3D Protein Alignment. Version 1.1

How to quickly begin to work with 3D Protein Alignment program.
---------------------------------------------------------------

After unzipping file "3dpaln.zip" you will obtain folder "3DPAln".
You can go to this "3DPAln" folder and run file "3dpaln.exe" in DOS 
or Windows 95/98/NT. 

Using the shell of 3D Protein Alignment program: 
Locate the cursor by arrows to first example file "Pdb1cc5.ent" 
(this is a file with 3D coordinates of Cytochrom C5 protein, 
ftp address ftp://ftp.rcsb.org/fullrelease/uncompressed_files/cc/) 
and press "Ins" on the keyboard. 

Then find second example file "Pdb351c.ent" (this is a file with 3D 
coordinates of Cytochrom C551 protein, ftp address ftp://ftp.rcsb.org/fullrelease/uncompressed_files/51/) 
and press "Ins". 

Then press "F6" to begin 3D alignment process. 

You should type "0" (zero) to choose the only subunit of 
"Pdbcc5.ent" file 
and type "1" to choose the only subunit in "Pdb351c.ent" file. 

When program writes:
"DO YOU WANT TO CHANGE SETTINGS?(Y/N)", type "N"

The program will begin to perform 3D alignment process.
In this example it will take a very short time. This is because of 
short length of example proteins. Of course then you can use this 
program with any proteins from PDB (Protein Data Bank) 
http://www.rcsb.org/

Some of this proteins are much more longer than in our example. 
In this case you can see this process in much more details. 
How to operate with long proteins see in this file below.


After the end of alignment procedure you can see the strings:
"FILE C:\3DPALN\PDBCC5.ENT CONTAINS ONE SUBUNIT:0
CHOOSE FIRST SUBUNIT TO COMPARE (INPUT ACCORDING NUMBER):".
You should type "0" (zero) and "Enter" to choose the only subunit 
of "Pdbcc5.ent" file for 3D visualization. 

Then you can see the strings:
"FILE aln.ent CONTAINS ONE SUBUNIT:1
CHOOSE SECOND SUBUNIT TO COMPARE (INPUT ACCORDING NUMBER):".
You should type "1" and "Enter" to choose the only subunit in 
"Pdb351c.ent" file for 3D visualization.

Then you can see the string:
"INPUT AMINO ACID MARK [1,2,3,5,10,0]:".
You should type "10" and "Enter" to obtain stereoscopic images of 
aligned subunits, with mark of each 10th amino acids by it's number 
in PDB file.

You can see stereoscopic image of aligned subunits (proteins) and 
evaluate the degree of it's 3D structure similarity by visual 
inspection. It is the only way in demo version to estimate 
3D structure similarity. In full retail version you can obtain 
file with coordinates of second protein shifted and rotated to 
the coordinates of first protein in PDB format. In this file you 
can find also a list of matched amino acids as a result of 
3D alignment process. You can use this file to see the result of 
3D alignment process next time. The second file that you can obtain 
in full retail version shows you the linear representation (as 
sequence alignment) of the resuly of 3D alignment process. In 
this file you can see a set of corresponding amino acids from both 
subunits that have Root Mean Square (RMS) deviations not more than 
given by user (in our example 3.5 angstroms). It is structure 
conservative regions. 

It is possible to see this stereoscopic image with the help of 
mirror stereo-glasses.

You can zoom ("F9"), unzoom ("F10") and rotate the image by arrow 
keys.

Press "Alt+X" to go from Visualizator to Shell of 3D Protein 
Alignment program.

Press "F10" to finish 3D Protein Alignment program. 



How to tune 3D Protein Alignment program to work with long (more 
than 400 amino acids) subunits (proteins) under the DOS and 
Windows 95/98/NT, see file "readme.txt" chapter Installation.


After Installation run 3dpaln.exe.

