabinopyranose biosynthetic process / UDP-glucuronate 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 / membrane
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 ...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
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsCaliseki M / Borucu U / Kadapalakere SY / Schaffitzel C / Kabasakal BV
Funding support United Kingdom, Turkey, 4 items
OrganizationGrant numberCountry
Other government118C225
Biotechnology and Biological Sciences Research Council (BBSRC)BB/P000940/1 United Kingdom
Other government210701/Z/18/Z
Other government Turkey
Citation
Journal: Acta Crystallogr D Struct Biol / Year: 2025
Title: Off-target structural insights: ArnA and AcrB in bacterial membrane-protein cryo-EM analysis.
Authors: Mehmet Caliseki / Ufuk Borucu / Sathish K N Yadav / Christiane Schaffitzel / Burak Veli Kabasakal /
Abstract: Membrane-protein quality control in Escherichia coli involves coordinated actions of the AAA+ protease FtsH, the insertase YidC and the regulatory complex HflKC. These systems maintain proteostasis ...Membrane-protein quality control in Escherichia coli involves coordinated actions of the AAA+ protease FtsH, the insertase YidC and the regulatory complex HflKC. These systems maintain proteostasis by facilitating membrane-protein insertion, folding and degradation. To gain structural insights into a putative complex formed by FtsH and YidC, we performed single-particle cryogenic electron microscopy on detergent-solubilized membrane samples, from which FtsH and YidC were purified using Ni-NTA affinity and size-exclusion chromatography. Although SDS-PAGE analysis indicated high purity of these proteins, cryo-EM data sets unexpectedly yielded high-resolution structures of ArnA and AcrB at 4.0 and 2.9 Å resolution, respectively. ArnA is a bifunctional enzyme involved in lipid A modification and polymyxin resistance, while AcrB is a multidrug efflux transporter of the AcrAB-TolC system. ArnA and AcrB, known Ni-NTA purification contaminants, were also consistently detected by mass spectrometry in Strep-Tactin affinity-purified samples, validating their presence independently of affinity-tag selection. ArnA, which is typically cytoplasmic, was consistently found in membrane-isolated samples, indicating an association with membrane components. Only 2D class averages corresponding to the cytoplasmic AAA+ domain of FtsH were observed; neither side views of full-length FtsH nor densities corresponding to an intact FtsH-YidC complex could be identified, due to the conformational flexibility of the FtsH complex and its transient interaction with YidC, which limited particle alignment and stable classification in cryo-EM data sets. Two-dimensional class averages revealed additional particles resembling GroEL and cytochrome bo oxidase. These results underscore the utility of cryo-EM in uncovering off-target yet structurally well defined complexes, which may reflect physiologically relevant interactions or purification biases during membrane-protein overexpression.
#1: Journal: Biorxiv / Year: 2025
Title: Off-Target Structural Insights: ArnA and AcrB in Bacterial Membrane Protein Cryo-EM Analysis
Authors: Caliseki M / Borucu U / Yadav SKN / Schaffitzel C / Kabasakal BV
History
DepositionMay 26, 2025-
Header (metadata) releaseJul 30, 2025-
Map releaseJul 30, 2025-
UpdateOct 8, 2025-
Current statusOct 8, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_64789.png

Downloads & links

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Map

FileDownload / File: emd_64789.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 360 pix.
= 378. Å		1.05 Å/pix.
x 360 pix.
= 378. Å		1.05 Å/pix.
x 360 pix.
= 378. Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00122
Minimum - Maximum-0.007120758 - 2.4883485
Average (Standard dev.)0.001695724 (±0.02875911)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 377.99997 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_64789_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_64789_half_map_2.map
Projections & Slices
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Sample components

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Entire : Hexameric Bifunctional polymyxin resistance protein ArnA

EntireName: Hexameric Bifunctional polymyxin resistance protein ArnA
Components
  • Complex: Hexameric Bifunctional polymyxin resistance protein ArnA
    • Protein or peptide: Bifunctional polymyxin resistance protein ArnA
    • Protein or peptide: Bifunctional polymyxin resistance protein ArnA

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Supramolecule #1: Hexameric Bifunctional polymyxin resistance protein ArnA

SupramoleculeName: Hexameric Bifunctional polymyxin resistance protein ArnA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Bifunctional polymyxin resistance protein ArnA

MacromoleculeName: Bifunctional polymyxin resistance protein ArnA / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
EC number: UDP-4-amino-4-deoxy-L-arabinose formyltransferase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 32.717416 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKTVVFAYHD MGCLGIEALL AAGYEISAIF THTDNPGEKA FYGSVARLAA ERGIPVYAPD NVNHPLWVER IAQLSPDVIF SFYYRHLIY DEILQLAPAG AFNLHGSLLP KYRGRAPLNW VLVNGETETG VTLHRMVKRA DAGAIVAQLR IAIAPDDIAI T LHHKLCHA ...String:
MKTVVFAYHD MGCLGIEALL AAGYEISAIF THTDNPGEKA FYGSVARLAA ERGIPVYAPD NVNHPLWVER IAQLSPDVIF SFYYRHLIY DEILQLAPAG AFNLHGSLLP KYRGRAPLNW VLVNGETETG VTLHRMVKRA DAGAIVAQLR IAIAPDDIAI T LHHKLCHA ARQLLEQTLP AIKHGNILEI AQRENEATCF GRRTPDDSFL EWHKPASVLH NMVRAVADPW PGAFSYVGNQ KF TVWSSRV HPHASKAQPG SVISVAPLLI ACGDGALEIV TGQAGDGITM QGSQLAQTLG LVQ

UniProtKB: Bifunctional polymyxin resistance protein ArnA

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Macromolecule #2: Bifunctional polymyxin resistance protein ArnA

MacromoleculeName: Bifunctional polymyxin resistance protein ArnA / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
EC number: UDP-4-amino-4-deoxy-L-arabinose formyltransferase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 39.744215 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MRVLILGVNG FIGNHLTERL LREDHYEVYG LDIGSDAISR FLNHPHFHFV EGDISIHSEW IEYHVKKCDV VLPLVAIATP IEYTRNPLR VFELDFEENL RIIRYCVKYR KRIIFPSTSE VYGMCSDKYF DEDHSNLIVG PVNKPRWIYS VSKQLLDRVI W AYGEKEGL ...String:
MRVLILGVNG FIGNHLTERL LREDHYEVYG LDIGSDAISR FLNHPHFHFV EGDISIHSEW IEYHVKKCDV VLPLVAIATP IEYTRNPLR VFELDFEENL RIIRYCVKYR KRIIFPSTSE VYGMCSDKYF DEDHSNLIVG PVNKPRWIYS VSKQLLDRVI W AYGEKEGL QFTLFRPFNW MGPRLDNLNA ARIGSSRAIT QLILNLVEGS PIKLIDGGKQ KRCFTDIRDG IEALYRIIEN AG NRCDGEI INIGNPENEA SIEELGEMLL ASFEKHPLRH HFPPFAGFRV VESSSYYGKG YQDVEHRKPS IRNAHRCLDW EPK IDMQET IDETLDFFLR TVDLTD

UniProtKB: Bifunctional polymyxin resistance protein ArnA

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statecell

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 60.6 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6pih

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 74212
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-9v5h:
cryo-EM structure of hexameric ArnA

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