biostudies-literatureLesovoy DSwedish Foundation for Strategic ResearchRSFCancer och Allergi FondenSwedish Cancer SocietySwedish Research Council8917https://www.ebi.ac.uk/biostudies/studies/S-EPMC12469643biostudies-literatureUnknown26(18)Conformational heterogeneity is essential for protein function, yet validating theoretical molecular dynamics (MD) ensembles remains a significant challenge. In this study, we present an approach that integrates free MD simulations, starting from an AlphaFold-generated structure, with refined experimental NMR-relaxation data to identify biologically relevant holistic time-resolved 4D conformational ensembles. Specifically, we select trajectory segments (RMSD plateaus) consistent with experimental observables. For the extracellular region of <i>Streptococcus pneumoniae</i> PsrSp, we found that only specific segments of the long MD trajectory aligned well with experimental data. The resulting ensembles revealed two regions with increased flexibility, both of which play important functional roles.International journal of molecular sciencesAccurate Protein Dynamic Conformational Ensembles: Combining AlphaFold, MD, and Amide &lt;sup&gt;15&lt;/sup&gt;N(&lt;sup&gt;1&lt;/sup&gt;H) NMR Relaxation.PMC12469643Cancer och Allergi Fonden 10399Swedish Research Council № 2021-05061; 2018-02874; 2023-03485; 2024-06251Swedish Cancer Society 21 1605 Pj 01 HRSF 24-13-00413Swedish Foundation for Strategic Research grant ITM17-0218Sala BMOrekhov VAgback PAchour ARoshchin KSandalova TLesovoy DAgback TfalseAccurate Protein Dynamic Conformational Ensembles: Combining AlphaFold, MD, and Amide &lt;sup&gt;15&lt;/sup&gt;N(&lt;sup&gt;1&lt;/sup&gt;H) NMR Relaxation.Conformational heterogeneity is essential for protein function, yet validating theoretical molecular dynamics (MD) ensembles remains a significant challenge. In this study, we present an approach that integrates free MD simulations, starting from an AlphaFold-generated structure, with refined experimental NMR-relaxation data to identify biologically relevant holistic time-resolved 4D conformational ensembles. Specifically, we select trajectory segments (RMSD plateaus) consistent with experimental observables. For the extracellular region of <i>Streptococcus pneumoniae</i> PsrSp, we found that only specific segments of the long MD trajectory aligned well with experimental data. The resulting ensembles revealed two regions with increased flexibility, both of which play important functional roles.2025-01-01T00:00:00Z2025 Sep2026-05-02T03:19:24.706Z2026-05-02T03:11:45.622ZS-EPMC124696434100948410.3390/ijms26188917