For example if you downloaded from PDB files "pdb2bmh.ent" 
(ftp address ftp://ftp.rcsb.org/fullrelease/uncompressed_files/bm/) 
and "pdb1oxa.ent" 
(ftp address ftp://ftp.rcsb.org/fullrelease/uncompressed_files/ox/)
with 3D structures of long proteins (Cytochromes P450) 
and want to make 3D alignment of first subunit of "pdb2bmh.ent" 
(this file contains two subunits that is two separate amino acid 
sequences) and the only subunit of "pdb1oxa".

Copy both files to "3DPAln" folder. 

Then you should go to "3DPAln" folder and run file "3dpaln.exe". 

Using the shell of this program, find file "pdb2bmh.ent" and press 
"Ins" on keyboard. 

Then find file "pdb1oxa.ent" and press "Ins". 

Then press "F6" to begin 3D alignment process. 

You should type "0" (zero) to choose first (A) subunit of 
"pdb2bmh.ent" file 

and type "2" to choose the only subunit in "pdb1oxa.ent" file. 

When program writes:
"DO YOU WANT TO CHANGE SETTINGS?(Y/N)", type "Y"
INPUT LENGTH RATIO   (0.010.0)   :, type "1"
INPUT GAP (1..1500) AMINOACID :, type "25"
INPUT MAX RMS (0..1000) ANGSTROM:, In this example you should type 
value "1.5" (see this file below)

INPUT FILENAME TO SAVE COORDS  : You should type "2bmh1oxa.ent" 
(extension ".ent" means that in this file will be coordinates 
of second protein shifted and rotated to the coordinates of first 
protein in PDB format (see details in this file below).

INPUT FILENAME TO SAVE ALIGNED STRINGS  : You should type 
"2bmh1oxa.315" (the first "3" in extension ".315" means that in 
this file will be sequence strings, as in sequence alignment, but 
it is the result of 3D alignment, the last "15" means that program 
will mark by asterisks pare of amino acids, that have 
Root Mean Square (RMS) Deviation not more than 1.5 angstroms (see 
details in this file below).

   WARNING. It is impossible to obtain files 2bmh1oxa.ent and 
   2bmh1oxa.315 in demo (unregistered) version of the program!!!

After the end of alignment procedure you should type "0" (zero) to 
choose first (A) subunit of "pdb2bmh.ent" file, 

and type "2" to choose the only subunit in "pdb1oxa.ent" file 

and type "10" to obtain stereoscopic images of aligned subunits, 
with mark of each 10th amino acids by it's number in PDB file.


It is possible to see this stereoscopic image with the help of 
mirror stereo-glasses.

You can zoom ("F9"), unzoom ("F10") and rotate the image by arrow keys.

Press "Alt+X" to go from Visualizator to Shell of 3D Protein Alignment program.

Press "F10" to finish 3D Protein Alignment program. 





HOT KEYS:
---------

Alt+F1 - selection of driver;

INSERT - (un)selection of Brookhaven file(s) for 3D-visualization or
         3D alignment;

         You should select:
         a) Two Brookhaven files (with extention *.ent) for 
            comparison of two protein structures; 
         b) One or two Brookhaven files for 3D-visualization of 
            proteins.  The choosen files with its full pathway 
            will displayed in the bottom of screen.  

F3     - text view of file in current position of cursor;

         For text view of files is used wpview.exe visualizator 
         of Norton Comander shell. It may be placed in the 
         3DALIGN program directory; otherwise you must add in 
         autoexec.bat file path to the directory where file 
         wpview.exe is located.  

F6     - 3D-alignment of two selected proteins;

F7     - continue 3D-alignment of two selected proteins;

F8     - fitting two structures;

F9     - 3D-visualization of selected proteins;

F10    - exit from program;





      3D alignment of tertiary structures of proteins (F6): 

The program executes the follow steps:

1. The program shows list of subunits contained in selected files 
   and requires to select subunits to compare.  

   For example:

   FILE pdb1fdh.ent CONTAINS SUBUNITS:
   A:0 G:1  
   FILE pdb1hds.ent CONTAINS SUBUNITS:
   A:2 B:3 C:4 D:5  

   CHOOSE FIRST  SUBUNIT TO COMPARE(INPUT ACCORDING NUMBER):5
 
   CHOOSE SECOND SUBUNIT TO COMPARE(INPUT ACCORDING NUMBER):0

   - in this case subunit D of pdb1hds.ent and subunit A of 
   pdb1fdh.ent will be compared. Coordinates of second protein 
   (pdb1fdh.ent) will changed to minimize RMS of CA-atoms of 
   selected subunits.  

