This page contains a tutorial for protein assignment using solid-state MAS NMR data and CCPNmr Analysis. It is hoped that it will be of use to those who are new to solid-state assignment and/or CCPNmr Analysis.
The tutorial uses data from the SH3 domain of chicken α-spectrin (Castellani et al. 2002; Castellani et al. 2003). The main text begins with a brief familiarisation with CCPNmr Analysis. It then shows how the threonine motifs in this protein can be assigned using spectra of uniformly [13C]-labelled protein and protein labelled using [1,3-13C] and [2-13C]-labelleld glycerol. This is based on the assignment method outlined in Higman et al. 2009. Several_figures provide some background information, e.g. about the glycerol labelling pattern and the 3D spectra used. For further information on CCPNmr Analysis see CCPNmr Analysis 2.x – Basics or the CCPN Wiki Site. The tutorial directory contains the spectra and a CCPNmr Analysis project (Version 2.0) in which the spectra and SH3 sequence have already been incorporated. Just send me a brief e-mail (vad5 (at) leicester.ac.uk) and I can send you a link where you can download the tutorial directory (about 800 Mb in size).
Unpacking the tutorial directory:
Type tar xvzf sh3_tutorial.tgz and the directory will unpack itself into a new sub-directory called sh3. This contains two further sub-directories: sh3_tutorial contains the CCPN project and ucsf contains the spectra used in the project in ucsf format.
Opening the Analysis Project:
After starting Analysis go to Project / Open Project and select the sh3_tutorial project. Analysis will then notice that you have moved directories. It should notice that the relative paths to the spectra are still the same and simply open the project without problems. However, sometimes it is not able to find the correct paths for the spectra in which case it shows a window in which the old paths are highlighted in red. Simply enter the correct path here (/a_path_on_your_computer/sh3/ucsf) and the red highlighting should go away. Close the window and the project should open without any further problems.
F. Castellani, B.-J. van Rossum, A. Diehl, M. Schubert, K. Rehbein and H. Oschkinat (2002) Nature 420 98-102. (Link to Article)
F. Castellani, B.-J. van Rossum, A. Diehl, K. Rehbein and H. Oschkinat (2003) Biochemistry 42 11476-11483. (Link to Article)
V. A. Higman, J. Flinders, M. Hiller, S. Jehle, S. Markovic, S. Fiedler, B.-J. van Rossum and H. Oschkinat (2009) J. Biomol. NMR 44 245-260. (Link to Article)