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- EMDB-53363: Cryo-electron tomogram of cryo-FIB milled E. coli cells lacking PBP1a -

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Basic information

Entry
Database: EMDB / ID: EMD-53363
TitleCryo-electron tomogram of cryo-FIB milled E. coli cells lacking PBP1a
Map dataDividing E. coli cell lacking PBP1a. Constriction and septation stage.
Sample
  • Cell: E. coli lacking PBP1a
KeywordsBacteria / Division / Tomography / PBPs / CELL CYCLE
Biological speciesEscherichia coli (E. coli)
Methodelectron tomography / cryo EM
AuthorsNavarro PP / Bernhardt TG
Funding support Switzerland, United States, European Union, 4 items
OrganizationGrant numberCountry
Swiss National Science FoundationTMSGI3_218251 Switzerland
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI083365 United States
Swiss National Science FoundationP500PB_203143 Switzerland
European Molecular Biology Organization (EMBO)ALTF_89-2019European Union
CitationJournal: bioRxiv / Year: 2025
Title: The aPBP-type cell wall synthase PBP1b plays a specialized role in fortifying the division site against osmotic rupture.
Authors: Paula P Navarro / Andrea Vettiger / Roman Hajdu / Virly Y Ananda / Alejandro López-Tavares / Ernst W Schmid / Johannes C Walter / Martin Loose / Luke H Chao / Thomas G Bernhardt
Abstract: A multi-protein system called the divisome promotes bacterial division. This apparatus synthesizes the peptidoglycan (PG) cell wall layer that forms the daughter cell poles and protects them from ...A multi-protein system called the divisome promotes bacterial division. This apparatus synthesizes the peptidoglycan (PG) cell wall layer that forms the daughter cell poles and protects them from osmotic lysis. In the model Gram-negative bacterium , PG synthases called class A penicillin-binding proteins (aPBPs) have been proposed to play crucial roles in division. However, there is limited experimental support for aPBPs playing a specialized role in division that is distinct from their general function in the expansion and fortification of the PG matrix. Here, we present cryogenic electron tomography data indicating that the aPBP-type enzyme PBP1b is required to produce a wedge-like density of PG at the division site. Furthermore, atomic force and live cell microscopy showed that loss of this structure weakens the division site and renders it susceptible to lysis. Surprisingly, we found that the lipoprotein activator LpoB needed to promote the general function of PBP1b was not required for normal division site architecture or its integrity. Additionally, we show that of the two PBP1b isoforms produced in cells, it is the one with an extended cytoplasmic N-terminus that functions in division, likely via recruitment by the FtsA component of the divisome. Altogether, our results demonstrate that PBP1b plays a specialized, LpoB-independent role in cell division involving the biogenesis of a PG structure that prevents osmotic rupture. The conservation of aPBPs with extended cytoplasmic N-termini suggests that other Gram-negative bacteria may use similar mechanisms to reinforce their division site.
History
DepositionApr 9, 2025-
Header (metadata) releaseApr 23, 2025-
Map releaseApr 23, 2025-
UpdateApr 30, 2025-
Current statusApr 30, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53363.png

Downloads & links

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Map

FileDownload / File: emd_53363.map.gz / Format: CCP4 / Size: 41.1 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationDividing E. coli cell lacking PBP1a. Constriction and septation stage.
Voxel sizeX=Y=Z: 21.64 Å
Density
Minimum - Maximum-128.0 - 125.0
Average (Standard dev.)16.117032999999999 (±4.3177333)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-104104-58
Dimensions720511117
Spacing511720117
CellA: 11058.04 Å / B: 15580.8 Å / C: 2531.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Dividing E. coli cell lacking PBP1a. Cytokinesis stage.

Fileemd_53363_additional_1.map
AnnotationDividing E. coli cell lacking PBP1a. Cytokinesis stage.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Dividing E. coli cell lacking PBP1a. Constriction and...

Fileemd_53363_additional_2.map
AnnotationDividing E. coli cell lacking PBP1a. Constriction and septation stage.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Dividing E. coli cell lacking PBP1a. Constriction and...

Fileemd_53363_additional_3.map
AnnotationDividing E. coli cell lacking PBP1a. Constriction and septation stage.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : E. coli lacking PBP1a

EntireName: E. coli lacking PBP1a
Components
  • Cell: E. coli lacking PBP1a

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Supramolecule #1: E. coli lacking PBP1a

SupramoleculeName: E. coli lacking PBP1a / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.5 / Details: Regular LB media
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: glow discharged for 30 seconds at 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
Details: Grids were plunge-frozen in liquid ethane62 with a FEI Vitrobot Mark IV (Thermo Fisher Scientific) at RT, 100 % humidity with a waiting time of 13 seconds, one-side blotting time of 13 ...Details: Grids were plunge-frozen in liquid ethane62 with a FEI Vitrobot Mark IV (Thermo Fisher Scientific) at RT, 100 % humidity with a waiting time of 13 seconds, one-side blotting time of 13 seconds and blotting force of 10. Customized parafilm sheets were used for one-side blotting..
DetailsOC600 = 0.3
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.1 / Focused ion beam - Duration: 600 / Focused ion beam - Temperature: 84 K / Focused ion beam - Initial thickness: 1200 / Focused ion beam - Final thickness: 200
Focused ion beam - Details: The value given for _em_focused_ion_beam.instrument is Aquilos 2. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 8.0 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 7.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 33000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 40

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