[Jmol-users] Finding protein chain termini

[Eric M. <em...@mi...>]

Finding the ends of a protein chain is easy for a human being. But 
writing code to find the ends of the protein chains in any PDB file in a 
general manner seems challenging. I have procrastinated dealing with 
this for years because of its seeming complexity. Here I lay out my 
ideas about how to do this. If anyone has other ideas, especially 
simpler methods, please let me know!

Why? I would like to have buttons in FirstGlance that zoom in on the 
terminal residues (with coordinates) of a protein chain for any PDB 
entry displayed. Also, in the /Charge/ view in FirstGlance, I would like 
to show whether the terminal amino and carboxy residues that have 
coordinates are charged -- that is, whether they are the termini of the 
experimental protein, or whether the actual terminal amino acids are 
missing coordinates. Or whether the terminal amino acids are blocked.

I prefer to rely on sequence numbers as LITTLE as possible because they 
are not required to increase monotonically between the N and C termini, 
may skip values, and may have insertion codes with the same sequence 
numbers. See examples in a new article I wrote recently: 
http://proteopedia.org/w/Unusual_sequence_numbering

For the C terminus, I expected that I could rely upon OXT (a C-terminal 
oxygen atom). Then if a protein chain has no OXT, it would mean that the 
C terminal residue(s) are missing coordinates. However, I have stumbled 
across several examples with zero missing residues, yet no OXT on the 
C-terminal residue. Rachel Kramer Green at RCSB has confirmed that there 
is no requirement for an OXT atom on the C-terminal residue. It is up to 
the authors of the PDB entry. Therefore one cannot rely on OXT.

Thus, when OXT is present, then that residue is indeed an end residue 
and is charged. But when OXT is absent, you don't know.

Quite recently, I realized that that Jmol's groupindex may offer the 
best solution. *If there is a better or simpler solution, I would like 
to know it!* Groupindex is not the sequence number. It is a 
group/residue number assigned by Jmol for internal use. Jmol assigns 
somewhat different groupindex numbering schemes to PDB format vs. mmCIF 
format files.

I will make two assumptions that appear to me to be true.

 1. In PDB files (including mmCIF format), residues within a chain are
    always listed in order from N terminus to C terminus.
 2. groupindex increases monotonically in the order in which residues
    are listed in the PDB file.

METHODS

*1. Minimal or maximal groupindex in each protein chain*

Let's try for the N terminal residue of chain A by finding the lowest 
groupindex:

AMin = {chain=A and protein}.groupindex.min;
select groupindex=AMin

In determining the minimum groupindex, one cannot exclude HETERO groups 
because termini may be non-standard amino acids, or not even amino acids 
(e.g. acetyl, ACE). Examples: the N-terminal residues of 12 of the 24 
chains in 4gxu are pyroglutamic acid [PCA]1, a hetero group. The 
N-terminal residues in 4mdh are ACE, acetyl.

*2. Is the end candidate peptide-bonded to the adjacent residue?*

Next we need to determine if the chain A residue with the lowest 
groupindex is peptide-bonded to the residue with the next higher 
groupindex. At the N-terminus I expect that it always will be but 
perhaps I will be surprised.

select groupindex=0 and *.c and connected(groupindex=1 and *.n)

But lets consider the C-terminus, where we will start with the chain A 
protein residue with the highest groupindex. Ligands and water are also 
assigned "chain A" and as far as I know, always have groupindices 
*higher* than the protein chain members. Water is excluded by limiting 
ourselves to protein. But ligands are not.

Some ligands are deemed "protein" by Jmol, presumaby because they have 
an alpha carbon (e.g. 3ES in 2xy9), but are not covalent members of the 
protein chain.

When the group with the highest index is not covalently peptide-bonded 
to the chain, our next candidate will be the next lower groupindex, and 
so forth until we find the highest groupindex that is peptide-bonded to 
the chain.

*3. Mono- and dipeptide ligands*

All chains of length 3 or more amino acids are represented with ATOM 
records, have distinct chain identifiers (A, B, C, etc.), and have 
SEQRES records.

However, single amino acid ligands (e.g. Gly308 in 4cpa) and dipeptides 
are by PDB rules deemed HETERO, have no SEQRES records (but HET and 
HETNAM records), and are assigned the same chain identifier as the chain 
to which they are bound. Rachel Kramer Green of RCSB has confirmed this 
rule. (There are a handful of cases deposited by PDBe that do not 
conform to this rule -- they will be remediated.)

