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- PDB-9i3n: Csu pilus rod type 1 stack -

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Basic information

Entry
Database: PDB / ID: 9i3n
TitleCsu pilus rod type 1 stack
ComponentsCsuA/B
KeywordsCELL ADHESION / pili / fimbriae / Acinetobacter baumannii pili / chaperone-usher pathway / archaic chaperone-usher pili / biofilm / 3D biofilm / adhesion / pathogenesis / pilus antiparallel binding junction
Function / homology: / Spore coat protein U / Spore Coat Protein U domain / Spore Coat Protein U domain / Spore coat protein U/FanG domain-containing protein
Function and homology information
Biological speciesAcinetobacter baumannii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.58 Å
AuthorsMalmi, H. / Pakharukova, N. / Zavialov, A.V.
Funding support Finland, 3items
OrganizationGrant numberCountry
Academy of Finland321762 Finland
Academy of Finland360760 Finland
Sigrid Juselius Foundation2023 Finland
CitationJournal: Nat Commun / Year: 2026
Title: Antiparallel stacking of Csu pili drives Acinetobacter baumannii 3D biofilm assembly.
Authors: Henri Malmi / Natalia Pakharukova / Bindusmita Paul / Minna Tuittila / Irfan Ahmad / Stefan David Knight / Bernt Eric Uhlin / Debnath Ghosal / Anton V Zavialov /
Abstract: Many Gram-negative nosocomial pathogens rely on adhesive filaments, known as archaic chaperone-usher pili, to establish stress- and drug-resistant, multi-layered biofilms. Here, we uncover the ...Many Gram-negative nosocomial pathogens rely on adhesive filaments, known as archaic chaperone-usher pili, to establish stress- and drug-resistant, multi-layered biofilms. Here, we uncover the mechanism by which these pili build three-dimensional (3D) biofilm architectures. In situ analyses of Acinetobacter baumannii biofilms using electron microscopy (EM) reveal an extensive network of ultrathin, flat stacks of archaic Csu pili interconnecting bacterial cells in 3D space. Cryo-EM structures of a single native pilus, pilus pairs, and two types of multi-pilus stacks show that the pili pack into antiparallel sheets, with their rods connected laterally by junctions at their zigzag corners. This antiparallel arrangement ensures that contacts form primarily between pili from interacting cells rather than pili from the same cell. With a remarkably short helical repeat, archaic chaperone-usher pili spontaneously establish a high density of junctions that determines the biofilm's 3D architecture. Our findings may help develop new therapies against multidrug-resistant bacterial infections by targeting pilus-pilus interactions.
History
DepositionJan 23, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 17, 2025Provider: repository / Type: Initial release
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Revision 1.0Dec 17, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.1Feb 18, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.1Feb 18, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A1: CsuA/B
B1: CsuA/B
C1: CsuA/B
D1: CsuA/B
E1: CsuA/B
F1: CsuA/B
G1: CsuA/B
H1: CsuA/B
I1: CsuA/B
J1: CsuA/B
K1: CsuA/B
L1: CsuA/B
M1: CsuA/B
N1: CsuA/B
O1: CsuA/B
P1: CsuA/B
Q1: CsuA/B
R1: CsuA/B
S1: CsuA/B
A2: CsuA/B
B2: CsuA/B
C2: CsuA/B
D2: CsuA/B
E2: CsuA/B
F2: CsuA/B
G2: CsuA/B
H2: CsuA/B
I2: CsuA/B
J2: CsuA/B
K2: CsuA/B
L2: CsuA/B
M2: CsuA/B
N2: CsuA/B
O2: CsuA/B
P2: CsuA/B
Q2: CsuA/B
R2: CsuA/B
S2: CsuA/B
A3: CsuA/B
B3: CsuA/B
C3: CsuA/B
D3: CsuA/B
E3: CsuA/B
F3: CsuA/B
G3: CsuA/B
H3: CsuA/B
I3: CsuA/B
J3: CsuA/B
K3: CsuA/B
L3: CsuA/B
M3: CsuA/B
N3: CsuA/B
O3: CsuA/B
P3: CsuA/B
Q3: CsuA/B
R3: CsuA/B
S3: CsuA/B


Theoretical massNumber of molelcules
Total (without water)915,97057
Polymers915,97057
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein ...
CsuA/B


