CONFLEX Tutorials

Amino acid residue substitution function

[Overview]

Some amino acid residues of proteins present in the body may be mutated due to some influence. Even the protein with mutations of only a few residues can have dramatic changes in function and activity. In order to study such the phenomena by a simulation, using pdb file of a protein which has amino acid sequence and can be got from Protein Data Bank (PDB), you should apply amino acid substitutions to the protein for making a mutated protein.

When you use the pdb file provided by the PDB, you may need to correct data in the file due to lack of data on side chains of amino acids. CONFLEX allows you to replace or supplement the lacking side chains of amino acids in the pdb file before starting structure optimization or other calculations. Furthermore, CONFLEX provides you information on the lacking data in the pdb file regardless of side chain and back bone.

[Substitution of side chain]

We use a pdb file having missing atoms, correct the data, and perform a calculation.

  1. Execution using original coordinates
  2. Execution by substitution of side chain

First, search “1OHR” from PDB web site, and download 1ohr.pdb file.
This structure is HIV-1 protease containing an inhibitor obtained by X-ray crystal structure analysis and is dimeric protein with 99 amino acid residues. In the 1ohr.pdb file, the line starting with “REMARK 470” contains information on the missing atoms.

Lines starting with “REMARK 470” in the 1ohr.pdb file

REMARK 470                                                                      
REMARK 470 MISSING ATOM                                                         
REMARK 470 THE FOLLOWING RESIDUES HAVE MISSING ATOMS (M=MODEL NUMBER;           
REMARK 470 RES=RESIDUE NAME; C=CHAIN IDENTIFIER; SSEQ=SEQUENCE NUMBER;          
REMARK 470 I=INSERTION CODE):                                                   
REMARK 470   M RES CSSEQI  ATOMS                                                
REMARK 470     GLN A   7    CD   OE1  NE2                                       
REMARK 470     LYS A  14    CE   NZ                                             
REMARK 470     GLU A  34    CD   OE1  OE2                                       
REMARK 470     GLU A  35    CD   OE1  OE2                                       
REMARK 470     ARG A  41    CG   CD   NE   CZ   NH1  NH2                        
REMARK 470     LYS A  43    CG   CD   CE   NZ                                   
REMARK 470     LYS A  45    CG   CD   CE   NZ                                   
REMARK 470     LYS A  55    CD   CE   NZ                                        
REMARK 470     GLN A  61    CG   CD   OE1  NE2                                  
REMARK 470     LYS A  70    CE   NZ                                             
REMARK 470     GLN B   7    CD   OE1  NE2                                       
REMARK 470     LYS B  14    CG   CD   CE   NZ                                   
REMARK 470     ARG B  41    CG   CD   NE   CZ   NH1  NH2                        
REMARK 470     LYS B  55    CE   NZ                                             
REMARK 470     GLN B  61    CD   OE1  NE2  

[REMARK 470     GLN A   7    CD   OE1  NE2] means that the Cδ, Oε1, and Nε2 of glutamine at the 7th residue of the A chain are missing. CONFLEX uses lines starting with “ATOM” to get structure data. Therefore, if you modify the data of “REMARK 470”, the modifications do not effect on the calculation.

Execute CONFLEX using the 1ohr.pdb file.

[Execution by Interface]

Open the 1ohr.pdb file by CONFLEX Interface. Interface 1OHR

Select [CONFLEX] in Calculation menu, and click Detail Settings in the calculation setting dialog displayed. Basic Settings

Next, click Edit & Submit at bottom right in the detail setting dialog. Detail Settings

After the click, a dialog with the keywords for the calculation settings is displayed. Edit and Submit

Delete all “PDB_CONECT=” keywords that automatically created by Interface, and add information of double bonds in the inhibitor by “PDB_CONECT=” keyword. In “PDB_CONECT=(i,j,n)”, i and j are serial number of atoms described in the pdb file, and n is bond order. When you complete the modifications, click Submit. The calculation will start.

[Execution by command line]

The calculation settings are defined by describing keywords in the 1ohr.ini file.

1ohr.ini file

MMFF94S
PDB_CONECT=(1768,1774,2)
PDB_CONECT=(1781,1782,2)
PDB_CONECT=(1783,1784,2)
PDB_CONECT=(1785,1786,2)
PDB_CONECT=(1787,1788,2)
PDB_CONECT=(1792,1793,2)
PDB_CONECT=(1794,1795,2)
PDB_CONECT=(1796,1797,2)

[MMFF94S] keyword means to use MMFF94s force field.
[PDB_CONECT=] keywords set information of double bonds in the inhibitor. In “PDB_CONECT=(i,j,n)”, the i and j are serial number of atoms described in the pdb file, and the n is bond order.

