Expression, purification, crystallization and preliminary crystallographic analysis of<i>Trypanosoma brucei</i>phosphofructokinase
Bibliographic record
Abstract
Phosphofructokinase from Trypanosoma brucei (TbPFK) was purified from a recombinant expression system in Escherichia coli by metal-affinity chromatography via its N-terminal His tag. The yield was 15-20 mg of pure enzyme per litre of culture. M(r) was shown to be 55 585 by mass spectrometry. Crystals suitable for X-ray diffraction analysis were obtained by the hanging-drop method of vapour diffusion with sodium formate as the precipitating agent. Monoclinic crystals of the apoenzyme grew within one week, as did orthorhombic crystals of PFK in the presence of enzymic reaction products or an active-site inhibitor. Initial attempts to solve the structure by molecular replacement with bacterial PFK structures as search models proved unrewarding, but a multiple-copy search with a polyalanine model was successful. In addition, heavy-atom soaking with platinum and mercury has yielded derivatives suitable for X-ray diffraction. A combination of the phase information from the molecular-replacement solution and the heavy-atom derivatives should allow structure solution of TbPFK. The availability of this first eukaryotic PFK structure will be of particular significance for structure-based drug design and will also provide important additional structural evidence for the allosteric control of PFK activity.
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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) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| 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 | 0.001 |
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".