Hierarchical autoclassification of cryo-EM samples and macromolecular energy landscape determination
Bibliographic record
Abstract
BACKGROUND AND OBJECTIVE: Cryo-electron microscopy using single particle analysis is a powerful technique for obtaining 3D reconstructions of macromolecules in near native conditions. One of its major advances is its capacity to reveal conformations of dynamic molecular complexes. Most popular and successful current approaches to analyzing heterogeneous complexes are founded on Bayesian inference. However, these 3D classification methods require the tuning of specific parameters by the user and the use of complicated 3D re-classification procedures for samples affected by extensive heterogeneity. Thus, the success of these approaches highly depends on the user experience. We introduce a robust approach to identify many different conformations presented in a cryo-EM dataset based on Bayesian inference through Relion classification methods that does not require tuning of parameters and reclassification strategies. METHODS: The algorithm allows both 2D and 3D classification and is based on a hierarchical clustering approach that runs automatically without requiring typical inputs, such as the number of conformations present in the dataset or the required classification iterations. This approach is applied to robustly determine the energy landscapes of macromolecules. RESULTS: We tested the performance of the methods proposed here using four different datasets, comprising structurally homogeneous and highly heterogeneous cases. In all cases, the approach provided excellent results. The routines are publicly available as part of the CryoMethods plugin included in the Scipion package. CONCLUSIONS: Our results show that the proposed method can be used to align and classify homogeneous and heterogeneous datasets without requiring previous alignment information or any prior knowledge about the number of co-existing conformations. The approach can be used for both 2D and 3D autoclassification and only requires an initial volume. In addition, the approach is robust to the "attractor" problem providing many different conformations/views for samples affected by extensive heterogeneity. The obtained 3D classes can render high resolution 3D structures, while the obtained energy landscapes can be used to determine structural trajectories.
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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 | 0.004 | 0.012 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.002 |
| Bibliometrics | 0.003 | 0.002 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.002 | 0.002 |
| Open science | 0.003 | 0.002 |
| Research integrity | 0.002 | 0.002 |
| Insufficient payload (model declined to judge) | 0.003 | 0.002 |
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".