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- PDB-9cn0: The cryo-EM structure of BamADE subcomplex from Neisseria gonorrhoeae -

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Basic information

Entry
Database: PDB / ID: 9cn0
TitleThe cryo-EM structure of BamADE subcomplex from Neisseria gonorrhoeae
Components
  • Outer membrane protein assembly factor BamA
  • Outer membrane protein assembly factor BamD
  • Outer membrane protein assembly factor BamE
KeywordsMEMBRANE PROTEIN / beta barrel assembly machinery / BAM complex / outer membrane protein / Neisseria / Neisseria gonnorhoeae / protein folding / beta barrel membrane protein
Function / homology
Function and homology information


establishment of competence for transformation / Bam protein complex / protein insertion into membrane / Gram-negative-bacterium-type cell outer membrane assembly / cell outer membrane / protein-macromolecule adaptor activity
Similarity search - Function
Outer membrane protein assembly factor BamE / Lipoprotein SmpA/OmlA / Outer membrane protein assembly factor BamE domain / Outer membrane protein assembly factor BamD / Outer membrane lipoprotein BamD-like / Outer membrane lipoprotein / BamE-like / Outer membrane protein assembly factor BamA / POTRA domain, BamA/TamA-like / Surface antigen variable number repeat ...Outer membrane protein assembly factor BamE / Lipoprotein SmpA/OmlA / Outer membrane protein assembly factor BamE domain / Outer membrane protein assembly factor BamD / Outer membrane lipoprotein BamD-like / Outer membrane lipoprotein / BamE-like / Outer membrane protein assembly factor BamA / POTRA domain, BamA/TamA-like / Surface antigen variable number repeat / Surface antigen D15-like / POTRA domain / POTRA domain profile. / Bacterial surface antigen (D15) / Omp85 superfamily domain / Prokaryotic membrane lipoprotein lipid attachment site profile. / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
Outer membrane protein assembly factor BamE / Outer membrane protein assembly factor BamD / Outer membrane protein assembly factor BamA
Similarity search - Component
Biological speciesNeisseria gonorrhoeae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.3 Å
AuthorsBillings, E.M. / Noinaj, N.
Funding support United States, 5items
OrganizationGrant numberCountry
American Heart Association909066 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM132024 United States
Simons FoundationSF349247 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129539 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM127884 United States
CitationJournal: Structure / Year: 2025
Title: Structural insights into outer membrane protein biogenesis in pathogenic Neisseria.
Authors: Evan Billings / Zixing Fan / Moloud Aflaki Sooreshjani / James C Gumbart / Nicholas Noinaj /
Abstract: N. gonorrhoeae (Ngo) causes the sexually transmitted infection gonorrhea with ∼106 million infections worldwide annually. Ngo infections can result in an increased risk of acquiring HIV, ...N. gonorrhoeae (Ngo) causes the sexually transmitted infection gonorrhea with ∼106 million infections worldwide annually. Ngo infections can result in an increased risk of acquiring HIV, infertility, and blindness. To combat Ngo infections, we report the cryoelectron microscopy (cryo-EM) structure of the Ngo β-barrel assembly machinery (NgBAM), which is responsible for the biogenesis of β-barrel outer membrane proteins (OMPs). NgBAM was observed in an inward-open state; however, the polypeptide transport-associated (POTRA) domains more closely match those found in the outward-open state in E. coli β-barrel assembly machinery (BAM). The barrel seam of NgBamA consists of partial pairing of strand β1 with β16; no outward-open state of NgBAM was observed. Molecular dynamics (MD) simulations reveal unique overall dynamics and interplay between the POTRA domains of NgBamA and NgBamD. We propose that in Ngo, initial recognition occurs in the inward-open state where the last strand of the OMP partially pairs with β1 of NgBamA and must compete off β16.
History
DepositionJul 15, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 3, 2025Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 3, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Sep 3, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.1Sep 10, 2025Group: Data collection / Database references / 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
A: Outer membrane protein assembly factor BamA
D: Outer membrane protein assembly factor BamD
E: Outer membrane protein assembly factor BamE


Theoretical massNumber of molelcules
Total (without water)135,2763
Polymers135,2763
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Outer membrane protein assembly factor BamA


Mass: 88025.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The endogenous BamA signal sequence was replaced with the pelB signal sequence.
Source: (gene. exp.) Neisseria gonorrhoeae (bacteria) / Strain: FA1090 / Gene: bamA, NGO_1801 / Production host: Escherichia coli (E. coli) / Strain (production host): T7Express / References: UniProt: Q5F5W8
#2: Protein Outer membrane protein assembly factor BamD / Competence lipoprotein ComL


Mass: 31334.385 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The endogenous lipidation signal sequence was replaced with the lipidation sequence from E. coli BamB.
Source: (gene. exp.) Neisseria gonorrhoeae (bacteria) / Strain: FA1090 / Gene: bamD, comL / Production host: Escherichia coli (E. coli) / Strain (production host): T7Express / References: UniProt: Q50985
#3: Protein Outer membrane protein assembly factor BamE


Mass: 15915.229 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The endogenous lipidation signal sequence was replaced with the lipidation signal from E. coli BamB. The C-terminus contains a 10x histidine tag.
Source: (gene. exp.) Neisseria gonorrhoeae (bacteria) / Strain: FA1090 / Gene: smpA, bamE, NCTC11421_03635, WHOF_00597, WHOF_02071 / Production host: Escherichia coli (E. coli) / Strain (production host): T7Express / References: UniProt: A0A1D3EW25
Has protein modificationN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: NgBamADE / Type: COMPLEX
Details: Structure of NgBamADE generated from a subclass of our NgBamACDE dataset.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 128 kDa/nm / Experimental value: NO
Source (natural)Organism: Neisseria gonorrhoeae (bacteria) / Strain: FA1090
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: T7Express
Buffer solutionpH: 7.5 / Details: 1xPBS pH 7.5, with 0.01% LMNG
Buffer component
IDConc.NameFormulaBuffer-ID
1137 mMsodium chlorideNaCl1
22.7 mMpotassium chlorideKCl1
310 mMsodium phosphateNa2HPO41
41.8 mMpotassium phosphateKH2PO41
50.01 %lauryl maltose neopentyl glycolLMNG1
SpecimenConc.: 4.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample used directly after purification over a Superdex200 size exclusion chromatography column.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R3.5/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blot force of 2, blot time of 15 seconds.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 9700 nm / Nominal defocus min: 2230 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.03 sec. / Electron dose: 41.87 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10825
Details: Data was collected at the National Center for Cryo-EM Access and Training, using one of the Titan Krios (Elizabeth).
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansWidth: 11520 / Height: 8184

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.2particle selection
2Leginonimage acquisition
4cryoSPARC3.2CTF correctionpatch motion and CTF correction
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinementReal space refine
12cryoSPARC3.2classification
13cryoSPARC3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4552267
3D reconstructionResolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27691 / Algorithm: FOURIER SPACE / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Chain-ID: A / Pdb chain-ID: A / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeInitial refinement model-ID
14K3B

4k3b

4K3B1
25WAQ

5waq

5WAQ2
35WAM

5wam

5WAM3
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048857
ELECTRON MICROSCOPYf_angle_d0.70311982
ELECTRON MICROSCOPYf_dihedral_angle_d5.3211221
ELECTRON MICROSCOPYf_chiral_restr0.0451299
ELECTRON MICROSCOPYf_plane_restr0.0051567

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