Energy-transducing proteins of «Escherichia coli»: lipid-based crystallization trials
Bibliographic record
Abstract
Gram-negative bacteria such as Escherichia coli possess a cell envelope composed of an outer membrane (OM) and a cytoplasmic membrane (CM). Widespread antibiotic resistance among these bacteria has necessitated the elucidation of novel therapeutic targets. Scarce nutrients such as iron and vitamin B12 cross the OM by energy-dependent transport. Since the OM lacks a source of energy, bacteria couple the energized CM to drive this process. The TonB-ExbB-ExbD integral membrane protein complex in the CM harnesses the proton gradient and transduces this energy to OM receptors. Despite decades of informative genetic and biochemical research, limited structural and functional information is available on this complex. In particular, only partial structures of the periplasmic domains of TonB and ExbD are known and all structural information is lacking for ExbB, considered to be the scaffold protein of the complex. Furthermore, the stoichiometry of the complex and the putative proton translocation pathway remain controversial. To gain insight into the molecular mechanisms of bacterial scarce nutrient transport, we produced, purified and characterized the ExbB and ExbD proteins before initiating lipid-based crystallization trials. The proteins were produced in milligram amounts and purified as a complex. The complex was found to be ~400 kDa and monodisperse by analytical size exclusion chromatography (SEC). Nuclear magnetic resonance-based detergent quantitation identified excess detergent that concentrated as a function of protein concentration. This led to greater apparent molecular weight of the complex by SEC. The excess detergent was able to be diminished by adsorbent beads or exchanged to a related, dialyzable detergent. Bicelle and in meso crystallization trials were initiated with purified ExbB-ExbD complexes, with promising leads identified. Optimization is ongoing to develop X-ray diffraction-quality crystals of ExbB-ExbD. These preliminary successes have formed the basis for the pursuit of ExbB-ExbD complex structure determination by lipid-based crystallization, a key to understand scarce nutrient import in Gram-negative 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 | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.001 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
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".