Solid-state NMR measurement at cryogenic temperature shows significant potential for biological analysis due to its advantage in sample stability and detection sensitivity. However, the commonly observed lineshape broadening results in low spectra resolution and limits the application in complex protein systems. Here, we explored the hypotheses for the broad linewidth at low temperatures using E. coli Dihydrofolate reductase (DHFR). Our results support the contribution of conformational heterogeneity by measuring the protein backbone torsion angle (Ψ) at 105 K. In a selectively enriched protein sample with only one amide 13C’-15N correlation expected, we identified three different conformations with distinct N chemical shift values accounting for the broadening observed in low temperature NMR spectra. The backbone torsion angle fluctuations on picosecond timescale at room temperature were connected with the ‘frozen out’ conformations at cryogenic temperature via MD simulations. The QM/MM predicted chemical shifts based on room temperature MD samplings show excellent agreement with the low temperature spectra, revealing the effect of protein conformational dynamics on NMR chemical shifts.