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- EMDB-70744: Cryo-EM structure of AaaA, a Pseudomonas Aeruginosa autotransporter -

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Basic information

Entry
Database: EMDB / ID: EMD-70744
TitleCryo-EM structure of AaaA, a Pseudomonas Aeruginosa autotransporter
Map dataCryo-EM map from the local refinement.
Sample
  • Complex: AaaA complex with Arginine
    • Protein or peptide: Autotransporter domain-containing protein
  • Ligand: ZINC ION
  • Ligand: ARGININE
KeywordsArginine aminopeptidase / Autotransporter / Pseudomonas Aeruginosa / Virulence factor / TRANSPORT PROTEIN
Function / homology
Function and homology information


autotransporter activity / outer membrane / metalloexopeptidase activity / aminopeptidase activity / cell motility / proteolysis
Similarity search - Function
Peptidase M28 family / Autotransporter beta-domain / Outer membrane autotransporter barrel / Autotransporter beta-domain / Autotransporter beta-domain profile. / Autotransporter beta-domain / Autotransporter beta-domain superfamily / Peptidase M28 / Peptidase family M28
Similarity search - Domain/homology
Autotransporter domain-containing protein
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.87 Å
AuthorsArachchige EJ / Rahman MS / Singendonk K / Kim KH
Funding support United States, 1 items
OrganizationGrant numberCountry
Not funded United States
CitationJournal: J Mol Biol / Year: 2025
Title: Structural and Functional Characterization of Pseudomonas aeruginosa Virulence Factor AaaA, an Autotransporter with Arginine-Specific Aminopeptidase Activity.
Authors: Erandi Jayawardana Arachchige / Md Shafiqur Rahman / Katharina S Singendonk / Kelly H Kim /
Abstract: AaaA is a virulence-associated outer membrane protein found in the Gram-negative pathogen Pseudomonas aeruginosa. Classified as both an autotransporter and a member of the M28 family of ...AaaA is a virulence-associated outer membrane protein found in the Gram-negative pathogen Pseudomonas aeruginosa. Classified as both an autotransporter and a member of the M28 family of aminopeptidases, AaaA has been shown to cleave N-terminal arginine residues from host-derived peptides. This activity has been demonstrated to enhance bacterial survival and suppress host immune responses by increasing local arginine availability. Here, we report the first successful purification and combined structural and biochemical characterization of full-length AaaA. We resolved its cryo-EM structure at 3.87 Å resolution, revealing the canonical three-domain architecture of autotransporters: a signal peptide, a passenger domain, and a translocator domain. Notably, the passenger domain adopts a compact globular fold characteristic of M28 aminopeptidases, which is less common than the extended or β-helical structures observed in the majority of autotransporters structurally characterized to date. The structure reveals a zinc-coordinated catalytic site and a negatively charged substrate binding pocket, consistent with specificity for positively charged N-terminal arginine residues. Mutagenesis of active site residues confirmed the molecular basis for arginine recognition. Functional assays demonstrated that AaaA exhibits zinc-dependent aminopeptidase activity across a broad pH (6-10) and temperature (20-60 °C) range. Together, these findings provide fundamental insights into the structure and function of AaaA and establish a framework for future efforts to develop targeted inhibitors that may attenuate P. aeruginosa virulence.
History
DepositionMay 20, 2025-
Header (metadata) releaseAug 13, 2025-
Map releaseAug 13, 2025-
UpdateAug 20, 2025-
Current statusAug 20, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70744.png

Downloads & links

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Map

FileDownload / File: emd_70744.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map from the local refinement.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.89 Å/pix.
x 288 pix.
= 255.168 Å		0.89 Å/pix.
x 288 pix.
= 255.168 Å		0.89 Å/pix.
x 288 pix.
= 255.168 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.886 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.7322572 - 1.096185
Average (Standard dev.)0.00019130485 (±0.020041937)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 255.168 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Cryo-EM halfmap2 of the local refinement.

Fileemd_70744_half_map_1.map
AnnotationCryo-EM halfmap2 of the local refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM halfmap1 of the local refinement.