Store the two files of 1ohr.pdb and 1ohr.ini in an one folder, and execute below command. The calculation will start.

C:\CONFLEX\bin\flex9a_win_x64.exe   -par   C:\CONFLEX\par   1ohrenter

The above command is for Windows OS. For the other OS, please refer to [How to execute CONFLEX].

Calculation results

After the calculation finished, if you check the bso file outputted, you will get the following messages.
The calculation has some errors. Make sure that these errors correspond to the missing atoms mentioned above.

Error messages in the bso file

 PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN A   7
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(GLN,A,7)
PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  14
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(LYS,A,14)
PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  34
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(GLU,A,34)
PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  35
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(GLU,A,35)
PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  41
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(ARG,A,41)
PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  43
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(LYS,A,43)
PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  45
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(LYS,A,45)
PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  55
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(LYS,A,55)
PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  61
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(GLN,A,61)
PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  70
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(LYS,A,70)
PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN B   7
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(GLN,B,7)
PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN B  14
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(LYS,B,14)
PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN B  41
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(ARG,B,41)
PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN B  55
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(LYS,B,55)
PDB_EXT/CHECK_AND_BUILD: ERROR -- INCLUDE UNKNOWN RESIDUE IN CHAIN B  61
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(GLN,B,61)

Here, there are two ways to avoid the errors and perform the calculation.

Execution using original coordinates

One of the ways is to execute the calculation using original coordinates.
In this case, add [PDB_NOMUTATE] keyword to the calculation setting. [PDB_NOMUTATE] means to directly use original data that has the missing atoms in the amino acid residues.

[Execution by Interface]

Add [PDB_NOMUTATE] keyword to the dialog displayed by click Edit & Submit. Edit and Submit NoMutate When you complete the modification, click Submit. The calculation will start.

[Execution by command line]

Add [PDB_NOMUTATE] keyword to the 1ohr.ini file.

1ohr.ini file

MMFF94S
PDB_NOMUTATE
PDB_CONECT=(1768,1774,2)
PDB_CONECT=(1781,1782,2)
PDB_CONECT=(1783,1784,2)
PDB_CONECT=(1785,1786,2)
PDB_CONECT=(1787,1788,2)
PDB_CONECT=(1792,1793,2)
PDB_CONECT=(1794,1795,2)
PDB_CONECT=(1796,1797,2)

Store the two files of 1ohr.pdb and 1ohr.ini in an one folder, and execute below command. The calculation will start.

C:\CONFLEX\bin\flex9a_win_x64.exe   -par   C:\CONFLEX\par   1ohrenter

The above command is for Windows OS. For the other OS, please refer to [How to execute CONFLEX].

Calculation results

After the calculation finished, if you check the bso file outputted, you will get the following messages. You can see that the error messages have changed to the warning ones.
* Note that it takes a very long time to finish the calculation because it is the large molecule.

Warning messages in the bso file

 PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN A   7
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(GLN,A,7)
PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  14
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(LYS,A,14)
PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  34
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(GLU,A,34)
PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  35
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(GLU,A,35)
PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  41
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(ARG,A,41)
PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  43
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(LYS,A,43)
PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  45
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(LYS,A,45)
PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  55
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(LYS,A,55)
PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  61
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(GLN,A,61)
PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN A  70
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(LYS,A,70)
PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN B   7
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(GLN,B,7)
PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN B  14
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(LYS,B,14)
PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN B  41
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(ARG,B,41)
PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN B  55
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(LYS,B,55)
PDB_EXT/CHECK_AND_BUILD: WARNING -- INCLUDE UNKNOWN RESIDUE IN CHAIN B  61
 *PLEASE SET KEYWORD(S) :PDB_MUTATE=(GLN,B,61)

Execution by substitution of side chain

The other way is to make up for the missing atoms and perform the calculation.
You can see “PDB_MUTATE=” keywords in the warning messages above. Delete “PDB_NOMUTATE” keyword, and add “PDB_MUTATE=” keywords shown above.

“PDB_MUTATE=” is a keyword for replacing or supplementing a side chain. The first element in the parenthesis is name of amino acid, the second one is ID of protein chain, and the third one is residue number. The name of amino acid supports 1-character, 3-characters, and full-characters notations (refer to below table). “PDB_MUTATE=(GLN,A,7)” means to replace the 7th residue of the A chain to glutamine.

