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- EMDB-15056: In situ cryo-electron tomogram of a T. kivui cell 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-15056
TitleIn situ cryo-electron tomogram of a T. kivui cell 2
Map dataCryo-electron tomogram of a T. kivui cell showing HDCR filament bundles. Tomogram was denoised using CRYO-CARE.
Sample
  • Cell: T. kivui cell
Biological speciesThermoanaerobacter kivui (bacteria)
Methodelectron tomography / cryo EM
AuthorsDietrich HM / Righetto RD / Kumar A / Wietrzynski W / Schuller SK / Trischler R / Wagner J / Schwarz FM / Engel BD / Mueller V / Schuller JM
Funding support Germany, European Union, 5 items
OrganizationGrant numberCountry
German Research Foundation (DFG)SCHU 3364/1-1 Germany
European Research Council (ERC)741791European Union
German Research Foundation (DFG)FOR 2092 Germany
Alexander von Humboldt Foundation Germany
German Research Foundation (DFG)20016/446 Germany
CitationJournal: Nature / Year: 2022
Title: Membrane-anchored HDCR nanowires drive hydrogen-powered CO fixation.
Authors: Helge M Dietrich / Ricardo D Righetto / Anuj Kumar / Wojciech Wietrzynski / Raphael Trischler / Sandra K Schuller / Jonathan Wagner / Fabian M Schwarz / Benjamin D Engel / Volker Müller / Jan M Schuller /
Abstract: Filamentous enzymes have been found in all domains of life, but the advantage of filamentation is often elusive. Some anaerobic, autotrophic bacteria have an unusual filamentous enzyme for CO ...Filamentous enzymes have been found in all domains of life, but the advantage of filamentation is often elusive. Some anaerobic, autotrophic bacteria have an unusual filamentous enzyme for CO fixation-hydrogen-dependent CO reductase (HDCR)-which directly converts H and CO into formic acid. HDCR reduces CO with a higher activity than any other known biological or chemical catalyst, and it has therefore gained considerable interest in two areas of global relevance: hydrogen storage and combating climate change by capturing atmospheric CO. However, the mechanistic basis of the high catalytic turnover rate of HDCR has remained unknown. Here we use cryo-electron microscopy to reveal the structure of a short HDCR filament from the acetogenic bacterium Thermoanaerobacter kivui. The minimum repeating unit is a hexamer that consists of a formate dehydrogenase (FdhF) and two hydrogenases (HydA2) bound around a central core of hydrogenase Fe-S subunits, one HycB3 and two HycB4. These small bacterial polyferredoxin-like proteins oligomerize through their C-terminal helices to form the backbone of the filament. By combining structure-directed mutagenesis with enzymatic analysis, we show that filamentation and rapid electron transfer through the filament enhance the activity of HDCR. To investigate the structure of HDCR in situ, we imaged T. kivui cells with cryo-electron tomography and found that HDCR filaments bundle into large ring-shaped superstructures attached to the plasma membrane. This supramolecular organization may further enhance the stability and connectivity of HDCR to form a specialized metabolic subcompartment within the cell.
History
DepositionMay 29, 2022-
Header (metadata) releaseJul 6, 2022-
Map releaseJul 6, 2022-
UpdateAug 10, 2022-
Current statusAug 10, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15056.png

Downloads & links

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Map

FileDownload / File: emd_15056.map.gz / Format: CCP4 / Size: 1.5 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-electron tomogram of a T. kivui cell showing HDCR filament bundles. Tomogram was denoised using CRYO-CARE.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
14.08 Å/pix.
x 464 pix.
= 6533.12 Å		14.08 Å/pix.
x 928 pix.
= 13066.24 Å		14.08 Å/pix.
x 928 pix.
= 13066.24 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: 14.08 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-5.958432 - 4.309724
Average (Standard dev.)0.00025374562 (±0.09051963)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions928928464
Spacing928928464
CellA: 13066.24 Å / B: 13066.24 Å / C: 6533.12 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Cryo-electron tomogram of a T. kivui cell showing...

Fileemd_15056_additional_1.map
AnnotationCryo-electron tomogram of a T. kivui cell showing HDCR filament bundles. Original reconstruction from IMOD.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : T. kivui cell

EntireName: T. kivui cell
Components
  • Cell: T. kivui cell

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Supramolecule #1: T. kivui cell

SupramoleculeName: T. kivui cell / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Thermoanaerobacter kivui (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
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.03 nA / Focused ion beam - Duration: 1800 sec. / Focused ion beam - Temperature: 91 K / Focused ion beam - Initial thickness: 2000 nm / Focused ion beam - Final thickness: 150 nm
Focused ion beam - Details: See https://bio-protocol.org/e1575 for detailed procedure.. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Aquilos FIB. This is not in a list of ...Focused ion beam - Details: See https://bio-protocol.org/e1575 for detailed procedure.. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Aquilos FIB. 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

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
DetailsDose-symmetric tilt-series were acquired (Hagen et al., 2017), starting at +10 degrees to match the pre-tilt of the lamella (i.e. from -50 to +70 degrees).
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.5 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: BACK PROJECTION / Software - Name: IMOD / Number images used: 60
CTF correctionSoftware: (Name: CTFFIND (ver. 4), IMOD, NOVACTF)

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