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- EMDB-13881: CS-TV2-reconstructed tomogram of a C. crescentus stalk covered by... -

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Basic information

Entry
Database: EMDB / ID: EMD-13881
TitleCS-TV2-reconstructed tomogram of a C. crescentus stalk covered by an S-layer
Map dataCS-TV2 reconstruction of a C. crescentus stalk covered by an S-layer of RsaA hexamers. The tomogram was cropped and rotated to reduce file size. Contrast may require manual adjustment.
Sample
  • Organelle or cellular component: C. crescentus stalk
KeywordsS-layer / C. crescentus / RsaA / STRUCTURAL PROTEIN
Biological speciesCaulobacter vibrioides (bacteria)
Methodelectron tomography / cryo EM
AuthorsBharat TAM / Boehning J / Collins SM
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
CitationJournal: Structure / Year: 2022
Title: Compressed sensing for electron cryotomography and high-resolution subtomogram averaging of biological specimens.
Authors: Jan Böhning / Tanmay A M Bharat / Sean M Collins /
Abstract: Cryoelectron tomography (cryo-ET) and subtomogram averaging (STA) allow direct visualization and structural studies of biological macromolecules in their native cellular environment, in situ. Often, ...Cryoelectron tomography (cryo-ET) and subtomogram averaging (STA) allow direct visualization and structural studies of biological macromolecules in their native cellular environment, in situ. Often, low signal-to-noise ratios in tomograms, low particle abundance within the cell, and low throughput in typical cryo-ET workflows severely limit the obtainable structural information. To help mitigate these limitations, here we apply a compressed sensing approach using 3D second-order total variation (CS-TV) to tomographic reconstruction. We show that CS-TV increases the signal-to-noise ratio in tomograms, enhancing direct visualization of macromolecules, while preserving high-resolution information up to the secondary structure level. We show that, particularly with small datasets, CS-TV allows improvement of the resolution of STA maps. We further demonstrate that the CS-TV algorithm is applicable to cellular specimens, leading to increased visibility of molecular detail within tomograms. This work highlights the potential of compressed sensing-based reconstruction algorithms for cryo-ET and in situ structural biology.
History
DepositionNov 16, 2021-
Header (metadata) releaseJan 26, 2022-
Map releaseJan 26, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing 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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Supplemental images

emd_13881.png

Downloads & links

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Map

FileDownload / File: emd_13881.map.gz / Format: CCP4 / Size: 976 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCS-TV2 reconstruction of a C. crescentus stalk covered by an S-layer of RsaA hexamers. The tomogram was cropped and rotated to reduce file size. Contrast may require manual adjustment.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.47 Å/pix.
x 71 pix.
= 317.157 Å		4.47 Å/pix.
x 1001 pix.
= 4471.467 Å		4.47 Å/pix.
x 3600 pix.
= 16081.2 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: 4.467 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum0.0 - 0.004115206
Average (Standard dev.)0.000043299762 (±0.00010632376)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin578458-124
Dimensions1001360071
Spacing3600100171
CellA: 16081.2 Å / B: 4471.467 Å / C: 317.157 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.4674.4674.467
M x/y/z3600100171
origin x/y/z0.0000.0000.000
length x/y/z16081.2004471.467317.157
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS458578-124
NC/NR/NS3600100171
D min/max/mean0.0000.0040.000

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Supplemental data

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Sample components

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Entire : C. crescentus stalk

EntireName: C. crescentus stalk
Components
  • Organelle or cellular component: C. crescentus stalk

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Supramolecule #1: C. crescentus stalk

SupramoleculeName: C. crescentus stalk / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: C. crescentus stalk, covered by an S-layer of RsaA hexamers.
Source (natural)Organism: Caulobacter vibrioides (bacteria) / Strain: CB15 / Location in cell: Stalk

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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
GridModel: Quantifoil R3.5/1 / Material: COPPER/RHODIUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
DetailsPlease see Bharat et al, Nature Microbiology 2017
Cryo protectantNone
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: CMC Utrecht / Diameter: 10 nm

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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 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 3.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 0.578 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: IMOD
Details: CS-TV2 reconstruction algorithm as described in manuscript was used.
Number images used: 121

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Atomic model buiding 1

RefinementProtocol: OTHER

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