## 3 Results  
### Implementation and Functionalities  
To run ssPINE-POKY, users may upload a POKY project containing the ssNMR experiments or generate a practice ssNMR dataset automatically, corresponding to a BMRB code, by running the POKY feature SIM-PRAC (two-letter-code _em_). The minimum set of experiments required to run ssPINE consists of 2D-CC, 2D-NCA, 2D-NCO, 3D-NCACX, 3D-NCOCX, and 3D-CAN(CO)CX, though additional experiments can improve assignment quality (2D-NCACB, 3D-NCACB, 3D-NCOCACB, 3D-NCOCAC, NCACO, CANCO, 3D-CAN(CO)CA, 3D-CAN(CO)CACB). For \({}^{1}\)H-detected ssNMR data, the user can use the I-PINE webserver and PINE-SPARKY.2 plugin [32]. Peak picking must be performed before running ssPINE, which can be automated using the integrated iPick program in POKY (two-letter-code _iP_[33]). Users can access ssPINE-POKY via the main window under _Poky > Automated assignment > Run SSPINE automated assignment (ssPINE-POKY)_, or by entering the two-letter code _EP_. Figure 2: Client–server architecture of ssPINE and ssPINE-POKY implemented around POKY. At the top of the ssPINE-POKY automation window, users can optionally enter their name and email address (Figure 3A). If provided, the server will use the email to notify users about job status and send the results upon completion. A zip file containing the results will be attached, along with a URL for accessing the visualized data in a web browser. Providing this information is not required; however, users will need the generated key (Figure 3J) to access their results. The sequence file input section (Figure 3B) supports three formats: (1) a three-letter code sequence file with indices (*.seq), (2) a one-letter code sequence file (*.fasta), or (3) a three-letter code sequence file without indices (*.txt). Similarly to PINE-SPARKY.2 ([PERSON] et al., 2018), the three-letter format file will be automatically set if the user has submitted the sequence to POKY using the Sequence Entry window (two-letter code _sq_). Users may optionally provide additional data to refine ssPINE results, such as pre-assignment (Figure 3C) and a PDB file (Figure 3H). Additionally, they can choose to run CS-Rosetta ([PERSON] et al., 2018; [PERSON] et al., 2018; [PERSON] et al., 2018) (Figure 3D) for protein structure prediction based on ssPINE outputs. The \"Spectra to run ssPINE/ssPINE-POKY\" section (Figure 3E) allows users to specify the spectra for ssPINE assignment. This is achieved by selecting a spectrum and an experiment type (Figure 3F), then clicking the \"Add\" button in the Button section (Figure 3G). Figure 3: Screenshot of ssPINE-POKY. The user-friendly graphical user interface (GUI) can be launched as a plugin in POKY using the two-letter-code _EP_. (**A**) Name and email fields (optional) for receiving results via email. (**B**) Sequence file input. (**C**) Option to use a pre-assignment. (**D**) Selection of CS-Rosetta for protein structure prediction based on ssPINE outputs. (**E**) List of user-added spectra. (**F**) Dropdown menus for spectrum type selection. (**G**) Row of functional buttons. (**H**) PDB file upload option to enhance ssPINE results. (**I**) Threshold values for chemical shift prediction. (**J**) Section for importing ssPINE results; the key is automatically generated upon clicking the “Submit” button. (**K**) Adjustable \({}^{15}\)N and \({}^{13}\)C tolerances and sample pH settings, the tolerance unit “ppm” can be visualized when the mouse hovers over on the box. Additional buttons in Figure 3G provide further functionality: the \"Delete\" button removes selected spectra, while the \"Clear\" button empties the spectra box. Once all required inputs are set, users can initiate job submission by clicking the \"Submit\" button, which sends the job to the ssPINE web server.