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- EMDB-67448: Cryo-EM structure of the E. coli ArnA hexamer -

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Basic information

Entry
Database: EMDB / ID: EMD-67448
TitleCryo-EM structure of the E. coli ArnA hexamer
Map dataSharpened map
Sample
  • Complex: Hexameric complex of ArnA
    • Protein or peptide: Bifunctional polymyxin resistance protein ArnA
KeywordsBifunctional enzyme / polymyxin resistance protein / antimicrobial resistance protein / 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 / membrane / 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 ...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: 3.23 Å
AuthorsJiang X / Kikkawa M / Yanagisawa H
Funding support Japan, 4 items
OrganizationGrant numberCountry
Japan Science and TechnologyJPMJER2202 Japan
Japan Society for the Promotion of Science (JSPS)JP21H05247 Japan
Japan Society for the Promotion of Science (JSPS)JP24KF0141 Japan
Japan Society for the Promotion of Science (JSPS)JP24K18106 Japan
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2026
Title: Cryo-EM reveals ArnA contamination during purification of a ciliary protein complex.
Authors: Xuguang Jiang / Masahide Kikkawa /
Abstract: Endogenous Escherichia coli proteins can co-purify with recombinant targets and dominate cryo-EM datasets, yet often escape detection during standard biochemical quality control. In parallel to the ...Endogenous Escherichia coli proteins can co-purify with recombinant targets and dominate cryo-EM datasets, yet often escape detection during standard biochemical quality control. In parallel to the recent report by Caliseki and coworkers [Caliseki et al. (2025), Acta Cryst. D81, 545-557], we independently identified ArnA contamination while purifying a soluble, low-yield KIF17-IFT70 complex, ultimately obtaining a 3.23 Å resolution cryo-EM structure of ArnA rather than the intended target. Our results reinforce that ArnA enrichment reflects general features of His-tag affinity purification and can become particularly problematic in cryo-EM workflows when the intended target is low in yield or conformationally heterogeneous. By comparing biochemical behavior and cryo-EM outcomes, we outline why ArnA may evade SDS-PAGE and size-exclusion chromatography, and be underappreciated in routine mass spectrometry-based quality control, yet becomes structurally dominant in cryo-EM. These findings broaden the scope of the original study and highlight the need for early cryo-EM screening and improved contaminant awareness in structural biology.
History
DepositionDec 4, 2025-
Header (metadata) releaseMar 25, 2026-
Map releaseMar 25, 2026-
UpdateMay 13, 2026-
Current statusMay 13, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_67448.png

Downloads & links

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Map

FileDownload / File: emd_67448.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 192 pix.
= 249.6 Å		1.3 Å/pix.
x 192 pix.
= 249.6 Å		1.3 Å/pix.
x 192 pix.
= 249.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.036624197 - 1.9625131
Average (Standard dev.)0.0036154198 (±0.039586116)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 249.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_67448_msk_1.map
Projections & Slices
AxesZYX

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Additional map: raw map

Fileemd_67448_additional_1.map
Annotationraw map
Projections & Slices
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Half map: Halfmap1

Fileemd_67448_half_map_1.map
AnnotationHalfmap1
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Histogram

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Half map: Halfmap2

Fileemd_67448_half_map_2.map
AnnotationHalfmap2
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Sample components

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Entire : Hexameric complex of ArnA

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

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Supramolecule #1: Hexameric complex of ArnA

SupramoleculeName: Hexameric complex of ArnA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 446 KDa

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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: 12 / Enantiomer: LEVO
EC number: UDP-4-amino-4-deoxy-L-arabinose formyltransferase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 74.385773 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 LVQGSRLNSQ PACTARRRTR VLI LGVNGF IGNHLTERLL REDHYEVYGL DIGSDAISRF LNHPHFHFVE GDISIHSEWI EYHVKKCDVV LPLVAIATPI EYTR NPLRV FELDFEENLR IIRYCVKYRK RIIFPSTSEV YGMCSDKYFD EDHSNLIVGP VNKPRWIYSV SKQLLDRVIW AYGEK EGLQ FTLFRPFNWM GPRLDNLNAA RIGSSRAITQ LILNLVEGSP IKLIDGGKQK RCFTDIRDGI EALYRIIENA GNRCDG EII NIGNPENEAS IEELGEMLLA SFEKHPLRHH FPPFAGFRVV ESSSYYGKGY QDVEHRKPSI RNAHRCLDWE PKIDMQE TI DETLDFFLRT VDLTDKPS

UniProtKB: Bifunctional polymyxin resistance protein ArnA

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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

Concentration0.01 mg/mL
BufferpH: 8
GridModel: Quantifoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 3000 / Average electron dose: 61.6 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: 1.6 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1811803
CTF correctionSoftware - Name: cryoSPARC (ver. v4.7.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: Ab-Initio Reconstruction
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.7.1) / Number images used: 67122
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.7.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.7.1)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

9v5h


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: BACKBONE TRACE
Output model

PDB-21ak:
Cryo-EM structure of the E. coli ArnA hexamer

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