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- PDB-9avi: TolQ inner membrane protein from Acinetobacter baumannii -

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Basic information

Entry
Database: PDB / ID: 9avi
TitleTolQ inner membrane protein from Acinetobacter baumannii
ComponentsTol-Pal system protein TolQ
KeywordsMEMBRANE PROTEIN / inner membrane protein complex / Structural Genomics / Center for Structural Biology of Infectious Diseases / CSBID
Function / homologyTol-Pal system, TolQ / : / bacteriocin transport / MotA/TolQ/ExbB proton channel / MotA/TolQ/ExbB proton channel family / protein import / cell division / plasma membrane / Tol-Pal system protein TolQ
Function and homology information
Biological speciesAcinetobacter baumannii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsQuade, B. / Otwinowski, Z. / Borek, D. / Center for Structural Biology of Infectious Diseases (CSBID)
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)75N93022C00035 United States
Citation
Journal: Sci Adv / Year: 2025
Title: Structural architecture of TolQ-TolR inner membrane protein complex from opportunistic pathogen .
Authors: Elina Karimullina / Yirui Guo / Hanif M Khan / Tabitha Emde / Bradley Quade / Rosa Di Leo / Zbyszek Otwinowski / D Peter Tieleman / Dominika Borek / Alexei Savchenko /
Abstract: Gram-negative bacteria harness the proton motive force (PMF) within their inner membrane (IM) to uphold cell envelope integrity, an indispensable aspect for both division and survival. The IM TolQ- ...Gram-negative bacteria harness the proton motive force (PMF) within their inner membrane (IM) to uphold cell envelope integrity, an indispensable aspect for both division and survival. The IM TolQ-TolR complex is the essential part of the Tol-Pal system, serving as a conduit for PMF energy transfer to the outer membrane. Here we present cryo-electron microscopy reconstructions of TolQ in apo and TolR-bound forms at atomic resolution. The apo TolQ configuration manifests as a symmetric pentameric pore, featuring a transmembrane funnel leading toward a cytoplasmic chamber. In contrast, the TolQ-TolR complex assumes a proton nonpermeable stance, characterized by the TolQ pentamer's flexure to accommodate the TolR dimer, where two protomers undergo a translation-based relationship. Our structure-guided analysis and simulations support the rotor-stator mechanism of action, wherein the rotation of the TolQ pentamer harmonizes with the TolR protomers' interplay. These findings broaden our mechanistic comprehension of molecular stator units empowering critical functions within the Gram-negative bacterial cell envelope.
#1: Journal: bioRxiv / Year: 2024
Title: Structural architecture of TolQ-TolR inner membrane protein complex from opportunistic pathogen .
Authors: Elina Karimullina / Yirui Guo / Hanif M Khan / Tabitha Emde / Bradley Quade / Rosa Di Leo / Zbyszek Otwinowski / D Tieleman Peter / Dominika Borek / Alexei Savchenko
Abstract: Gram-negative bacteria harness the proton motive force (PMF) within their inner membrane (IM) to uphold the integrity of their cell envelope, an indispensable aspect for both division and survival. ...Gram-negative bacteria harness the proton motive force (PMF) within their inner membrane (IM) to uphold the integrity of their cell envelope, an indispensable aspect for both division and survival. The IM TolQ-TolR complex is the essential part of the Tol-Pal system, serving as a conduit for PMF energy transfer to the outer membrane. Here we present cryo-EM reconstructions of TolQ in apo and TolR- bound forms at atomic resolution. The apo TolQ configuration manifests as a symmetric pentameric pore, featuring a trans-membrane funnel leading towards a cytoplasmic chamber. In contrast, the TolQ-TolR complex assumes a proton non-permeable stance, characterized by the TolQ pentamer's flexure to accommodate the TolR dimer, where two protomers undergo a translation-based relationship. Our structure-guided analysis and simulations support the rotor-stator mechanism of action, wherein the rotation of the TolQ pentamer harmonizes with the TolR protomers' interplay. These findings broaden our mechanistic comprehension of molecular stator units empowering critical functions within the Gram-negative bacterial cell envelope.
TEASER: Apo TolQ and TolQ-TolR structures depict structural rearrangements required for cell envelope organization in bacterial cell division.
History
DepositionMar 3, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 16, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Tol-Pal system protein TolQ
B: Tol-Pal system protein TolQ
C: Tol-Pal system protein TolQ
D: Tol-Pal system protein TolQ
E: Tol-Pal system protein TolQ


Theoretical massNumber of molelcules
Total (without water)126,9515
Polymers126,9515
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
Tol-Pal system protein TolQ


Mass: 25390.193 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acinetobacter baumannii (bacteria) / Gene: tolQ / Production host: Escherichia coli (E. coli) / References: UniProt: V5VAS0
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: TolQ inner membrane protein / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Acinetobacter baumannii (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C43(DE3)
Buffer solutionpH: 8
Buffer componentConc.: 50 mM / Name: TRIS
SpecimenConc.: 5.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 50mM Tris pH 8.0; 150mM NaCl; 0.5 mM TCEP
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: PROPANE / Humidity: 100 % / Chamber temperature: 277 K

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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: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1 nm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 4.4 sec. / Electron dose: 100 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 2718
Details: Images were collected as movies, with each movie containing 125 frames. The beam-image shift method, utilizing a 3x3 pattern, was used, with one image per grid hole.

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
12cryoSPARC3D reconstruction
Image processingDetails: Patch motion and patch CTF correction with binning to 0.833 A/pixel.
CTF correctionDetails: Patch motion and patch CTF correction with binning to 0.833 A/pixel.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 472724
Details: Manual picking to generate templates. Template based autopicking to get 472724 particles. Iterative 2D classification to get 246741 particles.
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 215170
Details: Map was post processed using DeepEMHancer to guide refinement. The deposited maps are pre-DeepEMHancer
Num. of class averages: 4 / Symmetry type: POINT
Atomic model buildingSource name: SwissModel / Type: in silico model
RefinementResolution: 3.02→119.95 Å / Cor.coef. Fo:Fc: 0.912 / SU B: 12.521 / SU ML: 0.209 / ESU R: 0.168
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.37114 --
obs0.37114 80832 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 128.73 Å2
Refinement stepCycle: 1 / Total: 1674
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.0131707
ELECTRON MICROSCOPYr_bond_other_d00.0151671
ELECTRON MICROSCOPYr_angle_refined_deg1.4471.6192311
ELECTRON MICROSCOPYr_angle_other_deg1.3331.5683827
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.465215
ELECTRON MICROSCOPYr_dihedral_angle_2_deg21.88922.56478
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.50615294
ELECTRON MICROSCOPYr_dihedral_angle_4_deg9.465158
ELECTRON MICROSCOPYr_chiral_restr0.0740.2227
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.021924
ELECTRON MICROSCOPYr_gen_planes_other0.0030.02400
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it11.05312.412863
ELECTRON MICROSCOPYr_mcbond_other11.04412.399862
ELECTRON MICROSCOPYr_mcangle_it16.89518.671077
ELECTRON MICROSCOPYr_mcangle_other16.88818.6871078
ELECTRON MICROSCOPYr_scbond_it13.60314.719844
ELECTRON MICROSCOPYr_scbond_other13.59514.73845
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other23.10921.2571235
ELECTRON MICROSCOPYr_long_range_B_refined32.4426986
ELECTRON MICROSCOPYr_long_range_B_other32.4426986
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.02→3.098 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.339 6022 -
obs--100 %

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