The scripts and input files that accompany this demo can be found in the demos/public directory of the Rosetta weekly releases.


Author: Kala Bharath Pilla (kalabharath at gmail dot com)
Protocol Validated by: : JKLeman (julia dot koehler1982 at gmail dot com)
PIs: Gottfied Otting, Thomas Huber (t dot huber at anu dot edu dot au)
Rosetta revision: 56658 from Oct 2014
Publication describing the method:

  • Pilla KB, Koehler Leman J, Otting G, Huber T (2015) Capturing Conformational States in Proteins Using Sparse Paramagnetic NMR Data. PLoS Comp Bio (In preparation)


The method described here is applicable to two-domain proteins, fusion proteins and proteins that exhibit distinct conformational states.where one-of-two domains/part of the structure is always constant and part of the structure which is variable due to induced conformational change. Experimentally Measured Pseudocontact Shifts (Bertini et al) are used as restraints in Rosetta's ab initio executables minirosetta/AbRelax protocol to direct the conformational sampling that fits the experimentally observed PCS data (Schmitz et al).

This protocol particularly uses PCS data from multiple metal centers in a manner analogous to GPS-satellites to accurately position the 3D corrdinates of the atoms, a.k.a GPS-Rosetta (Yagi et al).

Example System

To demonstrate this protocol the two domain (NS2B, NS3pro) Dengue viral protease is taken as an example system. The viral protease exhibits distinct conformational states (Open and closed) as found in crystal structures (PDB IDs: 2FOM, 3U1I). NS3pro remains unchanged, but NS2B adopts closed conformation when bound to a ligand and (PDBID 3U1I) and an open conformation in ligand free form as observed in crystal structure (PDBID: 2FOM)

The Protocol

Different conformational states for NS2B can be preferentially sampled only by changing input PCS data without changing the computational procedure. The protocol is demonstrated for each of the conformational states in seperate folders, namely 'open' & 'closed'.


  1. Pseudocontact shifts for protein backbone atoms from more than 2 metal centers (in '.npc' Numbat format)
  2. Starting model structure in pdb format.
  3. Rosetta's 9 and 3 mer fragment files.


  1. Bertini I, Luchinat C, Parigi G (2002) Magnetic susceptibility in paramagnetic NMR. Prog Nucl Magn Reson Spectrosc 40: 249–273. doi:10.1016/S0079-6565(02)00002-X.
  2. Schmitz C, Vernon R, Otting G, Baker D, Huber T (2012) Protein structure determination from pseudocontact shifts using ROSETTA. J Mol Biol 416: 668–677. doi:10.1016/j.jmb.2011.12.056
  3. Yagi H, Pilla KB, Maleckis A, Graham B, Huber T, et al. (2013) Three-dimensional protein fold determination from backbone amide pseudocontact shifts generated by lanthanide tags at multiple sites. Structure 21: 883–890. doi:10.1016/j.str.2013.04.001.
  4. Schmitz, C., Stanton-Cook, M.J., Su, X-C., Otting, G. and Huber, T. Numbat: an interactive software tool for fitting deltaX-tensors to molecular coordinates using pseudocontact shifts. J. Biomol. NMR (2008) 41 179-189
  5. de la Cruz L, Nguyen THD, Ozawa K, Shin J, Graham B, et al. (2011) Binding of low molecular weight inhibitors promotes large conformational changes in the dengue virus NS2B-NS3 protease: fold analysis by pseudocontact shifts. J Am Chem Soc 133: 19205–19215. doi:10.1021/ja208435s.