*Article* **Patterns in Protein Flexibility: A Comparison of NMR "Ensembles", MD Trajectories, and Crystallographic B-Factors**

**Christopher Reinknecht , Anthony Riga, Jasmin Rivera and David A. Snyder \***

Department of Chemistry, College of Science and Health, William Paterson University, 300 Pompton Rd, Wayne, NJ 07470, USA; reinknechtc1@student.wpunj.edu (C.R.); anthony\_riga@aol.com (A.R.); jaz\_x33@yahoo.com (J.R.)

**\*** Correspondence: snyderd@wpunj.edu

**Abstract:** Proteins are molecular machines requiring flexibility to function. Crystallographic B-factors and Molecular Dynamics (MD) simulations both provide insights into protein flexibility on an atomic scale. Nuclear Magnetic Resonance (NMR) lacks a universally accepted analog of the B-factor. However, a lack of convergence in atomic coordinates in an NMR-based structure calculation also suggests atomic mobility. This paper describes a pattern in the coordinate uncertainties of backbone heavy atoms in NMR-derived structural "ensembles" first noted in the development of FindCore2 (previously called Expanded FindCore: DA Snyder, J Grullon, YJ Huang, R Tejero, GT Montelione, *Proteins: Structure, Function, and Bioinformatics* 82 (S2), 219–230) and demonstrates that this pattern exists in coordinate variances across MD trajectories but not in crystallographic B-factors. This either suggests that MD trajectories and NMR "ensembles" capture motional behavior of peptide bond units not captured by B-factors or indicates a deficiency common to force fields used in both NMR and MD calculations.

**Keywords:** Friedman's test; backbone atom coordinate variances and uncertainties; superimposition
