We tested our extensions with the ubiquitin protein which consists of 76 residues. The following seven NMR experiments were performed.
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2D Experiments |
N-HSQC, C-HSQC |
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3D Experiments |
CBCA(CO)NH, CC(CO)NH, HBHA(CO)NH, HC(CO)NH, HCCH-TOCSY |
Outputs from the PINE server are saved in a native probabilistic format and you can check them out here.
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Native Probabilistic Backbone Output generated by PINE
Native Probabilistic Sidechain Output generated by PINE
We assigned ubiquitin with our extensions in one hour.
First, we used “Assigned the best by PINE” with threshold of 0.9 to assign. 92.5% of all possible peaks, 98.6% for backbone and 86.7% for sidechain. These assignments took only one minute.
Second, we used both “PINE Assigner” and “PINE Graph Assigner” to refine and perform further assignments. We assigned all the atoms of ubiquitin in one hour. All we needed to do was look at unassigned atoms from “PINE Graph Assigner” and assign them with “PINE Assigner” and “PINE Graph Assigner” by clicking on the few labels that had not yet been assigned.
Results of the first and second steps are compared to see how PINE-SPARKY works.
Step One: “Assign the best by PINE” with threshold of 0.9 was performed for all the spectra.
Step Two: “PINE Assigner” and “PINE Graph Assigner” were used to assign peaks that remained unassigned after Step One.
Result:
| Ideal assignments(%) | Automatically selected(%) | Manually selected(%) | Auto/Manual(%) | |
| All assignments | 590(100) | 520(88.136) | 562(95.254) | 520/562(92.527) |
| Backbone assignments | 287(100) | 272(94.774) | 276(96.167) | 272/276(98.551) |
| Sidechain assignments | 303(100) | 248(81.848) | 286(94.389) | 248/286(86.713) |
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Test result in text file format