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- EMDB-11124: Cryo-electron tomogram after FIB-milling of Tuwongella immobilis,... -

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Basic information

Entry
Database: EMDB / ID: EMD-11124
TitleCryo-electron tomogram after FIB-milling of Tuwongella immobilis, a species of the Planctomycetes phylum (high magnification, 33 000x)
Map dataCryo-electron tomogram after FIB-milling of Tuwongella immobilis, a Planctomycetes species
Sample
  • Cell: Tuwongella immobilis, whole cell cryo-electron tomogram after FIB-milling.
Biological speciesTuwongella immobilis (bacteria)
Methodelectron tomography / cryo EM
AuthorsSeeger C / Andersson SGE
Funding support Sweden, 3 items
OrganizationGrant numberCountry
Swedish Research Council349-2007-8732 Sweden
Knut and Alice Wallenberg Foundation2011.0148 Sweden
Swedish Research Council621-2014-4460 Sweden
CitationJournal: Genome Biol Evol / Year: 2020
Title: Evolutionary Remodeling of the Cell Envelope in Bacteria of the Planctomycetes Phylum.
Authors: Mayank Mahajan / Christian Seeger / Benjamin Yee / Siv G E Andersson /
Abstract: Bacteria of the Planctomycetes phylum have many unique cellular features, such as extensive membrane invaginations and the ability to import macromolecules. These features raise intriguing questions ...Bacteria of the Planctomycetes phylum have many unique cellular features, such as extensive membrane invaginations and the ability to import macromolecules. These features raise intriguing questions about the composition of their cell envelopes. In this study, we have used microscopy, phylogenomics, and proteomics to examine the composition and evolution of cell envelope proteins in Tuwongella immobilis and other members of the Planctomycetes. Cryo-electron tomography data indicated a distance of 45 nm between the inner and outer membranes in T. immobilis. Consistent with the wide periplasmic space, our bioinformatics studies showed that the periplasmic segments of outer-membrane proteins in type II secretion systems are extended in bacteria of the order Planctomycetales. Homologs of two highly abundant cysteine-rich cell wall proteins in T. immobilis were identified in all members of the Planctomycetales, whereas genes for peptidoglycan biosynthesis and cell elongation have been lost in many members of this bacterial group. The cell wall proteins contain multiple copies of the YTV motif, which is the only domain that is conserved and unique to the Planctomycetales. Earlier diverging taxa in the Planctomycetes phylum contain genes for peptidoglycan biosynthesis but no homologs to the YTV cell wall proteins. The major remodeling of the cell envelope in the ancestor of the Planctomycetales coincided with the emergence of budding and other unique cellular phenotypes. The results have implications for hypotheses about the process whereby complex cellular features evolve in bacteria.
History
DepositionJun 4, 2020-
Header (metadata) releaseAug 19, 2020-
Map releaseAug 19, 2020-
UpdateOct 14, 2020-
Current statusOct 14, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Movie viewer
Supplemental images

emd_11124.png

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Map

FileDownload / File: emd_11124.map.gz / Format: CCP4 / Size: 5.2 GB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationCryo-electron tomogram after FIB-milling of Tuwongella immobilis, a Planctomycetes species
Voxel sizeX=Y=Z: 4.37 Å
Density
Minimum - Maximum-20 - 11
Average (Standard dev.)-3.8185735 (±1.6227262)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-151515
Dimensions37103838391
Spacing38383710391
CellA: 16772.059 Å / B: 16212.699 Å / C: 1708.6699 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z4.36999973944764.36999973045824.37
M x/y/z38383710391
origin x/y/z0.0000.0000.000
length x/y/z16772.05916212.6991708.670
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS15-1515
NC/NR/NS38383710391
D min/max/mean-20.00011.000-3.819

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Supplemental data

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Sample components

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Entire : Tuwongella immobilis, whole cell cryo-electron tomogram after FIB...

EntireName: Tuwongella immobilis, whole cell cryo-electron tomogram after FIB-milling.
Components
  • Cell: Tuwongella immobilis, whole cell cryo-electron tomogram after FIB-milling.

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Supramolecule #1: Tuwongella immobilis, whole cell cryo-electron tomogram after FIB...

SupramoleculeName: Tuwongella immobilis, whole cell cryo-electron tomogram after FIB-milling.
type: cell / ID: 1 / Parent: 0
Details: Whole cell tomogram of Tuwongella immobilis revealing the invaginated cytoplasmic membrane, which is characteristic for many members of the phylum Planctomycetes.
Source (natural)Organism: Tuwongella immobilis (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: 7.4 / Component - Concentration: 10.0 mmol/L / Component - Name: Na-phosphate
Details: 10 mM Na-phosphate buffer, pH 7.4, 0.2 microm filtered. DO NOT use salt or any other components that increase osmolarity!
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
Details: Blot time: 6s, blot force: -5, wait time: 15s, 293K, 100% humidity.
DetailsBacterial cells grown on M1 agar plates at 32degrees celsius and kept at 20degress celsius until vitrification
Cryo protectantNo
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 93.0 K
Specialist opticsEnergy filter - Slit width: 20 eV
DetailsBefore tilt series acquisition, cryo focused ion beam (FIB) milling with gallium ion source was performed on a FEI Dual beam Scios (Thermo Fisher Scientific, Netherlands). Autogrids with vitrified cells were mounted onto a dedicated cryo holder and transferred onto a cooled (93K) cryo stage in the dual beam microscope. The lamellae were prepared with two parallel rectangular patterns at both sides (top and bottom) of the cells, at milling angle of 10-15deg, accelerating voltage of 30 kV and ion currents between 30-300 pA. The final thickness of the lamella (milled at the lowest ion current) was about 150-300 nm.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 61 / Average electron dose: 1.64 e/Å2
Details: Total dose tilt series: 100e-/angstrom; number of tilts: 61; dose per tilt: 1.64 e-/angstrom: motion correction for the acquired tilt series was performed by using motioncor2 of the IMOD package
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 33000 / Illumination mode: OTHER / Imaging mode: OTHER / 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 (ver. 4.9.10) / Number images used: 61

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