NOTE: A typical Bio3D-web session proceeds through five consecutive and dependent steps (see Figure 1 for a schematic representation). Each step is implemented as a consecutive navigation tab of the web application namely SEARCH, ALIGN, FIT, PCA, and eNMA.

1. Structure Search and Selection (SEARCH)

1. Input structure
   1. Obtain the PDB ID of adenylate kinase (Adk), e.g. by searching the PDB [http://www.rcsb.org/pdb]. Alternatively, obtain the protein amino acid sequence of interest, e.g. from UniProt [http://uniprot.org].
   2. Enter the four character long PDB ID for Adk (e.g. 1AKE), or paste a protein sequence, to the text box in the "Input structure or sequence" panel.
2. Hit selection
   1. Click the blue "Next" (Hit selection) button in the first panel or simply scroll down to panel B) "Hit selection" for further analysis.
   2. Make sure the "Limit total number of included structures" slider is set to its maximum value to include all structures above the cutoff.
   3. Lower the "Adjust the inclusion BitScore cutoff" to include more distantly related hits, or increase it to exclude.
3. Optional hit filtering
   1. Click the blue "Next" (Hit selection) button in the first panel or simply scroll down to panel C) "Optional filtering of related structures for further analysis".
   2. Make sure the selected hits represent relevant structures by inspecting details of the table, e.g. PDB name, species, and bound ligands.
   3. Manually refine the selected subset of structures if needed by clicking the rows of the table.
      ​NOTE: Rows highlighted with a blue color depict PDB IDs selected for further analysis in subsequent tabs.

2. Multiple Sequence Alignment Analysis (ALIGN)

1. Click the ALIGN tab to perform sequence alignment of the selected structures from the SEARCH tab.
2. Alignment summary
   1. Review the alignment summary in panel A) "Alignment summary". Make sure that the regions of interest are aligned and not masked by gaps in one or more structures.
   2. If needed, toggle the "Display alignment editing options" and remove unwanted PDB IDs, e.g. PDBs with missing residues.
3. Sequence alignment analysis
   1. Click the blue "Next" (Analysis) button to perform sequence-based clustering analysis of the collected structures.
   2. Select the plot option Dendrogram. Adjust the Cluster into K groups slider to partition the structures into k groups.
   3. Optionally change the clustering method if desired by toggling the More clustering and output options checkbox.
4. Residue conservation analysis
   1. Click the blue "Next" (Conservation) button to calculate the column-wise residue conservation.
   2. Select the Aligned structure sets to generate a plot of the residue conservation at each alignment position.
   3. Select Structures aligned with PFAM seed alignment to show conservation calculated with respect to the associated PFAM seed alignment containing representative members of the family.
5. Sequence alignment display
   1. Click the blue "Next" (Alignment) button to show the full sequence alignment with the in-browser alignment visualization tool.

3. Structure Fitting and Analysis (FIT)

1. Perform structure superimposition by entering the FIT tab.
2. Structure superposition
   1. Toggle the "Show PDBs" checkbox to visualize the aligned protein structures in-browser.
   2. Make sure the protein structures are superimposed to corresponding and relevant regions by visual inspections. Click and drag the mouse over the structures to rotate, and scroll to zoom.
   3. Adjust the coloring of the structures by clicking on the "Color options". Coloring options include alignment position, structural variability per position, RMSD cluster groups, sequence cluster groups, aligned regions and secondary structure.
   4. Download the superposed structures as either conventional PDB files or as a single PyMOL session file for visualization in a specialized molecular viewer program.
3. Structure analysis
   1. Click the blue "Next" (Analysis) button to perform structure-based clustering of the collected PDB structures.
   2. Toggle the RMSD Heatmap in the Plot options dropdown menu.
   3. Adjust the clustering options, including the clustering method itself, through toggling the "More clustering and output options" checkbox.
      NOTE: Pairwise RMSD data can also be visualized as a dendrogram, a histogram or a heat map.
4. Residue fluctuations
   1. Click the blue "Next" (RMSF) button to view the structural variability of each residue (shown as an RMSF plot) with major secondary structure elements shown in the marginal regions of the x-axis.
   2. Toggle the Show B-factors checkbox to overlay crystallographic B-factors of the reference structure onto the RMSF plot.

