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- EMDB-14817: Complex I from E. coli, LMNG-purified, under Turnover at pH 6, Op... -

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Basic information

Entry
Database: EMDB / ID: EMD-14817
TitleComplex I from E. coli, LMNG-purified, under Turnover at pH 6, Open-ready state, MD focused map
Map data
Sample
  • Complex: Complex I
Biological speciesEscherichia coli str. K-12 substr. MC4100 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.38 Å
AuthorsKravchuk V / Kampjut D / Sazanov L
Funding support Austria, 1 items
OrganizationGrant numberCountry
Not funded25541 Austria
CitationJournal: Nature / Year: 2022
Title: A universal coupling mechanism of respiratory complex I.
Authors: Vladyslav Kravchuk / Olga Petrova / Domen Kampjut / Anna Wojciechowska-Bason / Zara Breese / Leonid Sazanov /
Abstract: Complex I is the first enzyme in the respiratory chain, which is responsible for energy production in mitochondria and bacteria. Complex I couples the transfer of two electrons from NADH to quinone ...Complex I is the first enzyme in the respiratory chain, which is responsible for energy production in mitochondria and bacteria. Complex I couples the transfer of two electrons from NADH to quinone and the translocation of four protons across the membrane, but the coupling mechanism remains contentious. Here we present cryo-electron microscopy structures of Escherichia coli complex I (EcCI) in different redox states, including catalytic turnover. EcCI exists mostly in the open state, in which the quinone cavity is exposed to the cytosol, allowing access for water molecules, which enable quinone movements. Unlike the mammalian paralogues, EcCI can convert to the closed state only during turnover, showing that closed and open states are genuine turnover intermediates. The open-to-closed transition results in the tightly engulfed quinone cavity being connected to the central axis of the membrane arm, a source of substrate protons. Consistently, the proportion of the closed state increases with increasing pH. We propose a detailed but straightforward and robust mechanism comprising a 'domino effect' series of proton transfers and electrostatic interactions: the forward wave ('dominoes stacking') primes the pump, and the reverse wave ('dominoes falling') results in the ejection of all pumped protons from the distal subunit NuoL. This mechanism explains why protons exit exclusively from the NuoL subunit and is supported by our mutagenesis data. We contend that this is a universal coupling mechanism of complex I and related enzymes.
History
DepositionApr 22, 2022-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateOct 5, 2022-
Current statusOct 5, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14817.png

Downloads & links

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Map

FileDownload / File: emd_14817.map.gz / Format: CCP4 / Size: 488.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 504 pix.
= 534.24 Å		1.06 Å/pix.
x 504 pix.
= 534.24 Å		1.06 Å/pix.
x 504 pix.
= 534.24 Å

Surface

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Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.1953307 - 0.37006465
Average (Standard dev.)3.5003086e-06 (±0.004640835)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions504504504
Spacing504504504
CellA=B=C: 534.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_14817_half_map_1.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Half map: #2

Fileemd_14817_half_map_2.map
Projections & Slices
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Sample components

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Entire : Complex I

EntireName: Complex I
Components
  • Complex: Complex I

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Supramolecule #1: Complex I

SupramoleculeName: Complex I / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#13
Source (natural)Organism: Escherichia coli str. K-12 substr. MC4100 (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 6
Component:
ConcentrationFormulaName
20.0 mMC6H13NO4SMES
2.0 mMCaCl2calcium chloride
250.0 mMNaClsodium chloride
0.01 %C47H88O22LMNG
0.25 mg/mlC40H80NO8PE. coli lipid extract
0.1 %C32H58N2O7SCHAPS
2.0 mMC21H27N7O14P2NADH
0.8 mMC19H30O4DQ
GridModel: Quantifoil R0.6/1 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.9 nm
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Sampling interval: 5.0 µm / Number grids imaged: 1 / Number real images: 11316 / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
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

Particle selectionNumber selected: 4483166
CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4hea

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.38 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 317174
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

4hea

,

3rko

)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: ML

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