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- EMDB-50582: SOLIST native mouse heart muscle tomogram #2 -

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Entry
Database: EMDB / ID: EMD-50582
TitleSOLIST native mouse heart muscle tomogram #2
Map dataSOLIST native mouse heart muscle tomogram #2
Sample
  • Cell: In situ cryo-electron tomogram of native mouse heart muscle #2
Keywordsmuscle / motor protein / thick filament / thin filament
Biological speciesMus musculus (house mouse)
Methodelectron tomography
AuthorsErdmann PS / Nguyen HTD / Perone G / Klena N / Vazzana R / Kaluthantrige Don F / Silva M / Sorrentino S / Swuec P / Leroux F ...Erdmann PS / Nguyen HTD / Perone G / Klena N / Vazzana R / Kaluthantrige Don F / Silva M / Sorrentino S / Swuec P / Leroux F / Kalebic N / Coscia F
Funding support1 items
OrganizationGrant numberCountry
Other private
Citation
Journal: Structure / Year: 2023
Title: Single-particle cryo-EM analysis of the shell architecture and internal organization of an intact α-carboxysome.
Authors: Sasha L Evans / Monsour M J Al-Hazeem / Daniel Mann / Nicolas Smetacek / Andrew J Beavil / Yaqi Sun / Taiyu Chen / Gregory F Dykes / Lu-Ning Liu / Julien R C Bergeron /
Abstract: Carboxysomes are proteinaceous bacterial microcompartments that sequester the key enzymes for carbon fixation in cyanobacteria and some proteobacteria. They consist of a virus-like icosahedral shell, ...Carboxysomes are proteinaceous bacterial microcompartments that sequester the key enzymes for carbon fixation in cyanobacteria and some proteobacteria. They consist of a virus-like icosahedral shell, encapsulating several enzymes, including ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), responsible for the first step of the Calvin-Benson-Bassham cycle. Despite their significance in carbon fixation and great bioengineering potentials, the structural understanding of native carboxysomes is currently limited to low-resolution studies. Here, we report the characterization of a native α-carboxysome from a marine cyanobacterium by single-particle cryoelectron microscopy (cryo-EM). We have determined the structure of its RuBisCO enzyme, and obtained low-resolution maps of its icosahedral shell, and of its concentric interior organization. Using integrative modeling approaches, we have proposed a complete atomic model of an intact carboxysome, providing insight into its organization and assembly. This is critical for a better understanding of the carbon fixation mechanism and toward repurposing carboxysomes in synthetic biology for biotechnological applications.
#1: Journal: Nature Structural Biology / Year: 2024
Title: SOLIST cryo-tomogram of native left ventricle mouse heart muscle #1
Authors: Nguyen HTD / Perone G / Klena N / Vazzana R / Kaluthantrige Don F / Silva M / Sorrentino S / Swuec P / Leroux F / Kalebic N / Coscia F / Erdmann PS
History
DepositionJun 7, 2024-
Header (metadata) releaseJul 10, 2024-
Map releaseJul 10, 2024-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

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Map

FileDownload / File: emd_50582.map.gz / Format: CCP4 / Size: 256 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSOLIST native mouse heart muscle tomogram #2
Voxel sizeX=Y=Z: 15.62 Å
Density
Minimum - Maximum-56.617854999999999 - 27.153513
Average (Standard dev.)0.40812847 (±1.0222282)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-128
Dimensions512512256
Spacing512512256
CellA: 7997.44 Å / B: 7997.44 Å / C: 3998.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : In situ cryo-electron tomogram of native mouse heart muscle #2

EntireName: In situ cryo-electron tomogram of native mouse heart muscle #2
Components
  • Cell: In situ cryo-electron tomogram of native mouse heart muscle #2

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Supramolecule #1: In situ cryo-electron tomogram of native mouse heart muscle #2

SupramoleculeName: In situ cryo-electron tomogram of native mouse heart muscle #2
type: cell / ID: 1 / Parent: 0 / Details: cross-section of thick and thin filaments
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6 N / Organ: heart / Tissue: muscle

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Experimental details

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Structure determination

Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.5
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
Details: glow-discharged at 30 mA for 30 s (GloQube Plus Glow discharge system, Quorum Technologies)
DetailsSOLIST lamella preparation of native mouse heart muscle
High pressure freezingInstrument: OTHER
Details: Before use, 3 mm type A (100 micrometer well) and type B planchettes were cleaned by sonication for 5 min, 30 s on, 30 s off, 40% Amplitude on a Branson SFX 550. Type B lids were polished ...Details: Before use, 3 mm type A (100 micrometer well) and type B planchettes were cleaned by sonication for 5 min, 30 s on, 30 s off, 40% Amplitude on a Branson SFX 550. Type B lids were polished with 1 micrometer lapping paper and coated with 0.1% soy lecithin dissolved in chloroform.. The value given for _em_high_pressure_freezing.instrument is Leica EM ICE. This is not in a list of allowed values {'OTHER', 'BAL-TEC HPM 010', 'EMS-002 RAPID IMMERSION FREEZER', 'LEICA EM PACT2', 'LEICA EM HPM100', 'LEICA EM PACT'} so OTHER is written into the XML file.
Cryo protectant20% dextran, 5% sucrose
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.03 / Focused ion beam - Duration: 60 / Focused ion beam - Temperature: 80 K / Focused ion beam - Initial thickness: 4000 / Focused ion beam - Final thickness: 200
Focused ion beam - Details: SOLIST procedure. The value given for _em_focused_ion_beam.instrument is Aquilos 2. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 39 / Average electron dose: 3.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
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: eTomo (ver. 4.11.7) / Details: denoised with CryoCare 2.1 / Number images used: 39

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