High-resolution electron microscopy of energy-transducing proteins of «Escherichia coli»
Bibliographic record
Abstract
Transport of scarce nutrients such as iron and vitamin B12 across the outer membrane of Gram-negative bacteria depends on an integral cytoplasmic membrane protein complex: TonB-ExbB-ExbD. The proton motive force across the cytoplasmic membrane is transduced by this complex to TonB-dependent outer membrane receptors. High resolution structures of the C-terminal periplasmic domains of TonB and ExbD have been solved; structural information is lacking for the membrane-spanning regions of these two proteins and for ExbB. The stoichiometry of the complex, the native organization of TonB-ExbB-ExbD and the molecular mechanism of proton translocation remain undetermined. To investigate properties of ExbB-ExbD from Escherichia coli, we cloned exbB and exbD into different vectors and optimized expression of the recombinant proteins. By immobilized metal affinity chromatography in dodecyl maltoside, ExbB co-purified with His6-tagged ExbD, thereby demonstrating their interactions in vitro. Size-exclusion chromatography identified a protein-detergent complex with a molecular weight of approximately 350 kDa. The molecular weight of His6-tagged ExbB, also purified in dodecyl maltoside, was approximately 270 kDa. Negative-staining electron microscopy (EM) revealed uniform particles approximately 10 nm in diameter for the ExbB-ExbD complex. By low-dose single-particle analysis from high-resolution EM images, 2D averages and 3D reconstructions were generated for the ExbB-ExbD complex and for ExbB alone. A total of 45,321 particles for the ExbB-ExbD complex and 72,327 particles for ExbB alone were collected by a computational single-particle selection system (SIGNATURE). Computational single particle analysis for resolution extension (SPARX) refined the 3D reconstructions. His6-tagged ExbB showed particles approximately 9 nm in diameter and matched pentameric organization for the ExbB-ExbD complex. Preliminary analysis of the ExbB-ExbD complex by cryo-EM confirmed findings by negative-staining EM. Knowing the structural organization of the ExbB-ExbD complex will provide insights into the mechanism of energy-dependent nutrient import in bacteria.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".