View source for User:Wayne Decatur/1ig8 to 3b8a (hexokinase) morph methods

==MORPH==
* First I changed chain X of 3b8a to chain A to match 1ig8 using [http://dicsoft2.physics.iisc.ernet.in/pdbgoodies/ PDB Goodies] for the protein. I knew PDB Goodies ignored and left out the heteroatoms so I changed the few lines of the glucose ligand to chain a by hand. The heterotoms will not be used by the Yale Morph server but I need them in the original file in order to be able to use fit or compare to add them in later.
* Submitted 1gi8 and the altered 3b8a to the Yale Morph [http://molmovdb.mbb.yale.edu/cgi-bin/beta.cgi beta server]
* TURNS OUT THIS NEXT ISSUE WAS NOT INVOLVED IN PROBLEM SO CAN SKIP TO TWO STEPS BELOW WHERE I WAS ABLE TO GET RESULT. ---When I received nothing back after several minutes I looked at the end of the sequences to see if they matched identically and noticed they didn't have the same C-terminus or even N-terminus. In fact what I thought were the same protein, according to FASTA sequence obtained for each PDB file from PDBsum, differ quite a bit:

<small><font face = 'courier'>
<pre>
1ig8               1 ---DVPKELMQQIENFEKIFTVPTETLQAVTKHFISELEKGLSKKGGNIP     47
                        |||||||.:|...|.:|||.:|||:.|.||||.||.|||:|||||||
3b8a               1 SMADVPKELMDEIHQLEDMFTVDSETLRKVVKHFIDELNKGLTKKGGNIP     50

1ig8              48 MIPGWVMDFPTGKESGDFLAIDLGGTNLRVVLVKLGGDRTFDTTQSKYRL     97
                     |||||||:||||||||::|||||||||||||||||.|:.|||||||||:|
3b8a              51 MIPGWVMEFPTGKESGNYLAIDLGGTNLRVVLVKLSGNHTFDTTQSKYKL    100

1ig8              98 PDAMRTTQNPDELWEFIADSLKAFIDEQFPQGISEPIPLGFTFSFPASQN    147
                     |..||||::.:|||.|||||||.|:.||.... ::.:|||||||:|||||
3b8a             101 PHDMRTTKHQEELWSFIADSLKDFMVEQELLN-TDTLPLGFTFSYPASQN    149

1ig8             148 KINEGILQRWTKGFDIPNIENHDVVPMLQKQITKRNIPIEVVALINDTTG    197
                     ||||||||||||||||||:|.|||||:||.:|:||.:|||:|||||||.|
3b8a             150 KINEGILQRWTKGFDIPNVEGHDVVPLLQNEISKRELPIEIVALINDTVG    199

1ig8             198 TLVASYYTDPETKMGVIFGTGVNGAYYDVCSDIEKLQGKLSDDIPPSAPM    247
                     ||:||||||||||||||||||||||:|||.||||||:|||:||||.::||
3b8a             200 TLIASYYTDPETKMGVIFGTGVNGAFYDVVSDIEKLEGKLADDIPSNSPM    249

1ig8             248 AINCEYGSFDNEHVVLPRTKYDITIDEESPRPGQQTFEKMSSGYYLGEIL    297
                     |||||||||||||:||||||||:.:||:|||||||.||||:|||||||:|
3b8a             250 AINCEYGSFDNEHLVLPRTKYDVAVDEQSPRPGQQAFEKMTSGYYLGELL    299

1ig8             298 RLALMDMYKQGFIFKNQDLSKFDKPFVMDTSYPARIEEDPFENLEDTDDL    347
                     ||.|:::.::|.:.|:|||||..:|::||||||||||:|||||||||||:
3b8a             300 RLVLLELNEKGLMLKDQDLSKLKQPYIMDTSYPARIEDDPFENLEDTDDI    349

1ig8             348 FQNEFGINTTVQERKLIRRLSELIGARAARLSVCGIAAICQKRGYKTGHI    397
                     ||.:||:.||:.||||||||.||||.|||||:||||||||||||||||||
3b8a             350 FQKDFGVKTTLPERKLIRRLCELIGTRAARLAVCGIAAICQKRGYKTGHI    399

