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- EMDB-13670: isolated S-layer of Ca.M.Lanthanididphila -

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Basic information

Entry
Database: EMDB / ID: EMD-13670
Titleisolated S-layer of Ca.M.Lanthanididphila
Map dataSubtomogram average of the Ca.M.lanthanidiphila S-layer obtained from cryo-tomograms of isolated S-layer patches. 6-fold rotational symmetry has been applied.
Sample
  • Organelle or cellular component: isolated S-layer of Ca.M.lanthanidiphila
KeywordsS-layer / STRUCTURAL PROTEIN
Biological speciesCandidatus Methylomirabilis lanthanidiphila (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 21.0 Å
AuthorsGambelli L / Mesman R
Funding support5 items
OrganizationGrant numberCountry
European Research Council (ERC)339880
Netherlands Organisation for Scientific Research (NWO)024002002
European Research Council (ERC)669371
Netherlands Organisation for Scientific Research (NWO)192.001
European Research Council (ERC)803894
CitationJournal: Front Microbiol / Year: 2021
Title: The Polygonal Cell Shape and Surface Protein Layer of Anaerobic Methane-Oxidizing Bacteria.
Authors: Lavinia Gambelli / Rob Mesman / Wouter Versantvoort / Christoph A Diebolder / Andreas Engel / Wiel Evers / Mike S M Jetten / Martin Pabst / Bertram Daum / Laura van Niftrik /
Abstract: bacteria perform anaerobic methane oxidation coupled to nitrite reduction via an intra-aerobic pathway, producing carbon dioxide and dinitrogen gas. These diderm bacteria possess an unusual ... bacteria perform anaerobic methane oxidation coupled to nitrite reduction via an intra-aerobic pathway, producing carbon dioxide and dinitrogen gas. These diderm bacteria possess an unusual polygonal cell shape with sharp ridges that run along the cell body. Previously, a putative surface protein layer (S-layer) was observed as the outermost cell layer of these bacteria. We hypothesized that this S-layer is the determining factor for their polygonal cell shape. Therefore, we enriched the S-layer from cells and through LC-MS/MS identified a 31 kDa candidate S-layer protein, mela_00855, which had no homology to any other known protein. Antibodies were generated against a synthesized peptide derived from the mela_00855 protein sequence and used in immunogold localization to verify its identity and location. Both on thin sections of cells and in negative-stained enriched S-layer patches, the immunogold localization identified mela_00855 as the S-layer protein. Using electron cryo-tomography and sub-tomogram averaging of S-layer patches, we observed that the S-layer has a hexagonal symmetry. Cryo-tomography of whole cells showed that the S-layer and the outer membrane, but not the peptidoglycan layer and the cytoplasmic membrane, exhibited the polygonal shape. Moreover, the S-layer consisted of multiple rigid sheets that partially overlapped, most likely giving rise to the unique polygonal cell shape. These characteristics make the S-layer of a distinctive and intriguing case to study.
History
DepositionOct 4, 2021-
Header (metadata) releaseNov 24, 2021-
Map releaseNov 24, 2021-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.42
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 4.42
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13670.png

Downloads & links

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Map

FileDownload / File: emd_13670.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of the Ca.M.lanthanidiphila S-layer obtained from cryo-tomograms of isolated S-layer patches. 6-fold rotational symmetry has been applied.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.63 Å/pix.
x 240 pix.
= 630.314 Å		2.63 Å/pix.
x 240 pix.
= 630.314 Å		2.63 Å/pix.
x 240 pix.
= 630.314 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.62631 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.42 / Movie #1: 4.42
Minimum - Maximum-17.479994000000001 - 20.831
Average (Standard dev.)1.989011 (±1.5180178)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 630.31445 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.62630833333332.62630833333332.6263083333333
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z630.314630.314630.314
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-17.48020.8311.989

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Supplemental data

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Sample components

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Entire : isolated S-layer of Ca.M.lanthanidiphila

EntireName: isolated S-layer of Ca.M.lanthanidiphila
Components
  • Organelle or cellular component: isolated S-layer of Ca.M.lanthanidiphila

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Supramolecule #1: isolated S-layer of Ca.M.lanthanidiphila

SupramoleculeName: isolated S-layer of Ca.M.lanthanidiphila / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: S-layer was isolated by boiling disrupted flocks of Ca.M.Lanthanidiphila enrichment culture in 4% SDS.
Source (natural)Organism: Candidatus Methylomirabilis lanthanidiphila (bacteria)
Location in cell: on top of outer membrane

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation state2D array

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 20.0 kPa / Details: glow-discharged at 15 mA current
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
Details: 2 microlitre sample was mixed with 0.5 microlitre 10 nm ProteinA gold solution Grids were plunge frozen in liquid ethane, blot force 2, blot time 2.5-3 sec..
Detailsconcentrated isolated S-layer in ultrapure water

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 4.7 µm / Calibrated defocus min: 4.43 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 33000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMax: 93.0 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Average exposure time: 0.3 sec. / Average electron dose: 1.71538 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 2 / Number images used: 11274 / Method: random grid / Software - Name: PEET (ver. 1.14.0)
Final angle assignmentType: NOT APPLICABLE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: OTHER / Software - Name: PEET (ver. 1.14.0)
Details: Resolution of the Ca. M. lanthanidiphila S-layer sub-tomogram average was determined by FFT
Number subtomograms used: 8938

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