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- EMDB-53146: Tomogram of a Corynebacterium glutamicum cell-1 -

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Basic information

Entry
Database: EMDB / ID: EMD-53146
TitleTomogram of a Corynebacterium glutamicum cell-1
Map data
Sample
  • Cell: Corynebacterium glutamicum cell
KeywordsS-layer / PS2 / Corynebacterium glutamicum / RIBOSOMAL PROTEIN
Biological speciesCorynebacterium glutamicum ATCC 13058 (bacteria)
Methodelectron tomography / cryo EM
AuthorsIsbilir B / Bharat T
Funding support United Kingdom, France, European Union, 6 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_1201/31 United Kingdom
Wellcome Trust225317/Z/22/Z United Kingdom
Human Frontier Science Program (HFSP)RGY0074/2021 France
European Molecular Biology Organization (EMBO)YIP 2021European Union
Leverhulme TrustPhilip Leverhulme Prize United Kingdom
The Lister Institute of Preventive MedicineLister Prize United Kingdom
CitationJournal: PLoS Biol / Year: 2025
Title: Mapping the ultrastructural topology of the corynebacterial cell surface.
Authors: Buse Isbilir / Anna Yeates / Vikram Alva / Tanmay A M Bharat /
Abstract: Corynebacterium glutamicum is a diderm bacterium extensively used in the industrial-scale production of amino acids. Corynebacteria belong to the bacterial family Mycobacteriaceae, which is ...Corynebacterium glutamicum is a diderm bacterium extensively used in the industrial-scale production of amino acids. Corynebacteria belong to the bacterial family Mycobacteriaceae, which is characterized by a highly unusual cell envelope with an outer membrane consisting of mycolic acids, called mycomembrane. The mycomembrane is further coated by a surface (S-)layer array in C. glutamicum, making this cell envelope highly distinctive. Despite the biotechnological significance of C. glutamicum and biomedical significance of mycomembrane-containing pathogens, ultrastructural and molecular details of its distinctive cell envelope remain poorly characterized. To address this, we investigated the cell envelope of C. glutamicum using electron cryotomography and cryomicroscopy of focused ion beam-milled single and dividing cells. Our cellular imaging allowed us to map the different components of the cell envelope onto the tomographic density. Our data reveal that C. glutamicum has a variable cell envelope, with the S-layer decorating the mycomembrane in a patchy manner. We further isolated and resolved the structure of the S-layer at 3.1 Å-resolution using single particle electron cryomicroscopy. Our structure shows that the S-layer of C. glutamicum is composed of a hexagonal array of the PS2 protein, which interacts directly with the mycomembrane via an anchoring segment containing a coiled-coil motif. Bioinformatic analyses revealed that the PS2 S-layer is sparsely yet exclusively present within the Corynebacterium genus and absent in other genera of the Mycobacteriaceae family, suggesting distinct evolutionary pathways in the development of their cell envelopes. Our structural and cellular data collectively provide a topography of the unusual C. glutamicum cell surface, features of which are shared by many pathogenic and microbiome-associated bacteria, as well as by several industrially significant bacterial species.
History
DepositionMar 14, 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_53146.png

Downloads & links

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Map

FileDownload / File: emd_53146.map.gz / Format: CCP4 / Size: 2.5 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
8.51 Å/pix.
x 450 pix.
= 3830.4 Å		8.51 Å/pix.
x 1440 pix.
= 12257.28 Å		8.51 Å/pix.
x 1022 pix.
= 8699.264 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: 8.512 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-0.18763965 - 3.9812639
Average (Standard dev.)1.6815974 (±0.16040336)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions14401022450
Spacing10221440450
CellA: 8699.264 Å / B: 12257.28 Å / C: 3830.4001 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Corynebacterium glutamicum cell

EntireName: Corynebacterium glutamicum cell
Components
  • Cell: Corynebacterium glutamicum cell

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Supramolecule #1: Corynebacterium glutamicum cell

SupramoleculeName: Corynebacterium glutamicum cell / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Corynebacterium glutamicum ATCC 13058 (bacteria)

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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
GridModel: Quantifoil R1/4 / Material: GOLD / Mesh: 200 / Support film - Material: SILICON DIOXIDE / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
Details: 3.5 microlitre of cell suspension was apllied to Quantifoil R1 4 SiO2 Au 200 mesh grids and plunge frozen into liquid ethane using a Vitrobot Mark IV after wait time of 3 minutes with -7 ...Details: 3.5 microlitre of cell suspension was apllied to Quantifoil R1 4 SiO2 Au 200 mesh grids and plunge frozen into liquid ethane using a Vitrobot Mark IV after wait time of 3 minutes with -7 blot force and 13 seconds of blot time..
Cryo protectant2.5% glycerol
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: 1800 / Focused ion beam - Temperature: 93 K / Focused ion beam - Initial thickness: 700 / Focused ion beam - Final thickness: 150
Focused ion beam - Details: After loading the frozen specimen into the microscope, the grids were sputter coated with metallic platinum (0.1 mbar; 30 mA, 1V, 30 seconds), then mapped using the MAPs ...Focused ion beam - Details: After loading the frozen specimen into the microscope, the grids were sputter coated with metallic platinum (0.1 mbar; 30 mA, 1V, 30 seconds), then mapped using the MAPs software (v. 3.27; ThermoFisher Inc). A coating of organic platinum was then applied via gas injection system following previous reports (Lam & Villa, 2021) for 20 seconds, followed by a final sputter-coat, using the same settings as previously. AutoTEM (v. 2.4.3; ThermoFisher Inc) was set to rough mill the lamellae in three steps with currents of 0.5 nA, 0.3 nA and 0.1 nA and with milling angle of 10 degree. Lamellae polishing was performed at 50 pA and then finally at 30 pA current with a 0.5 degree overtilt to produce lamellae with a final 100-200 nm thickness.. The value given for _em_focused_ion_beam.instrument is TFS Aquilos 2 DualBeam FIB-SEM. 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
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Average exposure time: 0.377 sec. / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 8.0 µm / Calibrated defocus min: 5.0 µm / Calibrated magnification: 42000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 42000
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: ALGEBRAIC (ARTS) / Number images used: 52

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