A single amino acid is easily excluded since it is not peptide-bonded to 
the residue having one groupindex lower. However, the C-terminal residue 
of a dipeptide ligand is peptide-bonded to the  residue one groupindex 
lower, but is not part of the larger protein chain with the same chain 
identifier. Therefore we must require that any candidate for a terminal 
residue be peptide bonded to the adjacent residue, and that adjacent 
residue be peptide-bonded to the next adjacent residue (THREE peptide 
bonded residues).

For example 3deq contains chain B of length 345 amino acids, 341 of 
which have coordinates (2 missing at each end). Sequence numbers are 
3-343 and groupindices 436-776. Bound to chain B is a dipeptide (HETATM, 
also deemed "chain B") Ala411-Leu412. Jmol assigns this dipeptide 
groupindices 777-778 for the PDB format file, and 1366-1367 for the 
mmCIF file.

Taking the PDB file of 3deq, dipeptide groupindex 778 (the highest 
protein groupindex in chain B) is peptide-bonded to 777, but 777 is NOT 
peptide-bonded to 776, and thus 778 is REJECTED as the C-terminus of 
chain B. When we get to 776, we find it is peptide-bonded to 775, which 
is peptide-bonded to 774, and thus we ACCEPT 776 as the C-terminal residue.

*4. Is the terminal residue with coordinates charged?*

Now we have determined the terminal residues with coordinates in each 
chain. But are their amino or carboxy termini charged?*
*

*4A. Is the terminal residue missing coordinates?*

If the terminal residue is missing coordinates due to disorder, then the 
terminal residue with coordinates (determined by the above methods) is 
NOT charged.

If the C-terminal residue has an OXT atom, it is charged (and there 
cannot be a more-C-terminal residue that is missing coordinates).

For all N-terminal residues, and C-terminal residues lacking OXT, we 
need to ask whether the terminal residue in the experimental protein is 
missing coordinates. (Recall that authors often fail to put OXT atoms on 
C-terminal residues, and this is "legal".)

I am aware of two possible methods.

 1. Determine whether the next more-terminal *sequence number* (ATOM or
    HETERO) is missing.
 2. Examine the sequence alignment between SEQRES and ATOM records to
    determine whether the terminal candidate is indeed the terminus in
    SEQRES.

I plan to use method 1 although in rare cases where near-terminal 
sequence numbers are not sequential, this would fail.

(mmCIF files contain the SEQRES to ATOM sequence alignment, lacking in 
PDB format files, but I have not yet adapted FirstGlance to use mmCIF.)

Missing residues are listed in REMARK 465 by chain, residue name, and 
sequence number.

Take the case of 3deq. We have decided, using the methods above, that 
Lys343 (groupindex 776) is the C-terminal residue with coordinates in 
chain B. But we find that chain B sequence number 344 is listed as 
missing coordinates in REMARK 465. Therefore, we know that Lys343 is not 
the real C-terminus, and hence it has no carboxy-terminal charge.

Although I don't know of an example (and don't know how to search for 
one), there may be cases where the terminal residue is missing and is 
not protein. Presumably an N-terminal acetyl group that is missing 
coordinates would be listed in REMARK 465.

*4B. Is the terminal residue blocked?*

Fairly often, the N terminus may be acetylated with an ACE hetero group 
(e.g. 4mdh).

We can determine whether the N-terminus is blocked by asking whether its 
main chain nitrogen atom is covalently bonded (Jmol "connected" 
function) to two non-hydrogen atoms (three in the case of proline). If 
yes, then it is not charged. If no, and if the real N-terminal residue 
is not missing, then our N-terminus candidate is charged.

The same strategy can be used to determine whether the C terminus is 
blocked. Is the C-terminal main chain carboxy carbon, lacking OXT, 
covalently bonded to three non-hydrogen atoms?

In the case of chain C in 5fpk, the C-terminal Ala4 is amidated. So its 
main chain carboxy carbon is covalently bonded to 3 non-hydrogen atoms: 
the alpha carbon, the main chain oxygen, and a nitrogen with the group 
name NH2. Thus, the carboxy terminus of chain C is NOT charged.

*5. Missing main chain atoms**

*In rare cases, some atoms of an amino acid have coordinates but not all 
main chain atoms do. In such cases, the above methods will fail. I will 
try to make FirstGlance alert the user in all such cases.

1h3o: the C-terminal residues of chains A and C, Thr918, have only a 
single atom, N. Jmol manages to deem this atom protein, despite it 
lacking an alpha carbon.
**
*Whew!*

Comments? Suggestions?

Thanks, Eric