Preparing Protein Structures for Refinement

The refinement module can handle many types of structures, and can fix many structural problems. For standard residues, it fixes formal charges and bond orders, and corrects disparities between the sequence and the structure. For example, if a residue has the coordinates of ALA but is called SER, the 3HB is ignored and the OG and HG added during refinement. The standard residues are the 20 canonical amino acids; ACE and NMA; HOH; CYX (disulfide); and the acid/base variants ASH/AS1 (ASP), GLH/GL1 (GLU), ARN (ARG), LYN (LYS), HIE/HIP/HID (HIS), CYT (CYS), SRO (SER), TYO (TYR).

The input structures must meet certain conditions for a refinement job to be run successfully.

For all kinds of structures, the following condition must be met:

  • The structure must be an all-atom structure (i.e. with explicit hydrogens).

    This condition applies to the protein and any ligand, waters or cofactors present in the structure: all hydrogens must be present.

    You can add hydrogens to a structure by displaying the structure in the Workspace and clicking the Add hydrogens button in the 3D Builder.

    However, for structures containing nonstandard residues, you must correct bond orders and formal charges first.

For structures with nonstandard residues, the following conditions must be met:

  • No residue can consist of disconnected pieces (“fragmented residues”) without formal bonds. All atoms within a residue must be connected via bonds, including zero-order bonds in the case of metals.

  • Bond orders and formal charges must be corrected.

    For nonstandard residues the supplied structure will be used, and you must check for correct bond orders and formal charges. Metals and their ligating groups must be connected by zero-order bonds.

When you rename residues, you should be aware that OPLS_2005 or OPLS4 parameters are used for standard residues and nonstandard residues.

If your structure does not meet one or more of the conditions outlined above, you must fix it before you can successfully run a refinement. Some of the problems only become apparent when you have run a refinement job, so it can be a useful diagnostic procedure to run a Prime energy calculation first. This job is quick, and produces warnings in the log file, which you can check in the Monitor panel.

Many of the preparation tasks are performed automatically or interactively with the Protein Preparation Workflow. The Protein Preparation Workflow panel can be opened by clicking Protein Preparation Workflowon the Favorites toolbar, or Protein Preparation and Refinement → Protein Preparation Workflow. When the preparation has finished, you should check that all changes made are correct. You can fix any remaining errors with the procedures below. For more information on the Protein Preparation Workflow, see the Protein Preparation Guide — Contents.

Procedures for manually fixing bond orders, formal charges, and atom names are given below. These procedures use the 3d Builder panel and other tools. The first step in most of the procedures is to label the atoms with the relevant property. For this purpose, ensure that no atoms are selected so that the labels are applied to all atoms. The labeling is not necessary but may be useful for identification. You can also use the status bar to identify atoms, as the display is updated when the pointer is over an atom.

To assign bond orders:

1. Choose Edit → Assign → Bond Orders.
2. Inspect the residues in the structure for any remaining bond orders that are incorrect.
3. If there are still incorrect bond orders, select the bonds and click the Increment bond order or Decrement bond order button in the 3D Builder.

You can also right-click the bonds in the Workspace structure, and choose Increase Bond Order or Decrease Bond Order from the shortcut menu.

To view and change formal charges:

1. In the Style toolbox, click the arrow next to Apply Labels and choose Formal Charge.

Charges are displayed for atoms that are charged.

2. Select an incorrectly charged atom in the Workspace.
3. Click the Increment formal charge or Decrement formal charge button in the 3D Builder.

The formal charge is incremented or decremented by one unit for each click. If you select multiple incorrectly charged atoms, each click increments or decrements the charge on all the selected atoms.

To change PDB atom names:

  1. In the Style toolbox, click the arrow next to Apply Labels and choose PDB Atom Name.

  2. In the 3D Builder panel, click Other Edits and choose Change Other Properties.

    The Change Other Properties dialog box opens.

  3. From the Property option menu, choosePDB Atom Name.

  4. Ensure that Apply PDB atom name is selected.

  5. Zoom in on one of the residues (middle+right mouse buttons or mouse wheel).

  6. For each atom that needs renaming, click on the atom, then enter the desired PDB atom name in the text box and click Apply.

  7. Repeat the above two steps for each residue whose atoms need renaming.

To change residue names:

  1. In the Style toolbox, click the arrow next to Apply Labels and choose Residue Information.

  2. From the Property option menu, choose Residue / Chain Name.

  3. For each residue that needs renaming, select the residue in the Workspace, enter the desired PDB name in the Residue PDB name text box, and click Apply.

    You might need to zoom in to select the residues (middle+right mouse buttons or mouse wheel).