Cryo-EM of AKAP350 reveals fibrillar clusters and a potential association with DNA
Bibliographic record
Abstract
Abstract Protein kinase A (PKA) is a promiscuous serine/threonine kinase that phosphorylates a broad-spectrum of effectors involved in vital processes such as glucose, glycogen, and lipid metabolism. Its activity is thus tightly controlled by a family of eukaryotic scaffolding proteins known as the A-kinase anchoring proteins (AKAPs) that confine PKA signaling to specific compartments in the cell. AKAP350 (the protein encoded by AKAP9 ) is a massive scaffolding protein that anchors PKA to the Golgi apparatus and the centrosome where it nucleates macromolecular signaling hubs that control microtubule nucleation and dynamics. Here, we have expressed and purified full-length AKAP350 from HEK293F cells in a functional conformation. Electron cryo-microscopy (cryo-EM) of the purified protein revealed polydisperse particles forming fibrillar clusters around 50 nm in diameter, and long, thin filaments that reconstructed into double-stranded DNA. Tomographic reconstruction of a tilt series of the purified protein by electron cryo-tomography (cryo-ET) further elucidated these fibrillar clusters as 3D bundles of entangled filaments. Mass spectrometry and DNA sequencing confirmed the co-purification of DNA and DNA binding proteins such as nuclear factor 1 B (NFIB) and nucleolin (NCL). Pulldown of NFIB and NCL, but not of CEP290, CDK5RAP2, and CEP170 was diminished in the presence of DNase-I, suggesting that AKAP350 interaction with these two proteins is mediated by DNA. Overall, this study has achieved a quality purification of AKAP350 from which a previously uncharacterized interaction landscape with DNA and DNA binding proteins was discovered.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| 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 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 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 teacher head, 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".