Tracing a new structure

By clicking "TRACE" on the top level menu the trace page is made accessible. This page enables generation of a molecular model from an electron density map.

The Depp page menu block "MAIN_MENUN:trace.txt" carries the functionality. whereas the accompanyig menu blocks allow you to gain control over the process and manually intrefere with automated density interpretation and model generation including model rebuilding.

The role of a user is to monitor the progress and take decisions about the subsequent steps, whetaher to go on, interfere or step back. This enables a user to assist the program in making decission and thus enhace its auto tracing capabilities.

At first the starting point of model building ("TRC_ORIG") and check the quality of the map and a chain trace by skeleton generation ("GEN_SKEL") and proceed downwards. Skeleton should in general encompass at least an asymmetric unit though smaller boxes can used as well. Larger boxes prolong time.

"TRC_SKEL" is the cruical point, where decisions have to be made about the contouring level at which skeleton has been generated (change the level and "RE_SKEL" to obtain a new skeleton) and about the places where the program needs assistance. Skeleton editing is perfomed by the "DELE_BON", "DELE_ATO", "MAKE_BON" and "MAKE_ATO" items. Usually false connectivites must be broken, therefore the "MAKE" items are only seldomly applicable. After skeleton has been improved, "TRC_SKEL" has to be repeated. Chain trace of your molecule is along the yellow and green parts of the skeletonbelonging to secondary structure elements and connections between them, whereas the blue parts represent side chains or unassigned parts.

There is no specific resolution limit to the current procedure. The only requirenment is that a few branches of side chains are picked by the skeleton - which allows to build partial models approximately to 3.5 A resolution. Clearly the success decreases with lower resolution.

From here continue to "TRC_HYBR", which is an atomic model, which you can rebuild, minimize, refine and use for phase combination and from there on "TRC_ALA", which generates a polyala chain along the hybrid models. If the density indicates that side chains could be build, try it with "TRC_SIDE". "TRC_SIDE" will perform side chain interpretation based on map and geometric restraints and not sequence.

Before going into refinement or phase combination the model should not encompass more than an asymmetric unit. "TRC_SYMM" and a following "TRC_MERG" wil take care of that. Alternatively one can start with selected portions of the skeleton, which do not extend beyond a asymmetric unit - a topic covered in the MAIN_MENU:map_mask.html.

Most of the automated model building tools are included in the menu block MAIN_MENU:trace.html. Some of the items need to be configured via "" scripts.

The general outline is to click subsequent items in the direction from the top to bottom. The procedure is iterative, so it makes sense almost at any stage to jump of a few steps backwards and transform the model to some other form. The procedures are written the way that each provides input for the next step. Most of them use the WORK_SEGM variable, whereas some, as "GEN_SKEL" use key "active" as input. The final outcome are atoms with the segment name "HELP". This segment you should save to a file and continue as in the cases where you already have the model including renaming of the segment names.

Trace origin

Trace origin makes a search in the score map for neighboring peaks closest to the coordinate system origin. The search requires a local macro "map_mask_score_map.cmds", which should be configured with a high cutoff for solvent content ("0.93"), method "FFT", and radius of at least 10.0.
-h|--help) prints this message with available options and current status -c|--content solvent content [0.93] -i|--input) input density map [1] -m|--method) solvent flattening method [WANG/SOLOMON] [WANG] -r|--result) resulting score map [3] -s|--sphere) solvent flattening sphere radius [10.0] -w|--way) calculation way [REAL_SPACE/FFT] [FFT] -o|--output) set file name of the created macro [map_mask_score_map.cmds]

   --doit)    create the macro

Resulting atoms are input for "GEN_SKEL".

Map skeletonization

Skeleton is trace of a map in which skeleton atoms are connected by covalent bonds.

Skeleton of a map is generated around an extended box around a chosen set of atoms (see MAIN_MENU:use_atoms.html). The currently active map ("MAP_ACT?") and its current contouring level ("MAP_CONT") define inside which map contour level a skeleton will be genarated.

Repeated clicks on "GEN_SKEL" will extpand the box of skeletonized density map for 15 grid points. Each time the current skeleton atoms will be deleted and new generated.

Map skeleton can help you to build your model (to visualize the fold of your molecule) to recognize your asymmetric unit (for averaging or other density modification purposes) and define molecular envelope or masks.

Skeleton atoms are input to the hybridization step.

Hybridization of an input model

Along the skeleton mainchain trace hybrid fragments are introduced and fitted to the active density map using chemical as well as secondary structure constraints. After fitting, the fragemnts are combined into a covalent bond network of a polycarbon model. The resulting structure is composed of chiral four atom SP3 and SP3R fragments. (R in SP3R stands for reverse direction).

Note that any regular protein structure can be hybridized at well.

Chain tracing

Each input atom is origin point for potential chain extension. From each atom up to three branches can originate. Each branch can form a fork with a chemically reasonable geometry. Chain trace atoms are SP3 atoms. After each chain extension possible chain merges are tested. After a few steps of chain traceing (five) the model is minimized an the procedure can either go back to the hybridization step ("TRC_HYBR") or is repeated.

Symmetry mates removal

Shifts around input atoms applying crystal symmetry operations. Atoms are moved to the position which is the closest to the center of the molecule. If two atoms connected by a covalent bond are moved by two different symmetry operations, the bond is broken.

Default center is the arythmetic middle of input atoms, centers can, however, be specified manually and are not limited by a number: Copy the file MAIN_CMDS:trace_symm_overlaps.cmds to the working directory and add new centers explicitly:

 find symm sele segm name SEGMENTS end map MAP_ONE \
           center coor 34.9162   56.6384   18.5048 \
           center coor 44.3816   25.7612   28.7371

More centers are mandatory when asymmetric unit is composed of more subunits or when a molecule is elongated.

Output of "TRC_SYMM" is input for "TRC_HYBR" only.

Conversion of polycarbon model to amino acids

Based on geometry and model connectivity main chain atoms are assigned and named "TRY_NCAC". The new chain trace is drawn as a stick model. Inspect the model and when necessary improve it by making ("MAKE_BON") or breaking ("DELE_BON") bonds or deleting atoms ("DELE_ATO") and repeat the step.

Once the main chain trace is accepted it can be used in further model building and rebuilding steps, including "SAVE_FIL", side chain ("NAME_SID", "TRC_SIDE"), sequence assignment and refinement ("REFINE") and map calculation ("PHAS_CMB").

Merge traces into the best possible trace

The T

Trace side chains

Traces side chains based on the current map contoruing level using the CA-CB starting point. The procedure tries to fit all topologically unique side chains and keeps the one with the best score. It does not check any sequence information.

Currently only amino acid residues are implemented, nucleic acids are one of the next steps.