Journal: bioRxiv

Article Title: Sampling Function-Related Metastable States of Proteins With DASH

doi: 10.64898/2025.12.24.696448

Figure Lengend Snippet: a. Problem description and physical function for seed selection. b. An example of sampling the active conformation (orange) from inactive conformation (blue). Upper: change of conformation coverage RMSD to active state along iterations. Lower: change of ensemble along iterations. c. Comparison of minimum relative RMSD (rRMSD) to active state by different sampling methods (DASH, GaMD, REST2 and CMD). d. Comparison of normalized conformational coverage among different sampling methods. In brief, conformations were reduced to 2-D using collective variables (CVs), and the area in 2-D space is defined as the conformational coverage. Three types of CVs were used, including CTMAE-generated CVs, RMSD-Rg and physical CVs (case by case). Outliers were shown in markers, suggesting that GaMD or REST2 sample broader conformations in these cases. Circle: ARF6. Dimond: ARGT. Square: RAG5. Colors of the markers is the same to the corresponding sampling method. e. Comparison of transition barrier between cases that GaMD or REST performs better (orange) and worse (blue). Transition barriers were estimated on three types of CV-space. f. An example of larger conformational coverage but not boosted toward the active site in GaMD and REST2. g. Ablation studies. ADK: Removal of fragmentation. MURD: Removal of marginal structure hopping. ALGQ1: Use CTMAE or AutoEncoder for CV calculations. ARF6: Removal of seed selection. h. Bar plot: Operation time of sampling MURD by DASH (30 round × 10 ns, 1 RTX-3080 Ti), GaMD (500 ns, 1 RTX-3080 Ti) and REST2 (40 replicas × 200 ns, 4 RTX-3080 Ti). Pie plot: Time cost of each part in DASH. i. Orange: Average transition ratios and energy of conformations in transition state (TS) bin (see transition paths in Supplementary information and Methods section). The marker is square if the TS is at the end of the transition path and circle otherwise. Blue: Average transition ratio and energy for the bin whose transition ratio is closest to 0.25 on 10 cases. Right: A proposed mechanism of protein activation. Transition barriers were estimated on three types of CV-space. j. Sampling the active state conformation of SIRT6 from inactive state. Conformations are projected using CVs generated by CTMAE and reweighted by MSM. The contour plot (left) is colored by estimated free energy. The scatter plot is colored by the attack distance (see cartoon plot in the left) related to catalysis. k. comparison of the activator site in input conformation and the sampled conformation. l. Dose-dependent response of activators screened from the sampled conformation. m. Comparison of rankings in virtual screening using different conformations. Error bars represent standard deviations. Statistics analysis are analyzed using Student’s t-test (normal and homo-variance) or Mann-Whitney test (otherwise). N=10.

Article Snippet: Library for screening SIRT6 activators were downloaded from https://www.targetmol.com/compound-library/High_Solubility_Fragment_Library .

Techniques: Selection, Sampling, Comparison, Generated, Marker, Activation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Sampling Function-Related Metastable States of Proteins With DASH

doi: 10.64898/2025.12.24.696448

Figure Lengend Snippet: a. Fragmentation doesn’t affect cases with only one domain moved. b. Top 10 selected compounds by virtual screening on the selected conformation of SIRT6. Docking poses of active compounds (blue) are given. A previous activator (white), MDL-800 is given for location reference.

Article Snippet: Library for screening SIRT6 activators were downloaded from https://www.targetmol.com/compound-library/High_Solubility_Fragment_Library .

Techniques:

Journal: bioRxiv

Article Title: Sampling Function-Related Metastable States of Proteins With DASH

doi: 10.64898/2025.12.24.696448

Figure Lengend Snippet: a. Problem description. b. Illustration of method combination. A further force is applied on helix CV during seed hopping. c,e,g. Free energy surface of KRas4B C-terminal ( c ), LBX2 C-terminal ( e ) and SIRT6 C-terminal ( g ) sampled, all reweighted by MSM. d,f,h. Transition path of helix formation of KRas4B C-terminal ( d ), LBX2 C-terminal ( f ) and SIRT6 C-terminal ( h ). Local minima are labeled by blue dots and the representative structures are shown in cartoon. Transition states are labeled by orange dots and energies are shown. i. Circular dichroism spectrum of KRAS4B C-terminal, LBX2 C-terminal and SIRT6 C-Terminal with different ratio of trifluoroethanol (TFE). j. Relationship between normalized signal (helix degree) and ratio of TFE.

Article Snippet: Library for screening SIRT6 activators were downloaded from https://www.targetmol.com/compound-library/High_Solubility_Fragment_Library .

Techniques: Labeling, Circular Dichroism