Mass: 16069.642 Da / Num. of mol.: 57
Source method: isolated from a genetically manipulated source
Details: Csu pilus rods are homopolymers of subunit CsuA/B. Structure depicts three Csu pilus rods forming type 1 stack architecture through antiparallel interactions.
Source: (gene. exp.) Acinetobacter baumannii (bacteria) / Strain: 19096 / Gene: ATCC19606_12570 / Plasmid: pBAD-Csu / Cell (production host): Planktonic bacteria / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): AI / References: UniProt: A0A6F8TDQ5
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Csu pilus rod type 1 stack / Type: COMPLEX
Details: Csu pilus rods are homopolymers of subunit CsuA/B. The sample contains Csu pilus rods self-assembled into type 1 and type 2 stack architectures through antiparallel interactions. The stacks ...Details: Csu pilus rods are homopolymers of subunit CsuA/B. The sample contains Csu pilus rods self-assembled into type 1 and type 2 stack architectures through antiparallel interactions. The stacks accumulate slowly over time as a gel-like substance at the bottom of a sample tube containing purified Csu pili.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 16.05974 kDa/nm / Experimental value: NO
Source (natural)Organism: Acinetobacter baumannii (bacteria) / Strain: 19096 / Cellular location: Outer membrane / Organelle: Outer membrane / Tissue: Planktonic bacteria
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21-AI / Plasmid: pBAD-Csu
Buffer solutionpH: 7.4
Details: Sample is a fraction taken from an ion exchange column elution gradient, so the NaCl concentration may vary.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2170 mMsodium chlorideNaCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Csu pilus rods are homopolymers of subunit CsuA/B. The sample contains Csu pilus rods self-assembled into type 1 and type 2 stack architectures through antiparallel interactions. The stacks ...Details: Csu pilus rods are homopolymers of subunit CsuA/B. The sample contains Csu pilus rods self-assembled into type 1 and type 2 stack architectures through antiparallel interactions. The stacks accumulate slowly over time as a gel-like substance at the bottom of a sample tube containing purified Csu pili.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.79 sec. / Electron dose: 59.848 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7941
Details: Grid squares and holes were selected manually as the pilus stacks were visible in the atlas.
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv4.5.3particle selectionFilament Tracer and Template Picker
2EPUv3.60image acquisition
4cryoSPARCv4.5.3CTF correctionPatch CTF Correction
7UCSF Chimera1.15model fittingUCSF Chimera was used to fit the subunits into the map and adjust their relative angles, and it was also used to adjust map pixel spacing to match model dimensions.
8Coot0.8.9.2 ELmodel fittingCoot was used to adjust side chain and loop positions.
10PHENIX1.20.1_4487:model refinementPhenix was used to fit the type 1 stack model into the map.
11cryoSPARCv4.5.3initial Euler assignmentHomogeneous Refinement and Heterogeneous Refinement
12cryoSPARCv4.5.3final Euler assignment
13cryoSPARCv4.5.3classificationHeterogeneous Refinement
14cryoSPARCv4.5.33D reconstructionLocal Refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 30605819
Details: Two different types of Csu pilus stack architectures were solved from the same particle set. Particle picking failed to distinguish between the two, so the initial number of particles is the ...Details: Two different types of Csu pilus stack architectures were solved from the same particle set. Particle picking failed to distinguish between the two, so the initial number of particles is the same for both stack architectures.
3D reconstructionResolution: 7.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 504940
Details: Local Refinement allowed generating a single map without individual particles flipping and settling into pseudosymmetric orientations.
Symmetry type: POINT
Atomic model buildingB value: 668.1 / Protocol: RIGID BODY FIT / Space: REAL
Details: UCSF Chimera was used to first fit the subunits into the single native pilus rod map (PDB Accession ID: 9I37) and average their relative angles, and it was also used to adjust pixel spacing ...Details: UCSF Chimera was used to first fit the subunits into the single native pilus rod map (PDB Accession ID: 9I37) and average their relative angles, and it was also used to adjust pixel spacing of all maps to match model dimensions. Coot was used to adjust side chain and loop positions. A pair of antiparallel pili with subunit angles adjusted to 3-turns-per-7-subunits symmetry was overlapped with a 2D class to adjust the binding junction angle. Finally, Phenix was used to fit the type 1 stack model into the map.
Atomic model buildingPDB-ID: 7ZL4

7zl4


Accession code: 7ZL4 / Chain residue range: 24-178
Details: The initial model consisted of a single pilus rod fragment of the same type. The initial model in turn is based on a crystal structure of CsuA/Bsc with PDB accession code 6FM5.
Pdb chain residue range: 24-178 / Source name: PDB / Type: experimental model

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