Manual

Start

The FragFit start display has two different panels. In the upper left corner you find the general controls for the NGL viewer, located in dropdown menus. The right panel contains the FragFit controls: a button to start new jobs or to load an example.

FragFit job

Start search To start a new FragFit job you have to provide a coordinate file of a protein structure in PDB format and an cryo-EM map in MRC or CCP4 (MAP) format. To reduce the computation time it is highly recommended to upload a map containing only the direct vicinity of the missing fragment. The two stem residues flanking a missing segment or a segment that should be remodeled have to be typed in a form or selected from the NGL visualization by mouse selection (e.g. 135:A). To clear the stem residues from the form use the 'clear stems' button, afterwards, they can be filled by mouse selection again. The order is not important but the chains must be the same. Finally, the amino acids sequence of the missing segment and the resolution of the cryo-EM density map have to be provided, excluding the stem residues. Additionally, the membrane can be calculated by applying informations from TMDET [5] and some further processing. Depending on fragment length, a fragment search may take up to 2 minutes. When the job is finished, the results are displayed automatically. Modify search If no suitable loop was found a possible solution is to extend the loop search in N- or C-terminal direction. To do this push one of the extension buttons. The stem residue will be changed by one into the chosen direction and the search sequence will be updated accordingly. To modify the search parameters manually change the form and press submit. The result table will be updated after the search is completed. To start a completely new job please reload the web-site. Old results will be lost.

Results

The top candidate loop is automatically displayed in the NGL Viewer together with the uploaded protein (model) and the user provided cryo-EM density map. Other loops can be selected from the list of up tp 100 candidates by visualization or by using the information provided in the table at the right panel for guidance. This table includes the following information for each candidate loop: an ID, a correlation score, a score describing the steric fit of the segment, a clash score, the PDB ID and the position of the fragment in the template protein and a comparison of the user-defined input sequence with the template sequence (sequence similarity). Sequences that are underlined contain prolines or glycines. By selecting a result, the loop is displayed together with the uploaded protein. The complete list of loop candidates can be displayed simultaneously to visualize the conformational space of the loop. The loop can be colored accordingly to its score, correlation score, sequence identity or clash score by selecting the corresponding color scheme from the dropdown menu. Additionally, the representation of the structure and the loop (collapsed underneath the result table) can be changed as explained separatly in the documentation of the NGL Viewer. The completed structure (initial model plus selected loop) can be downloaded by clicking the download symbol. Alternatively, the complete list of loops can be downloaded for further analyses. The loops contain sidechains, which are not minimized and also not used during density fit but can be utilized during visual comparison and selection.

Example sets

Cryo-EM structure of the human gamma-secretase complex at 3.4 angstrom resolution.

1. Download the map from EMDB: EMD-3061 and upload it to the server via the form
2. Downlaod PDB file: 5a63 and upload it to the server via the form
3. Type in the stem residues: Stem residue 1: 473:A Stem residue 2: 482:A
4. Type in the missing sequence: EDLNFVT
5. Add the map resolution: 3.4
6. Press start.
7. Visualize results.

Download further example data sets here:
5oby
6bo8

A detailed youtube tutorial can also be found here.

Validation

A validation can be done by using MolProbity. For the given example sets we found that Fragfit could improve the MolProbity Score. It can happen that the downloaded structure from Fragfit can not be uploaded to MolProbity, if that happens do the following: Copy the atoms of the loop from the Fragfit result into the original pdb and delete the original loop, then upload this file.