Xfit Automated Fitting Features

by Mark Israel and Duncan McRee

Xfit v3.5 has a new menu on the Tool Bar, Auto Fit, which incorporates features for automated fitting procedures that used to be done one step at a time. The new menu allows very fast fitting in the parts of the map that are clear allowing you to concentrate on the difficult bits without having to doing the repetitive fitting tasks on most of the map. By clear we mean that the main-chain density should be mostly connected at positive contour levels and the side-chains should be visible.

The auto fit menu is shown above. Note that most menu items have a character in single quotes that indicate a keyboard shortcut for the operation. After you become accustomed to the keys you will find this much quicker than using the menu. Before we explain how to use the menu we need to discuss some notation. MRK - A residue with a single CA atom. It is used to mark the positions of residues and treated as an amino-acid. Another useful special residue is a VEC which has a CA and a CB atom to indicate the direction of the side-chain. Both residues are part of the standard xfit dictionary found in $XTALVIEWHOME/data/dict.pdb. If you don't have a dictionary being automatically loaded read the beginning of the xfit man page before continuing. You must have a dictionary before automated fitting can proceed.

Fragment/Chain - the basic unit of fitting is a string of residues connected together. The criteria for connectedness for MRK residues (i.e. CA's) is two successive ones be within 4.5 Angstroms of each other. Besides the functions on the auto fit menu several functions that deal with chains have also been added to the Model window. In particular, the model window automatically numbers each fragment and marks the chain termini. Also the Rename button has been expanded into a Sequence menu that includes the ability to reverse chains, reorder them, name them and match them to a sequence.

Focus Residue - This is the last picked residue either on the canvas or on the Model Window. It shows up as highlighted in Model window residue list and on the lower right footer of the canvas. All of the commands discussed here start with the focus residue and use it to identify the chain to be acted upon.

Current map - the map used in the auto fit commands is the one enumerated in the Refine window in the map number field. The default is map 1. If another is desired it must be changed here before using the auto fit commands.

Auto Fit Menu

Add CA(MRK) Before - Adds a MRK residue to the N-terminal end of the current focus residue (see above). The "<" key is a shortcut for this operation. If there is already a residue here the user is warned and given the opportunity to cancel. If there is more than one residue in the fragment the vector between the last two residues and the density of the current map (see above) is used to find the position of the residue. The 5 most likely positions are saved and can be cycled through by pressing the space bar or by the "Cycle CA" option on the menu. For small adjustments or if none of the positions looks correct, the middle mouse button moves the CA atom in refine while fitting mode keeping the distance 3.8 Angstroms to the focus residue. The MRK residue is automatically numbered one less than the focus residue. Lastly the command makes the added residue the focus residue so that the next invocation of this command will add another residue to the end of the chain before the new one. In maps with clear main-chain density the command finds the next CA position and no further fitting is needed. Occasionally the side chain density is stronger than the main chain and in this case pressing the space-bar will find the correct choice.

Add CA (MRK) After - Similar to the Add MRK(CA) to Nter except that it adds to the C-terminal end of the chain/fragment starting at the focus residue. The ">" key is the shortcut.

Next CA Choice - Cycles through the CA choices for the Add MRK commands. The spacebar is a shortcut to this command.

Cancel Baton mode - Accepts the CA position and ends the refinement mode. The ";" key is the keyboard shortcut. It is good practice to always issue this command when finished adding to the ends of a fragment otherwise a later press of the spacebar may have surprising results. Note this command and the ";" key will accept any fitting operation and any refinement mode so it can be used in other fitting/refinement operations.

Go to Fragment Nter - moves to the N-terminal end of the fragment/chain containing the focus residue and makes it the new focus residue. Use it as a precursor to the Add to Nter command.

Go to Fragment Cter - moves to the C-terminal end of the fragment/chain containing the focus residue and makes it the new focus residue. Use it as a precursor to the Add to Cter command.

Poly Ala Fragment - Turns all the MRK or VEC residues in the fragment/chain containing the focus residue into alanines using overlapping pentamer commands. If the best pentamer contains either a glycine or proline it is kept as such instead of an alinine to prevent inserting alanines in bad positions.

Set Sequence - Allows changing the sequence of a fragment with a sequence file. First a residue is picked and then the command issued which brings up a window with the sequence. Choose the corresponding position in the sequence.

Auto Fit Strategy

Starting a new model - After locating a stretch of density center the position of a CA atom on the cross in the center of the screen and Open the Model window. Set the active model to a unused number, the residue type to MRK, enter a likely residue number and select New model from the Insert menu. Select this residue to make it the focus residue and then use the ">" key to add a second MRK residue. Since there is an ambiguity as to where this residue should go the program leaves it up to you to move it with the middle mouse button. If you have some indication of the chain direction (i.e. the side-chains of helices point to the N-terminal end and the curl of a tight-turn indicates the chain direction) then you can place the residue in that direction. Otherwise, just guess and reverse the chain direction later if needed. To place the CA atom use the middle mouse button and drag the residue until is in the desired position. Use the ">" key again to place the third residue automatically. The program finds the position of the next residue by considering the map density and the direction between the first two residues. In considering the map density the program tries to eliminate strong side-chain paths by considering a second move and adds the two probabilities together to find the final probability. Thus short side-chains are eliminated because they will not provide a good position for the second move. If the program still finds a side-chain press the spacebar to cycle through the other 4 choices. Pressing the spacebar 5 times brings you back to the original choice. You can also move the CA position with the middle mouse button as you did for the second position. In a medium resolution map it is not possible to precisely determine the CA position so instead concentrate on getting the path right and use the pentamer fitting followed by real-space refinement to better position the fragment.

After a fragment has been built as MRK's it can be changed into poly-ala with the "Poly Ala Fragment" command if there at least 5 residues. Just pick a residue in the fragment and issue the command. You will see the fragment being built from overlapping pentamers.

Next set the sequence with the "Set Sequence" command which brings up the window above. If you haven't loaded a sequence file (see below) then do so with the Files... window. Pick a residue that you believe you know the sequence of and then pick the corresponding sequence entry. Then Pick "Change and Fit to Density" and the sequence will be changed in both directions and then the side-chains positioned to the density by a rotomer search. If you change your mind about the sequence you can redo it later in the same manner.

Sequence File Format

This a new file format for this version of the program. The format is one letter code in a free format. Numbers and white space are ignored. A record starting with a '!', ';' or '#' is treated as a comment and ignored. An example: cvccp.seq.

Back to Index