Limitations of the refolding pipeline for de novo protein design
Open accessPublished version of a preprint
Abstract
With the emergence of powerful deep learning‐based tools, computational protein design has become a widely accessible technique. Nowadays, it is possible to perform both sequence and structure design in a matter of minutes, making the technology attractive to the broader scientific community. In protein design campaigns, one of the most common in silico strategies to evaluate how well a sequence encodes a target structure is the so‐called self‐consistency or refolding pipeline. In this approach, a structure prediction model is used to refold the designed sequence to probe whether it is compatible with the intended structure, and is evaluated via two metrics linked to experimental success: the confidence score of the predicted structure (predicted local distance difference test) and the self‐consistency root‐mean‐square deviation, which measures how closely the refolded structure matches the target. In this work, we systematically evaluate how different models and structure prediction settings impact these metrics, and to what extent they can be used to reliably filter sequence design candidates. We show that evolutionary information can obscure folding models' abilities to assess sequence‐structure compatibility, reducing the predictive performance of refolding metrics for experimental success, particularly for designs that share homology with natural sequences. We further highlight limitations of refolding metrics, including their sensitivity to structural features, such as flexibility. Our findings raise awareness of potential pitfalls in refolding‐based evaluation and support more informed use of these metrics in protein design campaigns.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
Highly accurate protein structure prediction with AlphaFold
Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., et al. (2021) · Nature · doi:10.1038/s41586-021-03819-2 · Cited by 43,764 · see citing works
Evolutionary-scale prediction of atomic level protein structure with a language model
Lin, Z., Akin, H., Rao, R., Hie, B., Zhu, Z., Lu, W., Smetanin, N., Verkuil, R., Kabeli, O., Shmueli, Y., et al. (2022) · doi:10.1101/2022.07.20.500902 · Cited by 5,523 · see citing works
Watson, J. L., Juergens, D., Bennett, N. R., Trippe, B. L., Yim, J., Eisenach, H. E., Ahern, W., Borst, A. J., Ragotte, R. J., Milles, L. F., et al. (2022) · doi:10.1101/2022.12.09.519842 · Cited by 2,369 · see citing works
De novo design of protein structure and function with RFdiffusion
Watson, J. L., Juergens, D., Bennett, N. R., Trippe, B. L., Yim, J., Eisenach, H. E., Ahern, W., Borst, A. J., Ragotte, R. J., Milles, L. F., et al. (2023) · Nature · doi:10.1038/s41586-023-06415-8 · Cited by 2,325 · see citing works
Robust deep learning based protein sequence design using ProteinMPNN
Dauparas, J., Anishchenko, I., Bennett, N., Bai, H., Ragotte, R. J., Milles, L. F., Wicky, B. I. M., Courbet, A., de Haas, R. J., Bethel, N., et al. (2022) · doi:10.1101/2022.06.03.494563 · Cited by 2,107 · see citing works
Robust deep learning–based protein sequence design using ProteinMPNN
Dauparas, J., Anishchenko, I., Bennett, N., Bai, H., Ragotte, R. J., Milles, L. F., Wicky, B. I. M., Courbet, A., de Haas, R. J., Bethel, N., et al. (2022) · Science · doi:10.1126/science.add2187 · Cited by 2,070 · see citing works
A structural biology community assessment of AlphaFold2 applications
Akdel, M., Pires, D. E. V., Pardo, E. P., Jänes, J., Zalevsky, A. O., Mészáros, B., Bryant, P., Good, L. L., Laskowski, R. A., Pozzati, G., et al. (2022) · Nature Structural & Molecular Biology · doi:10.1038/s41594-022-00849-w · Cited by 675 · see citing works
AlphaFold and Implications for Intrinsically Disordered Proteins
Ruff, K. M., & Pappu, R. V. (2021) · Journal of Molecular Biology · doi:10.1016/j.jmb.2021.167208 · Cited by 635 · see citing works
Local fitness landscape of the green fluorescent protein
Sarkisyan, K. S., Bolotin, D. A., Meer, M. V., Usmanova, D. R., Mishin, A. S., Sharonov, G. V., Ivankov, D. N., Bozhanova, N. G., Baranov, M. S., Soylemez, O., et al. (2016) · Nature · doi:10.1038/nature17995 · Cited by 591 · see citing works
Illuminating protein space with a programmable generative model
Ingraham, J. B., Baranov, M., Costello, Z., Barber, K. W., Wang, W., Ismail, A., Frappier, V., Lord, D. M., Ng-Thow-Hing, C., Van Vlack, E. R., et al. (2023) · Nature · doi:10.1038/s41586-023-06728-8 · Cited by 444 · see citing works
Using AlphaFold to predict the impact of single mutations on protein stability and function
Pak, M. A., Markhieva, K. A., Novikova, M. S., Petrov, D. S., Vorobyev, I. S., Maksimova, E. S., Kondrashov, F. A., & Ivankov, D. N. (2023) · PLOS One · doi:10.1371/journal.pone.0282689 · Cited by 322 · cited 1× · see citing works
“These findings extend the widely held view that structure prediction models cannot reliably infer the effects of single mutations on stability or fold (Buel & Walters, 2022; Pak et al., 2023).”
Language models generalize beyond natural proteins
Verkuil, R., Kabeli, O., Du, Y., Wicky, B. I. M., Milles, L. F., Dauparas, J., Baker, D., Ovchinnikov, S., Sercu, T., & Rives, A. (2022) · doi:10.1101/2022.12.21.521521 · Cited by 181 · cited 1× · see citing works
“…2022) and our mutagenesis experiment indicated that AF2 struggles to predict accurate structures of natural sequences in single sequence mode, whereas pMPNN or protein language model designs yield high-confidence models (Dauparas et al., 2022; Goverde et al., 2024; Verkuil et al., 2022).”
De novo protein design by inversion of the <scp>AlphaFold</scp> structure prediction network
Goverde, C. A., Wolf, B., Khakzad, H., Rosset, S., & Correia, B. E. (2023) · Protein Science · doi:10.1002/pro.4653 · Cited by 103 · see citing works
Flagged first, then by citations
- Authors
- Korbeld, K. T., Viliuga, V., & Fürst, M. J. L. J.
- Affiliations
- University of Groningen (NL)
- Stockholm University (SE)
- Corresponding author
- Fürst, Maximilian J. L. J.
- Venue
- Protein Science
- Publisher
- Wiley
- Published
- 2026-05-06
- Volume
- 35
- Issue
- 6
- Pages
- e70613-e70613
- ISSN
- 0961-8368 (ISSN-L)
- 1469-896X
- OpenAlex
- W4417262331
- Cited by
- 5
- References
- 13
- Citations by year
- 2026: 5
- FWCI
- 10.33
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- Protein Science (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
- PubMed (publishedVersion)
- PubMed Central (publishedVersion, cc-by)
- University of Groningen research database (University of Groningen / Centre for Information Technology) (publishedVersion, cc-by)
- MPG.PuRe (Max Planck Society) (submittedVersion, cc-by)
- Funders
- European Commission
- Vetenskapsrådet (2021‐29)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Physical Sciences › Computer Science › Computational Theory and Mathematics › Computational Drug Discovery Methods
- Keywords
- de novo protein design, self-consistency, RMSD, evolutionary information, homology, protein flexibility
- MeSH terms
- Amino Acid Sequence, Models, Molecular, Protein Conformation, Proteins, Protein Engineering, Protein Folding, Computational Biology, Protein Refolding
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