4. Principal Component Analysis (PCA)

1. Perform principal component analysis by entering the "PCA" tab.
2. Visualization of the principal components
   1. Toggle the "Show PC Trajectory" checkbox to visualize motions described by the PCs with the in-browser visualization tool.
   2. Make sure "Principal Component 1" is chosen from the first drop down menu.
   3. To visualize the motions described by other PCs, choose the desired PC from the "Choose Principal Component" drop down menu.
   4. Change the coloring of the trajectory from the "Color options" drop down menu.
   5. Choose "Variability per position" from the "Color options" to color by displacement magnitude.
   6. Click the "Download PDB trajectory" button in the "Principal Component Visualization" panel to obtain a trajectory view of the motion described by the PCs.
   7. Click the button "Download PyMOL" session file to generate a PyMOL session file giving the motions as a vector field.
3. Conformer analysis
   1. Project the individual structures onto two selected PCs by clicking on the blue "Next" (Plot) button.
   2. Make sure "PC on X-axis" is set to 1, and "PC on Y-axis" to 2. To project the structures onto other PCs, adjust the PC numbering accordingly.
   3. Choose "Cluster by PC Subspace" to color the structures in the plot by PC-based clustering; "RMSD" to color by "RMSD-based" clustering; and "Sequence" to color by sequence based clustering.
   4. Click on any individual points in the plot to label the structures. Alternatively, highlight one or more structures in the table "PCA conformer plot annotation" below the plot.
   5. Slide the PCs in subspace slider to including more/less PCs for the clustering algorithm.
4. Residue contributions
   1. Calculate the residue contributions to the individual PCs by clicking the blue "Next" (Residue contributions) button.
   2. Plot the contributions for additional PCs by including the PC number in the "Choose Principal Component" text box.
   3. Toggle the "Spread lines" checkbox avoid plotting the residue contributions on top of each other.
   4. Toggle off the "Multiline plot" checkbox to plot the residue contributions in separate plots.
   5. Toggle the "Show RMSF" to include the RMSF values (from the FIT tab).

5. Ensemble Normal Mode Analysis (eNMA)

1. Click the eNMA tab to initiate normal modes (NMs) calculation.
2. Filter structure
   1. Adjust the number of structures by lowering or increasing the "Cutoff" for structure inclusion/exclusion.
   2. Click the green "Run Ensemble NMA" to start the NMA calculation.
3. Normal modes visualization
   1. Scroll down to the second panel of the eNMA tab (Normal Modes Visualization) for visualization of the NMs.
      ​NOTE: By default, the NM with the highest overlap (similarity) to PC-1 is displayed in the visualization window.
   2. To visualize the motions described by other NMs or other PDB structures, choose the desired NM and structure from the "Choose Mode" and "Show NMs for structure" drop down menus, respectively.
4. Residue fluctuations
   1. Click the blue "Next" (Fluctuations) button to calculate the residue-wise fluctuations of structures selected for eNMA.
   2. Toggle the "Cluster by RMSD" to color the fluctuation profiles by RMSD-based clustering.
   3. Toggle the "Cluster by RMSIP" to color the fluctuation profiles by RMSIP-based clustering.
   4. Toggle the "Spread lines" checkbox to plot the grouped fluctuation profiles apart from each other.
5. Comparing NMA and PCA
   1. Click the blue "Next" (PCA-vs-NMA) button to calculate the similarity between the individual NMs and PCs.
   2. Select a PDB ID from the "Compare NMs of structure" drop down menu to calculate the similarity between the NMs of this structure to the PCs calculated in the PCA tab.
6. Overlap analysis
   1. Click the blue "Next" (Overlap analysis) button to calculate the overlap between calculated NMs and the structure difference vector between two selected structures.
   2. Select a 'reference' PDB ID from the "Compare NMs of structure" drop down menu and or one or more PDB IDs in the structure table for the pairwise comparison with the reference PDB.
7. Clustering analysis
   1. Click the blue "Next" (Clustering) button to perform structure clustering based on pair-wise NM similarity (RMSIP).