2. The program proposes default pararmeters that may be changed:

   DEFAULT SETTINGS:

   GAP            :25  AMINOACID
   MAXRMS         :3.5 ANGSTREM
   LENGTH RATIO   :1
   FILE TO SAVE COORDS           :ALN.ENT
   FILE TO SAVE ALIGNED STRINGS  :ALN.STR

   DO YOU WANT TO CHANGE SETTINGS?(Y/N)


   ALN.ENT - file where new coordinates of second 
             protein (pdb1fdh.ent) and information about results 
             of fitting will be placed .   

             File contains matching fields:
             MATCH    1       3       0.343
             MATCH    2       4       0.254
             MATCH    3       5       0.264
             ...
             first columnb - aminoacid numbers of first pattern 
                             (pdb1hds.ent subunit D); 
             second - aminoacid numbers of second pattern 
                      (pdb1fdh.ent subunit A); 
             third  - distances between corresponding CA-atoms; 


   ALN.STR - file where aligned strings of compared subunits and 
             information about the results of fitting will be 
             placed. Linear representation of results of 3D 
             alignment. 

   WARNING. It is impossible to obtain files aln.ent and aln.str 
   in demo (unregistered) version of the program!!!

   GAP     - defines threshold value of deletion length that can 
             ocurr in patterns. It may be vary from 0 to the 
             length of subunit. The more is this parameter the 
             more accurate will be alignment, but it will need 
             more time. The optimal value is 25.

   MAXRMS  - displays pares of aminoacids  in  file  ALN.STR  that 
             have   RMS  not  more  than  MAXRMS  by  "*"  (marks 
             structure conservative regions): 

             ACDFDGDFSGTRUEYI
               ***   ********
             ACNFDGDFSGTRUEYI


   LENGTH RATIO - defines ratio between length of frame and rms.
                  If you want to find more global and more rough 
                  alignment you should increase this parameter.
                  If you want to find more local and more 
                  accurite alignment you should decrease this 
                  parameter.  

 
3. The program displays:

   First You  can  see  the  3D  automatic  scanning  each  other 
   procedure of two protein 3D structures (stereo image).  

   Then  You  can  see  the  distance  map of alignment procedure 
   process of  two  protein  3D  structures.  The  darker  points 
   coorespond to the shorter distances between amino acids.  

   You can see the progress of  current  iteration  of  alignment 
   process  with  thin vertical red line that grow from bottom to 
   top on the left edge of distanse matrix.  

   The diagonal red line on the map is accepted  set  of  matched 
   aminoacids in the previous iteration.  

   To end alignment on current iteration, press <ESC>. 
   To exit program press F10. 


4. Then the program show the result of 3D alignment in stereo.



                   CONTINUE 3D-ALIGNMENT (F7):

   WARNING. It is impossible to continue 3D alignment process 
   in demo (unregistered) version of the program!!!

The parameters used during 3D-alignement may be changed to inprove
the result. Use the output file 3D-align program containing match 
fields:
MATCH    1       3       0.343
MATCH    2       4       0.254
MATCH    3       5       0.264
...

You can change proposed default parameters to obtain 
possibly another aminoacid matching. 




           FITTING (SUPERIMPOSITION) OF PROTEINS (F8):

One   output  file  of  structure  comparison  program  (ALN.ENT) 
contains coordinates and matching fields: 

MATCH    1       3       0.343
MATCH    2       4       0.254
MATCH    3       5       0.264
...
first columnb - aminoacid numbers of first  pattern  (pdb1hds.ent 
                subunit D); 
second - aminoacid numbers of second pattern (pdb1fdh.ent subunit 
         A); 
third  - distances between corresponding CA-atoms; 

This set may be corrected by deleting(adding) matches and replacing 
aminoacid numbers. To fit new set, select output file(ALN.ENT) and
file contained first pattern(pdb1hds.ent), then press F8.