1ig8             398 AADGSVYNRYPGFKEKAANALKDIYGWTQTSLDDYPIKIVPAEDGSGAGA    447
                     ||||||||:||||||.||..|:||||||..:..| ||.||||||||||||
3b8a             400 AADGSVYNKYPGFKEAAAKGLRDIYGWTGDASKD-PITIVPAEDGSGAGA    448

1ig8             448 AVIAALAQKRIAEGKSVGIIGA    469
                     ||||||::||||||||:|||||
3b8a             449 AVIAALSEKRIAEGKSLGIIGA    470
</pre>
<br>
</font></small><br/>
Turns out they are not the same protein; one is yeast hexokinase PI (3b8a) and the other is hexokinase PII (1ig8).

According to help page for the Yale Morph server this task was submitted, this shouldn't matter because it will do a ClustalW alignment. Plus Yale Morph [http://molmovdb.mbb.yale.edu/cgi-bin/beta.cgi beta server] says on the main page of the beta server, "this server will handle mutations and homologous sequences (only for proteins)." Frustratingly, though it does seem to matter. In the past, sometimes the files needed to be identical to me for anything to work on some versions of the Yale Morph servers. And unfortunately when it fails, it doesn't tell you why it failed or even that it failed. A lot of stuff they talk about never got implemented and maybe the alignment was part of it?<--NOPE, IT WAS A SERVER GLITCH. SEE NEXT TO SECTIONS.

*THIS WHOLE AVENUE WAS UNNECESSARY BUT IT HELPED LEAD TO ISSUE.---To get so they were the same two proteins, I submitted the FASTA sequence of 3b8a (without amino acids 15-17) (that i got from PDBsum) to SwissModel and told it to homology model it to 1ig8. Got that result and called 3b8amodeledto1ig8.pdb
** Submitted 3b8amodeledto1ig8.pdb and the 3b8achainA file to every version of the Yale Morph server I could find. However, I got nothing back for any attempt and when I looked at the new file I realized it had renumbered to start at 1, and so to get equal chains, I submitted the  FASTA sequence of 3b8a (without amino acids 15-17) (that i got from PDBsum) to SwissModel and told it to model it to 3b8aASchainA.pdb. This should renumber like it did when I submitted it to model against 1ig8. By doing the chain A version, I hope it helps insure I stay dealing with that chain only.
**Submitted the set of files on every version and it still failed to give me any completed job message.
**Then I tried some things that I knew worked in the past and they failed to elicit e-mails and so I realized server had problems and contacted them. They fixed something and I still wasn't receiving e-mail replies of completion. Decided to use an old job complete message I fortunately had and changed the URL to include the new submission confirmation IDs and I was able to get the test files.
**I informed the server people that maybe e-mails weren't being sent beyond Yale and they said that seemed to be problem and would work on it but I had a work around for now of updating old URL with new submissions.

*So I resubmitted 1gi8 and the altered 3b8a (altered to chain A) to the Yale Morph [http://molmovdb.mbb.yale.edu/cgi-bin/beta.cgi beta server] and noted the confirmation number. Later tried th confirmation number in the URL (<nowiki>http://www.molmovdb.org/cgi-bin/morph.cgi?ID=b049347-636</nowiki>) and result was there. 
**Downloaded result.
**Edited model numbers to go from 1-12 instead of 0 to 11 since the 0 model number causes issues when I try to call it in Jmol. --> file = 1ig8TO3b8a.pdb.
**Opened "3b8aASchainA.pdb" and copied SO4 and glucose from this file and added it to end of last frame (12th) in "1ig8TO3b8a.pdb"  and saved as "1ig8TO3b8aplusheteroatomstolastframe.pdb"
**In preparation for adding glucose falling into active site, I copied ""1ig8TO3b8aplusheteroatomstolastframe.pdb" and renamed it to "1ig8TO3b8aplusheteroatomsto11n12frames.pdb" and then copied glucose from frame 12 to end of frame 11. Now I need to move glucose in frame 11 out and rotate slightly to look like out of the active site a tiny bit relative to frame 12.
***To do that, issued in console:
set dragSelected ON;
set PICKING select molecule;
set allowRotateSelected ON;
select model=11 and [BGC];
AND AFTER THAT COMMAND TO PICK GLUCOSE held down alt and shift and clicked on glucose molecule and pulled molecule out of active site a little and holding alt along rotated it slightly.