Fileemd_70744_half_map_2.map
AnnotationCryo-EM halfmap1 of the local refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : AaaA complex with Arginine

EntireName: AaaA complex with Arginine
Components
  • Complex: AaaA complex with Arginine
    • Protein or peptide: Autotransporter domain-containing protein
  • Ligand: ZINC ION
  • Ligand: ARGININE

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Supramolecule #1: AaaA complex with Arginine

SupramoleculeName: AaaA complex with Arginine / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: The full length AaaA contain two domains: a globular passenger domain and a beta barrel translocator domain.
Source (natural)Organism: Pseudomonas aeruginosa (bacteria) / Strain: ATCC 15692 / Location in cell: Membrane
Molecular weightTheoretical: 70.36 KDa

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Macromolecule #1: Autotransporter domain-containing protein

MacromoleculeName: Autotransporter domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria) / Strain: ATCC 15692
Molecular weightTheoretical: 72.329414 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: ASAWSHPQFE KGGGSGGGSG GSAWSHPQFE KENLYFQGYQ YGEYAGETLE RLITDYPGRY RGTASFAGAS KLMQSRLGFG YQTSRQDFT WAGNRSSQNV IASAPGSSGK FLVLGAHYDT YYGRPTLQGL DDNASGAAVL TEIARNLGGI ALENGLEVVG F GAEEEGLR ...String:
ASAWSHPQFE KGGGSGGGSG GSAWSHPQFE KENLYFQGYQ YGEYAGETLE RLITDYPGRY RGTASFAGAS KLMQSRLGFG YQTSRQDFT WAGNRSSQNV IASAPGSSGK FLVLGAHYDT YYGRPTLQGL DDNASGAAVL TEIARNLGGI ALENGLEVVG F GAEEEGLR GSRAYVESLD ASQRANLLGM INLDSLVTGD KMYAHAGSNS VSNPALGAYR EQILRIAREL DIPLFTNPGL NA EYPAGTG CCSDGESFNG MDIPVLFIEA TNWELGDLDG YEQTDNPAIP GGSTWHDPAE DNKEVLTNAL GQERIEQRMR DFS RLLTRL VLEQTNADLL ASTASGGALA RQMEDQLQRQ HQALTRLHDR RWLTLLGSNR PVGSFDGEVG AEGEVSPDSG FDMP GNPES RRAGVHLLGD YRYSEALTLG GSLAFQRSRD KLDHGGRIEG DTWQLGLFGL YNDGGPEWLA GELNLGHTRY DSKRS VYLQ AAGGPVLLDQ RLSGDTSAWS WGARLEGGYD FSFGELRSGP LAGLDYMHYR IDDFREDEAL RTALGYEKQD YDSLEA SLG WRLRGELALG ARMRLQPYAS LRWVRELADG RLDDMDLTSR GDGRVRVADM GGVDKDFGRA QLGAQLAITE QLGVFAE AN SRFAHSEGNQ AGYSLGVNWQ F

UniProtKB: Autotransporter domain-containing protein

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: ARGININE

MacromoleculeName: ARGININE / type: ligand / ID: 3 / Number of copies: 1 / Formula: ARG
Molecular weightTheoretical: 175.209 Da
Chemical component information

ChemComp-ARG:
ARGININE

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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

Concentration7.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMSodium ChlorideNaCl
0.05 %n-dodecyl -D-maltoside

Details: During solubilization of protein from membrane, 1% DDM was used. Subsequent purification buffers contain 0.05% DDM.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 1200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.034 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: During vitrification 3 ul of sample placed on a grid and then vitrified with 10s hold and 4s blot time..
DetailsThe protein sample was concentrated from the single peak fractions of size exclusion chromatography.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 77.0 K / Max: 100.0 K
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 7084 / Average electron dose: 44.71 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3009424
Details: Particles picked using topaz train following blob and template picking in cryoSPARC.
CTF correctionSoftware - Name: cryoSPARC (ver. 4.4.1) / Details: Patch CTF in CryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio reconstruction in cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Resolution.type: BY AUTHOR / Resolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1)
Details: Final map reconstructed from local refinement following non-uniform refinement to improve the region of interest excluding the detergent belt in cryoSPARC. Mask of the region of interest ...Details: Final map reconstructed from local refinement following non-uniform refinement to improve the region of interest excluding the detergent belt in cryoSPARC. Mask of the region of interest used to do the local refinement.
Number images used: 241938
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1) / Details: Local refinement following non-uniform refinement.
Final 3D classificationNumber classes: 3 / Avg.num./class: 97882 / Software - Name: RELION (ver. 4.0.1)
Details: A total of 293,645 particles were imported into RELION from cryoSPARC. 3D classification into 3 classes was performed in RELION using a regularization parameter (T) of 100, without image ...Details: A total of 293,645 particles were imported into RELION from cryoSPARC. 3D classification into 3 classes was performed in RELION using a regularization parameter (T) of 100, without image alignment. The best-resolved class was selected for further refinement in cryoSPARC.

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Atomic model buiding 1

Initial modelChain - Residue range: 23-647 / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9oqa:
Cryo-EM structure of AaaA, a Pseudomonas Aeruginosa autotransporter

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