Select  subunits that was previevsly compared:

FILE ALN.ENT CONTAINS SUBUNITS:
A:0 G:1  
FILE pdb1hds.ent CONTAINS SUBUNITS:
A:2 B:3 C:4 D:5  

CHOOSE FIRST  SUBUNIT TO COMPARE(INPUT ACCORDING NUMBER):5

CHOOSE SECOND SUBUNIT TO COMPARE(INPUT ACCORDING NUMBER):0


input new MAXRMS,  filename to save string and filename  to  save 
coords: 

INPUT MAXRMS(0-10): 0.3
INPUT FILENAME TO SAVE COORDS: aln1.ent
INPUT FILENAME TO SAVE ALINED STRINGS: aln1.str

   WARNING. It is impossible to obtain files aln1.ent and aln1.str 
   in demo (unregistered) version of the program!!!

The  program  fits patterns according new set,  then demonstrates 
fitted (superimposed) subunits and distatces map.  
   


             VISUALISATION OF PROTEIN STRUCTURE (F9): 

You  can  see  stereoimage  of one or two 3D aligned proteins and 
operate them: 

1. Operation switches:   

   F1  - switch Off and On visualization of first protein.  
   F2  - switch Off and On visualization of second protein.  
   F3  - switch Off and On operation with first protein.  
   F4  - switch Off and On operation with second protein.
   TAB - switch between rotation and moving state.

2. Moving and rotation:

   a Rotation state:                                          
     Left  Arrow - rotation round "Y" axes;         
     Right Arrow - rotation round "Y" axes;
     Up    Arrow - rotation round "X" axes; 
     Down  Arrow - rotation round "X" axes;
     C           - rotation round "Z" axes;
     V           - rotation round "Z" axes.    

   b Moving state:
     Left  Arrow - moving LEFT;         
     Right Arrow - moving RIGHT;         
     Up    Arrow - moving UP; 
     Down  Arrow - moving DOWN;

3. Zoom:
   F9  - increase (zoom)
   F10 - decrease (unzoom)


4. Moving stereo cursor (white ball) along protein chain:

   F5 - moving cursor along protein chain of first protein;
   F6 - moving cursor along protein chain of first protein back;
   F7 - moving cursor along protein chain of second protein;
   F8 - moving cursor along protein chain of second protein back;
   - this keys active for "on" and "off" operation switches.

   Home - moving cursor to the beginning of protein chain.
   End  - moving cursor to the end of protein chain.


5. Selection  parts of proteins:
   a. Ctrl+{F5,F6,F7,F8,HOME,END} - marking (unmarking) the parts 
      of protein chains.  
   b. ESC    - unmarking all parts of protein chains.
   c. Enter  - make (in)vizible marked parts of proteins.

6. Shading: S

7. Acceleration:
   Shift+OPERATION - accelerates Operations.

8. Exit: ALT+X


   It is possible to interrupt the alignment process pressing ESC 
   The program will save files aln.ent and aln.str.

   WARNING. It is impossible to obtain files aln.ent and aln.str 
   in demo (unregistered) version of the program!!!

   To continue the alignment process you should type next command

   3dpaln.exe file1.ent aln.ent (or file2.ent) aln.ent cont

   program ask all parameters and continue alignment process.


   To check the new MAXRMS with the same result of alignment you 
   should type next command:

   3dpaln.exe file1.ent aln.ent (or file2.ent) aln.ent fot

   In both command strings the first file aln.ent (second parameter 
   in command string) is the source of coordinates for continue 
   alignment process or calculate new RMS. The second file aln.ent 
   (third parameter in command string) is the source of matching 
   information for same purpose.