Unavailability of experimental 3D structural data on protein folding dynamics and necessity for a new generation of structure prediction methods in this context

Aydin Wells¹, Khalique Newaz², Jennifer Morones¹, Jianlin Cheng³, Tijana Milenković^1,*

¹Department of Computer Science and Engineering, University of Notre Dame, USA
²Institute for Computational Systems Biology, University of Hamburg, Germany
³Department of Electrical Engineering and Computer Science, University of Missouri, Columbia, USA
^*Corresponding author (contact: tmilenko@nd.edu)

This paper is currently under review. Also, it is submitted to arxiv (details to be added).

1. The existing studies with experimentally determined 3D structures of protein folding intermediates

This section lists the studies that our literature search has identified which report 3D structures of experimentally identified post-translational or co-translational folding intermediates. We provide direct links to the published studies and the PDB entries for the structures of intermediates used in our analyses.

• Post-translational intermediates (Section 2.1 in the main paper):
  • Neudecker et al. (2012): Fyn SH3 domain
    • PDB IDs: 2L2P (intermediate 1), 2L25 (intermediate 2)
  • Zhou et al. (2008): RNase H
    • PDB IDs: 2RPI (intermediate 1), 1RIL (intermediate 2)
• Co-translational intermediates (Section 2.2 in the main paper):
  • Agirrezabala et al. (2022): Cold shock protein A (CspA)
    • PDB IDs: 7NWW (intermediate 1a), 7OIF (intermediate 1b), 7OIG (intermediate 1c), 7OT5 (intermediate 2a), 7OII (intermediate 2b), 1MJC (intermediate 2c)
  • Hanazono et al. (2018): Lambda repressor
    • PDB IDs: 5ZCA (intermediate 1), 3WOA (intermediate 1), 1LMB (intermediate 1)
  • Hanazono et al. (2016): WW-domain
    • PDB IDs: 5B3X (intermediate 1), 5BMY (intermediate 2), 5B3Y (intermediate 3), 5B3Z (intermediate 4)
  • Cabrita et al. (2016): FLN5+FLN6
    • PDB IDs: 6G4A (intermediate 1), 2N62 (intermediate 2)

2. The existing studies on computational prediction of 3D structures of protein folding intermediates

Here we highlight the computational studies on 3D structural prediction/analysis of intermediates that we discuss in our paper.

• Outeiral et al. (2023) (Section 3.1 in the main paper)
• Huang et al. (2023) (Section 3.1 in the main paper)
• Wang et al. (2025) (Section 4 in the main paper)

3. The existing software that we use in our paper

This section links to the external software tools and resources used in our analyses.

• TM-score web server (Section 2 and later in the main paper)
• AlphaFold2 (ColabFold) (Section 3.2.1 in the main paper)
• "Proxy" intermediates (Section 3.2.2 in the main paper)
• The original paper on "proxy" intermediates: Newaz et al. (2022)

4. Download for our predicted 3D structures of intermediates

You can click here to download (.zip) the AlphaFold2-predicted co-translational intermediates for all four considered proteins, as well as the "proxy" co-translational intermediates for two of the four considered proteins, as discussed in our paper.