Biophysical characterization and crystallization of the membrane protein Etk
Bibliographic record
Abstract
The inner membrane protein E. coli tyrosine kinase (Etk) is part of a large protein complex that assembles and exports capsular polysaccharide (CPS) in Gram-negative bacteria. Etk interacts with an outer membrane protein channel, YccZ (1), and regulates CPS export through autophosphorylation of a tyrosine cluster in its C-terminal tail. Previous work resulted in the structure of the isolated Etk C-terminal kinase domain (2). In the present study, the full-length protein has been purified and characterized in vitro. When purified in n-dodecyl-β-D-maltoside (DDM), Etk full length retains autophosphorylation activity, but is not suitable for crystallization because it severely aggregates and degrades. Using the main degradation product, a truncation containing the N-terminal domain (interacts with YccZ) and both transmembrane helices was designed. Truncated Etk does not further degrade and exists as a mixture of monomers and dimers when solubilized by five detergents as determined by size-exclusion chromatography and analytical ultracentrifugation. Crystals have been successfully grown when the protein is solubilized in DDM or n-decyl-β-D-maltoside (DM). The most promising crystals (DDM, 0.1 M MES pH 6.0, 1-5% PEG 3000, 20-30% PEG 200) have been reproduced and optimized with the assistance of a colorimetric assay (3). This assay relies on a reaction between 2,6-dimethylphenol, sulfuric acid, and the sugar head group of certain detergents to accurately quantify detergent in crystallization samples with minimal sample loss. Additive screening also revealed that MgCl2 improves crystallization. Optimization of this crystallization condition has significantly improved reproducibility of these crystals, but x-ray diffraction is limited to 6.5 Å. Current work is focused on reproducing and optimizing a second crystallization lead (DM, 0.1 M KH2PO4 pH 7.5, 32% PEG 400, 0.1 M KCl).
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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 | 0.001 |
| 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 | 0.001 | 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".