User's Guide 1 A guided example 2 User's Guide 1 A guided example. ============================================================= There is an example under ./example directory. If you finish this example, you know most of this program. There are several files in this directory: * fabh.faa is a file of protein sequences in FASTA format. The description line of the first sequence is '>FABH_ECOLI|pdb|1HND|A', which means that this sequence has a 3D PDB structure with PDB ID 1HND and this sequence is corresponding to chain A. * pdb1HND.ent is the PDB file for the first sequence. * 1HND.pocket is a file listing residues of binding pocket of pdb1HND.ent. Please follow the instructions below a) Type 'cd ./example' b) Start this program by '../vissa.pl&', the main window will be open. c) Start a new job from menu bar File -> new. A window will be open for you to enter a job ID and the file name of input protein sequences. * Input a name for job ID, * Check the fasta box, * Browse and input fabh.faa as input file name * Hit Ok button A new window will be open for you to input the PDB file for the first sequence. Please browse and input pdb1HND.ent, and hit Ok. Now the sequences will be displayed in the main window. d) Hit the alignment button The sequence will be aligned in the main window. Run secondary structure calculation from menubar->tool->DSSP, then secondary structure will be displayed. e) Mouse operations On the left panel: * CLICK on text to select or de-select a single sequence * Double-CLICK on text to show a sequence * Shift-CLICK on text to select a range of sequences if there are other sequences in selection On the right panel: * CLICK on background to select or de-select a single column * Double-CLICK on '+' or '-' turn on/off the display of residue numbers * Shift-CLICK on background to select a range of columns if there are other columns in selection f) Making selection and some basic operation Selection of proteins and selection of aligned residues can be made by mouse click or from menubar->select and menubar->select columns. Then you can display just selected proteins, selected residues from menubar->display g) Define a site A site is a set of residues of interests, can be a binding pocket, a structural motif, or a surface patch etc. Hit 'Site' button to open a window to define a site. You can define a site by doing one of the followings: * File 1HND.pocket contains a list of residues I picked up. Open 1HND.pocket and paste the residues into the residue text box, add some comments in the Remark text box, then hit Add button. * Hit Read button to read in residues in 1HND.pocket, add some comments in the Remark text box, then hit Add button. * Select residues of interest in main window by mouse, add some comments in the Remark text box, then hit "Add selected" button You can display this site by menubar->display->display site h) Display structure Hit "View structure" button, a rasmol window will be open to display your PDB structure. You may try following things (read notice to windows users on very end of this file) * Set display options from menubar->view * Highlight selected residues * Highlight defined site i) Export image Hit "Export image" button, it will open another window, in this window the sequence can be wrapped into multiple segments. It can be used to make publication quality alignment. With this window, you can take snap shot to JPEG file, or, you may export to HTML file. j) Edit alignment You can edit the names of proteins for display from menubar->edit. You can also manully edit the alignment. k) Save, export, open You can save everything from menubar->save, the results is in a .slp file. This file can be opened later. You can also export alignment. 2 User Guide ============================================================= 2.1 Prepare input files 2.2 Start new job 2.3 Making alignment 2.4 Mouse function 2.5 Program functions 2.1 Prepare input files Input sequences need to be in following formats: FASTA, FASTA alignment, or ClustalW alignment. The sequences that have a 3D PDB structure, they need to have the following pattern in their description: (see above example) pdb|XXXX|X, where XXXX is pdb id and X is the chain name If there is no chain name, X should be a space ' '. You can make a fake pdb id if your 3D structure is not in PDB databank. You can have zero or more than one sequences that have PDB structures. 2.2 Start new job If you run the above example, you should already know how to start a new job. Each job has a job id. When you save your result, it will be saved as job_id.slp. 2.3 Making alignment If you start new job from FASTA alignment, or from ClustalW alignment, you should have alignment displayed in the main window. Otherwise, just hit alignment button, which will run Clustalw. You can also use Clustalx for alignment from menubar->tool->clustalx. In that case, you can change the parameters within Clustalx. You can also make alignment on selected proteins from menubar->tool->align selected. If an alignment is made, alignment will be color at the consensus columns. Default cutoff of consensus is 60%, means that 60% of residues of certain column are same. You can change this cutoff. 2.4 Mouse Just see the above example. 2.5 Program functions 2.5.1 File functions Menubar->File have several basic file functions. * Open: open saved alignment * New: start a new job * Save: save current job, which will be saved as job_id.slp * Close: close current job * Rename: rename current job id * Export: export all or selected sequences or alignments * Exit: exit the program 2.5.2 Making selection of proteins Protein sequences can be selected by mouse click and from menubar->select. Some function work on selected proteins. You can also just display selected proteins only. 2.5.3 Making selection of aligned residues Aligned residue columns can be selected by mouse click and from menubar -> select columns. Many functions work on selected columns. 2.5.4 Display functions Menubar->display has functions to control graphic display. * refresh: redraw current window. You usually don't need to use it, but in case that the program doesn't update the window after you make some changes, use this function. * All sequence: display all sequences * Selected sequence: display only selected sequences * All columns: display all columns * Selected columns: display only selected columns * Consensus columns: display only the consensus columns * Font: change font size * color none: don't color amino acid, but consensus color can be on * color any: color all amino acid * color group: color all amino acid by group * consensus: turn on/off consensus coloring 2.5.5 Tool functions * clustalw alignment: run Clustalw alignment * align selected: run Clustalw alignment for only selected alignment * clustalx alignment: run Clustalx alignment * clustalx selected: run clustalx for only selected alignment * compute consensus: as it says * cluster: cluster the sequences based on sequence similarity * DSSP: run DSSP program on the PDB structures 2.5.6 Site function It is used to define a structural site for PDB sequence, see the example above. 2.5.7 View function (read notice to windows users on very end of this file) The functions here are used to open and control Rasmol windows. A basic function is to highlight the residues of interest on 3D rasmol window. * new: open a new rasmol window * Show protein/site/ligand as: how to render them * Protein/site/ligand color: how to color them * Single chain only: if more than 1 chains in PDB, just show the one with defined chain ID * Show ligand: turn on/off ligand. ligand in PDB is defined by rasmol command "select ligand" * highlight consensus: highlight consensus residues * highlight site: highlight site * highlight selected: highlight selected residues Within the rasmol control window, you can also edit / input your rasmol command if you know rasmol well. Then hit the command button. 2.5.8 Function buttons * Alignment: same as menubar->tool->clustalw * View structure: same as menubar->View->new rasmol * Site: same as menubar->Site->view * Export image: see 2.5.9 * Consensus cutoff: change consensus cutoff 2.5.9 Export alignment image and HTML file You can use it to make publication quality alignment in either jpeg image file or HTML file. The alignment in the export window will be same as the alignment displayed in the main window, except that it is wrapped into more segments. There is a pull down menu within this window. You need to use its own refresh function to update the display after you change the wrapping length, or after you change the font size from the main window, or after you make any other change of alignment of main window. After you export it to HTML file, you may able to further edit it with text editor. Try to open the .html file with your Microsoft Word, You will have an alignment that formated and colored like in the program. Then, you can edit it for publication. =========================================== Notice to Windows users On Windows system, when you apply the highlight function, the program can not do it automatically, because I can not control windows rasmol as I control linux rasmol. But I write a rasmol script, so please load this script within rasmol window each time after you apply the highlight function.