Name of amino acid 1-Character 3-Characters Full name
Alanine A ALA ALANINE
Cysteine C CYS CYSTEINE
Aspartic acid D ASP ASPARTIC_ACID
Glutamic acid E GLU GLUTAMIC_ACID
Phenylalanine F PHE PHENYLALANINE
Glycine G GLY GLYCINE
Histidine H HIS HISTIDINE
Isoleucine I ILE ISOLEUCINE
Lysine K LYS LYSINE
Leucine L LEU LEUCINE
Methionine M MET METHIONINE
Asparagine N ASN ASPARAGINE
Proline P PRO PROLINE
Glutamine Q GLN GLUTAMINE
Arginine R ARG ARGININE
Serine S SER SERINE
Threonine T THR THREONINE
Valine V VAL VALINE
Tryptophan W TRP TRYPTOPHAN
Tyrosine Y TYR TYROSINE

[Execution by Interface]

Add [PDB_MUTATE] keywords to the dialog displayed by click Edit & Submit. Edit and Submit When you complete the modification, click Submit. The calculation will start.

[Execution by command line]

Add [PDB_MUTATE] keywords to the 1ohr.ini file

1ohr.ini file

MMFF94S
PDB_MUTATE=(GLN,A,7)
PDB_MUTATE=(LYS,A,14)
PDB_MUTATE=(GLU,A,34)
PDB_MUTATE=(GLU,A,35)
PDB_MUTATE=(ARG,A,41)
PDB_MUTATE=(LYS,A,43)
PDB_MUTATE=(LYS,A,45)
PDB_MUTATE=(LYS,A,55)
PDB_MUTATE=(GLN,A,61)
PDB_MUTATE=(LYS,A,70)
PDB_MUTATE=(GLN,B,7)
PDB_MUTATE=(LYS,B,14)
PDB_MUTATE=(ARG,B,41)
PDB_MUTATE=(LYS,B,55)
PDB_MUTATE=(GLN,B,61)
PDB_CONECT=(1768,1774,2)
PDB_CONECT=(1781,1782,2)
PDB_CONECT=(1783,1784,2)
PDB_CONECT=(1785,1786,2)
PDB_CONECT=(1787,1788,2)
PDB_CONECT=(1792,1793,2)
PDB_CONECT=(1794,1795,2)
PDB_CONECT=(1796,1797,2)

Store the two files of 1ohr.pdb and 1ohr.ini in an one folder, and execute below command. The calculation will start.

C:\CONFLEX\bin\flex9a_win_x64.exe   -par   C:\CONFLEX\par   1ohrenter

The above command is for Windows OS. For the other OS, please refer to [How to execute CONFLEX].

Calculation results

After the calculation finished, if you check the bso file outputted, you will get the following messages.

Information on "MUTATE RESIDUE" in the bso file

PDB_EXT: MUTATE RESIDUE FROM    7GLN TO GLN
PDB_EXT: MUTATE RESIDUE FROM   14LYS TO LYS
PDB_EXT: MUTATE RESIDUE FROM   34GLU TO GLU
PDB_EXT: MUTATE RESIDUE FROM   35GLU TO GLU
PDB_EXT: MUTATE RESIDUE FROM   41ARG TO ARG
PDB_EXT: MUTATE RESIDUE FROM   43LYS TO LYS
PDB_EXT: MUTATE RESIDUE FROM   45LYS TO LYS
PDB_EXT: MUTATE RESIDUE FROM   55LYS TO LYS
PDB_EXT: MUTATE RESIDUE FROM   61GLN TO GLN
PDB_EXT: MUTATE RESIDUE FROM   70LYS TO LYS
PDB_EXT: MUTATE RESIDUE FROM    7GLN TO GLN
PDB_EXT: MUTATE RESIDUE FROM   14LYS TO LYS
PDB_EXT: MUTATE RESIDUE FROM   41ARG TO ARG
PDB_EXT: MUTATE RESIDUE FROM   55LYS TO LYS
PDB_EXT: MUTATE RESIDUE FROM   61GLN TO GLN

In order to correct the missing atoms, you should specify name of the original amino acid by PDB_MUTATE keyword. If you want to change the sequence of amino acids in the protein, you should specify name of a different amino acid from the original by PDB_MUTATE keyword.