The NMR SPECTROSCOPY Team was headed by John Markley, PhD, and was responsible for NMR condition optimization, data collection and processing, and structure determination. This group took advantage of the facilities available at the National Magnetic Resonance Facility at Madison (NMRFAM) and the Medical College of Wisconsin (MCW).
The Teams' streamlined approach employed cryogenic probes and automated analysis to rapidly and efficiently determine three-dimensional protein structures by NMR. For proteins of up to 25 kDa in effective molecular weight that were soluble (> 0.5 mM), folded and stable, we acquire a complete data set consisting of 17 2D and 3D experiments in 10–14 days. Time-domain data were processed with NMR Pipe program, and converted to the XEASY format.
Data analysis was carried out in a semi-automated manner using software from various academic sources. Signals in all 3D experiments were detected automatically and integrated using the SPSCAN program. GARANT or PINE was used for automated assignment of backbone and chemical shifts, and side chain assignments are completed manually in XEASY or CARA. Backbone torsion angle restraints were predicted empirically from chemical shift values using the TALOS package and included in the initial round of structure calculations.
Initial protein structures were generated using an iterative and fully automatic methodology for assignment of NOESY cross peaks provided by the NOEASSIGN module of CYANA. The final stages of structure refinement were accomplished through manual optimization of NOE assignments, NOE intensity-to-distance calibration functions, and backbone torsion angle restraints. Torsion angle dynamics structures that meet a set of objective criteria for agreement with experimental constraints and coordinate precision were subjected to molecular dynamics calculations in explicit solvent using the XPLOR-NIH package before final validation and deposition in the PDB and BMRB databases.
Structures were interrogated using bioinfomatic methods that utilized structure and sequence based comparison tools, such as FATCAT, VAST, FFAS03, and Pfam. Bioinformatic analysis can generate a testable functional hypothesis, and experimental validation of our findings led to the identification of a new family of membrane-associated ubiquitin fold proteins, the MUBs. This modular pipeline strategy relied on a predefined framework for data management that enables an efficient workflow even when multiple personnel participate at various stages of the process. The self-contained nature of individual steps allows for substitution of improved software tools as new technology becomes available.