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- EMDB-27485: Cryo-electron tomogram of cryo-FIB milled E. coli with deletion o... -

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Entry
Database: EMDB / ID: EMD-27485
TitleCryo-electron tomogram of cryo-FIB milled E. coli with deletion of envC and nlpD. Low-passed. Septation stage.
Map dataCryo-electron tomogram of cryo-FIB milled E. coli with deletion of envC and nlpD. Low-passed. Septation stage.
Sample
  • Cell: Dividing E. coli ftsN with deletion of SPOR domain
KeywordsCell division / CELL CYCLE
Biological speciesEscherichia coli (E. coli)
Methodelectron tomography / cryo EM
AuthorsNavarro PP / Vettiger A / Ananda VY / Montero Llopis P / Allolio C / Bernhardt TG / Chao LH
Funding support Switzerland, European Union, United States, 5 items
OrganizationGrant numberCountry
Swiss National Science FoundationP2BSP3_188112 Switzerland
Swiss National Science FoundationP400PB_199252 Switzerland
Swiss National Science FoundationP500PB_203143 Switzerland
European Molecular Biology Organization (EMBO)ALTF_89-2019European Union
Howard Hughes Medical Institute (HHMI) United States
Citation
Journal: Nat Microbiol / Year: 2022
Title: Cell wall synthesis and remodelling dynamics determine division site architecture and cell shape in Escherichia coli.
Authors: Paula P Navarro / Andrea Vettiger / Virly Y Ananda / Paula Montero Llopis / Christoph Allolio / Thomas G Bernhardt / Luke H Chao /
Abstract: The bacterial division apparatus catalyses the synthesis and remodelling of septal peptidoglycan (sPG) to build the cell wall layer that fortifies the daughter cell poles. Understanding of this ...The bacterial division apparatus catalyses the synthesis and remodelling of septal peptidoglycan (sPG) to build the cell wall layer that fortifies the daughter cell poles. Understanding of this essential process has been limited by the lack of native three-dimensional views of developing septa. Here, we apply state-of-the-art cryogenic electron tomography (cryo-ET) and fluorescence microscopy to visualize the division site architecture and sPG biogenesis dynamics of the Gram-negative bacterium Escherichia coli. We identify a wedge-like sPG structure that fortifies the ingrowing septum. Experiments with strains defective in sPG biogenesis revealed that the septal architecture and mode of division can be modified to more closely resemble that of other Gram-negative (Caulobacter crescentus) or Gram-positive (Staphylococcus aureus) bacteria, suggesting that a conserved mechanism underlies the formation of different septal morphologies. Finally, analysis of mutants impaired in amidase activation (ΔenvC ΔnlpD) showed that cell wall remodelling affects the placement and stability of the cytokinetic ring. Taken together, our results support a model in which competition between the cell elongation and division machineries determines the shape of cell constrictions and the poles they form. They also highlight how the activity of the division system can be modulated to help generate the diverse array of shapes observed in the bacterial domain.
#1: Journal: Biorxiv / Year: 2021
Title: Cell wall synthesis and remodeling dynamics determine bacterial division site architecture and cell shape
Authors: Navarro PP / Vettiger A / Ananda VY / Llopis PM / Allolio C / Bernhardt TG / Chao LH
History
DepositionJul 3, 2022-
Header (metadata) releaseSep 14, 2022-
Map releaseSep 14, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27485.png

Downloads & links

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Map

FileDownload / File: emd_27485.map.gz / Format: CCP4 / Size: 252.6 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationCryo-electron tomogram of cryo-FIB milled E. coli with deletion of envC and nlpD. Low-passed. Septation stage.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
20.52 Å/pix.
x 360 pix.
= 7387.2 Å		20.52 Å/pix.
x 720 pix.
= 14774.4 Å		20.52 Å/pix.
x 511 pix.
= 10485.721 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 20.52 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-1528.0 - 937.0
Average (Standard dev.)84.338904999999997 (±62.928710000000002)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-104104-180
Dimensions720511360
Spacing511720360
CellA: 10485.721 Å / B: 14774.4 Å / C: 7387.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Cryo-electron tomogram of cryo-FIB milled E. coli with...

Fileemd_27485_additional_1.map
AnnotationCryo-electron tomogram of cryo-FIB milled E. coli with deletion of envC and nlpD. Septation stage.
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: Cryo-electron tomogram of cryo-FIB milled E. coli with...

Fileemd_27485_additional_2.map
AnnotationCryo-electron tomogram of cryo-FIB milled E. coli with deletion of envC and nlpD. Cytokinesis stage.
Projections & Slices
AxesZYX

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Additional map: Cryo-electron tomogram of cryo-FIB milled E. coli with...

Fileemd_27485_additional_3.map
AnnotationCryo-electron tomogram of cryo-FIB milled E. coli with deletion of envC and nlpD. Constriction stage. Double constriction.
Projections & Slices
AxesZYX

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Histogram

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Additional map: Cryo-electron tomogram of cryo-FIB milled E. coli with...

Fileemd_27485_additional_4.map
AnnotationCryo-electron tomogram of cryo-FIB milled E. coli with deletion of envC. Double constriction.
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Additional map: Cryo-electron tomogram of cryo-FIB milled E. coli with...

Fileemd_27485_additional_5.map
AnnotationCryo-electron tomogram of cryo-FIB milled E. coli with deletion of envC. Cytokinesis stage.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Cryo-electron tomogram of cryo-FIB milled E. coli with...

Fileemd_27485_additional_6.map
AnnotationCryo-electron tomogram of cryo-FIB milled E. coli with deletion of envC and nlpD. Septation stage.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Cryo-electron tomogram of cryo-FIB milled E. coli with...

Fileemd_27485_additional_7.map
AnnotationCryo-electron tomogram of cryo-FIB milled E. coli with deletion of envC and nlpD. Low-passed. Constriction stage.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Cryo-electron tomogram of cryo-FIB milled E. coli with...

Fileemd_27485_additional_8.map
AnnotationCryo-electron tomogram of cryo-FIB milled E. coli with deletion of envC and nlpD. Low-passed. Septation stage.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Cryo-electron tomogram of cryo-FIB milled E. coli with...

Fileemd_27485_additional_9.map
AnnotationCryo-electron tomogram of cryo-FIB milled E. coli with deletion of envC and nlpD. Low-passed. Cytokinesis 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 : Dividing E. coli ftsN with deletion of SPOR domain

EntireName: Dividing E. coli ftsN with deletion of SPOR domain
Components
  • Cell: Dividing E. coli ftsN with deletion of SPOR domain

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Supramolecule #1: Dividing E. coli ftsN with deletion of SPOR domain

SupramoleculeName: Dividing E. coli ftsN with deletion of SPOR domain / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MG1755

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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: 9
GridModel: C-flat-2/2 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 294.15 K / Instrument: FEI VITROBOT MARK IV
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 5 / Focused ion beam - Current: 0.5 / Focused ion beam - Duration: 600 / Focused ion beam - Temperature: 88 K / Focused ion beam - Initial thickness: 1000 / Focused ion beam - Final thickness: 150
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) / Average electron dose: 1.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.5 µm / Nominal defocus min: 3.5 µm / Nominal magnification: 36000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: IMOD / Number images used: 54

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