Ignacio Sanchez

Christian Landry

eLife assessment
This work presents the application of protein language models in combination with current methods for the detection of distant evolutionary relationships. While the results are important, they are supported by incomplete, preliminary evidence. A more comprehensive benchmark and clarification of technical details may turn this exploratory study into an algorithm ready for wide use.

anonymous

Reviewer #1 (Public Review):
Summary:
This work describes a new method for sequence-based remote homology detection. Such methods are essential for the annotation of uncharacterized proteins and for studies of protein evolution.
Strengths:
The main strength and novelty of the proposed approach lies in the idea of combining state-of-the-art sequence-based (HHpred and HMMER) and structure-based (Foldseek) homology detection methods with recent developments in the field of protein language models (the ESM2 model was used). The authors show that features extracted from high-dimensional, information-rich ESM2 sequence embeddings can be suitable for efficient use with the aforementioned tools.
The reduced features take the form of amino acid occurrence probability matrices estimated from ESM2 masked-token predictions, or structural descriptors predicted by a modified variant of the ESM2 model. However, we believe that these should not be called "embeddings" or "representations". This is because they don't come directly from any layer of these networks, but rather from their final predictions.
The benchmarks presented suggest that the approach improves sensitivity even at very low sequence identities &lt;20%. The method is also expected to be faster because it does not require the computation of multiple sequence alignments (MSAs) for profile calculation or structure prediction.
Weaknesses:
The benchmarking of the method is very limited and lacks comparison with other methods. Without additional benchmarks, it is impossible to say whether the proposed approach really allows remote homology detection and how much improvement the discussed method brings over tools that are currently considered state-of-the-art.

Reviewer #2 (Public Review):
Summary:
The authors present a number of exploratory applications of current protein representations for remote homology search. They first fine-tune a language model to predict structural alphabets from sequence and demonstrate using these predicted structural alphabets for fast remote homology search both on their own and by building HMM profiles from them. They also demonstrate the use of residue-level language model amino acid predicted probabilities to build HMM profiles. These three implementations are compared to traditional profile-based remote homology search.
Strengths:
- Predicting structural alphabets from a sequence is novel and valuable, with another approach (ProstT5) also released in the same time frame further demonstrating its application for the remote homology search task. 
- Using these new representations in established and battle-tested workflows such as MMSeqs, HMMER, and HHBlits is a great way to allow researchers to have access to the state-of-the-art methods for their task. 
- Given the exponential growth of data in a number of protein resources, approaches that allow for the preparation of searchable datasets and enable fast search is of high relevance.
Weaknesses:
- The authors fine-tuned ESM-2 3B to predict 3Di sequences and presented the fine-tuned model ESM-2 3B 3Di with a claimed accuracy of 64% compared to a test set of 3Di sequences derived from AlphaFold2 predicted structures. However, the description of this test set is missing, and I would expect repeating some of the benchmarking efforts described in the Foldseek manuscript as this accuracy value is hard to interpret on its own. 
- Given the availability of predicted structure data in AFDB, I would expect to see a comparison between the searches of predicted 3Di sequences and the "true" 3Di sequences derived from these predicted structures. This comparison would substantiate the innovation claimed in the manuscript, demonstrating the potential of conducting new searches solely based on sequence data on a structural database. 
- The profile HMMs built from predicted 3Di appear to perform sub-optimally, and those from the ESM-2 3B predicted probabilities also don't seem to improve traditional HMM results significantly. The HHBlits results depicted in lines 5 and 6 in the figure are not discussed at all, and a comparison with traditional HHBlits is missing. With these results and presentation, the advantages of pLM profile-based searches are not clear, and more justification over traditional methods is needed. 
- Figure 3 and its associated text are hard to follow due to the abundance of colors and abbreviations used. One figure attempting to explain multiple distinct points adds to the confusion. Suggestion: Splitting the figure into two panels comparing (A) Foldseek-derived searches (lines 7-10) and (B) language-model derived searches (line 3-6) to traditional methods could enhance clarity. Different scatter markers could also help follow the plots more easily. 
- The justification for using Foldseek without amino acids (3Di-only mode) is not clear. Its utility should be described, or it should be omitted for clarity. 
- Figure 2 is not described, unclear what to read from it.

Sensitive remote homology search by local alignment of small positional embeddings from protein language models

Figures and data

Schematics of embedding models and the experimental design.

Logos related to the example test sequence YBGC_HELPY__14-90 from the 4HBT family.

Homology detection accuracy.

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