Then issued:
select all;
write COORDS PDB "1ig8TO3b8aplusheteroatomsto10thru12frames.pdb"

Opened "1ig8TO3b8aplusheteroatomsto10thru12frames.pdb" in text editor and copied the molecule of glucose from frame 11 to 10.
Now I need to repeat the pulling and rotating of the glucose molecule in frame 10 so looks a little more like drifted out of active site relative next two frames.
REALIZED AFTER STARTING THIS APPROACH THAT ALTHOUGH IT WOULD PRODUCE A COOL ANIMATION, IT MIGHT NOT BE VERY CHEMICALLY ACCURATE AND SO MAYBE FOR NEEDS I HAVE BETTER TO HAVE GLUCOSE AND SULFATE ION JUST AT ENDPOINTS, WITH AN UNBOUND REPRESENTATION OF GLUCOSE FOR 1ig8.
* To add sulfate ion from 1ig8 back to first frame of "1ig8TO3b8a.pdb":<br/>
**First made file of just first frame of "1ig8TO3b8a.pdb", then loaded that into Jmol.<br/>
**Then used open with append option to open the original 1ig8 pdb fle.
**Then issued in the console:<br/>
compare {2.1}{1.1} rotate translate;
select 2.1;
write COORDS PDB "heteroatomsforfirstframeof1ig8TO3b8a.pdb";
**Opened that new file and copied sulfate ion and pasted into the morph file "1ig8TO3b8aplusheteroatomstolastframe.pdb", renumbered atoms by adding another 20 so don't overlap with heteroatoms in last frame, and saved as 1ig8TO3b8aplusheteroatomstofirstnlastframe.pdb . (Actually added this sulfate ion to the first frame of the last intermediate I made, "1ig8TO3b8aplusheteroatomsto10thru12frames.pdb", because may end up using those at some point.)<br/>
**I added the glucose I had from frame 10 of "1ig8TO3b8aplusheteroatomsto10thru12frames.pdb" to the end of frame 1 of "1ig8TO3b8aplusheteroatomsto10thru12frames.pdb", renumbered to be after sulfate ion atom numbers I had there already, and saved as "1ig8TO3b8aplusglucose.pdb ".
**Opened "1ig8TO3b8aplusglucose.pdb " and set so viewing first frame (model) and issued following commands in console:<br/>
set dragSelected ON;
set PICKING select molecule;
set allowRotateSelected ON;
select model=1 and [BGC];
AND AFTER THAT COMMAND TO PICK GLUCOSE held down alt and shift and clicked on glucose molecule and pulled molecule out of active site.

Then issued in the console:
select all;
write COORDS PDB "1ig8TO3b8aplusglucose.pdb";
RENAMED file to "1ig8TO3b8aplusglucoseENDPT.pdb" since glucose only at end points.
*Now that I had glucose at end points, thought I'd see how it looked adding glucose back to each frame. Going back to "1ig8TO3b8aplusheteroatomsto10thru12frames.pdb" where I had added sulfate ion to frame 1, I continued the process of adjusting glucose in higher number frames, copying to previous frame and adjusting successively to be further from active site. Due to sulfate ion in frame 1, I had to re-adjust each of earlier frames to keep glucose away from it. Did this by using buttons in Jmol application to click through to frame I needed to adjust glucose in and issued command like used above with that model number and adjusted. For frame 1, I found in fact I needed to temporarily remove sulfate ion atoms from pdb file while I was working on glucose or Jmol was connecting glucose and sulfate because they were so close. Eventually seemed to be away from that area and so placed sulfate ion back in frame 1 and saved file as "1ig8TO3b8aplusglucoseALLframes.pdb" with:
select all; write COORDS PDB "1ig8TO3b8aplusglucoseALLframes.pdb";