This page describes how to use programs of the SPASM package together with O to find and inspect proteins that share a structural motif with your own protein. It uses the following programs:
Reference: Kleywegt, G.J. (1999). Recognition of spatial motifs in protein structures. J Mol Biol 285, 1887-1897. (MEDLINE)
Contents:
The first thing you need to do is to put the residues you want to look for in a small PDB file. In this example, we will use residues Trp A279 and Met B33 of the complex between acetylcholinesterase and the snake toxin fasciculin II (MEDLINE). You can cut these residues out of the PDB entry 1FSS, or cut and paste them from here (put them in a PDB file called 1fss.pdb):
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- ATOM 2144 N TRP A 279 10.520 24.780 31.275 1.00 53.83 1FSS2369 ATOM 2145 CA TRP A 279 9.591 25.831 30.895 1.00 53.83 1FSS2370 ATOM 2146 C TRP A 279 8.589 26.213 31.972 1.00 53.83 1FSS2371 ATOM 2147 O TRP A 279 7.863 27.194 31.833 1.00 53.83 1FSS2372 ATOM 2148 CB TRP A 279 8.936 25.480 29.566 1.00 53.83 1FSS2373 ATOM 2149 CG TRP A 279 9.955 25.461 28.453 1.00 53.83 1FSS2374 ATOM 2150 CD1 TRP A 279 10.577 24.364 27.912 1.00 53.83 1FSS2375 ATOM 2151 CD2 TRP A 279 10.491 26.601 27.772 1.00 53.83 1FSS2376 ATOM 2152 NE1 TRP A 279 11.464 24.759 26.937 1.00 53.83 1FSS2377 ATOM 2153 CE2 TRP A 279 11.429 26.126 26.830 1.00 53.83 1FSS2378 ATOM 2154 CE3 TRP A 279 10.266 27.981 27.864 1.00 53.83 1FSS2379 ATOM 2155 CZ2 TRP A 279 12.138 26.985 25.988 1.00 53.83 1FSS2380 ATOM 2156 CZ3 TRP A 279 10.969 28.830 27.029 1.00 53.83 1FSS2381 ATOM 2157 CH2 TRP A 279 11.893 28.330 26.103 1.00 53.83 1FSS2382 ATOM 4499 N MET B 33 4.078 25.286 24.096 1.00 17.83 1FSS4724 ATOM 4500 CA MET B 33 5.102 24.475 23.450 1.00 17.83 1FSS4725 ATOM 4501 C MET B 33 4.918 22.975 23.595 1.00 17.83 1FSS4726 ATOM 4502 O MET B 33 4.592 22.477 24.676 1.00 17.83 1FSS4727 ATOM 4503 CB MET B 33 6.455 24.846 24.022 1.00 17.83 1FSS4728 ATOM 4504 CG MET B 33 6.516 24.705 25.534 1.00 17.83 1FSS4729 ATOM 4505 SD MET B 33 7.770 25.770 26.209 1.00 17.83 1FSS4730 ATOM 4506 CE MET B 33 8.499 26.448 24.607 1.00 17.83 1FSS4731 END 1FSS5000 ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Make sure that SPASM is installed (and that the names of the PDB files in the SPASM database have been changed such that they point to your local copy of the PDB).
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- % 713 gerard sarek 21:05:46 spasm/sites > run spasm [...] SPASM database file ? (/home/gerard/lib/spasm.lib) [...] Which PDB file ? (0xyz.pdb) 1fss.pdb [...] Four-character ID for this run ? (1FSS) [...] Max superpositioning RMSD ? ( 1.500) [...] Max CA-CA distance mismatch ? ( 2.000) 3 Max SC-SC distance mismatch ? ( 2.000) 1 [...] Resolution cut-off (A) ? ( 999.900) [...] [do not allow any residue substitutions :] Substitution option ? ( 3) 1 [...] Conserve sequence directionality ? (N) [...] Conserve neighbouring residues ? (N) [...] Conserve sequence gaps ? (N) [...] Print distance matrices ? (N) [...] Print operators ? (N) [...] Extensive output ? (N) [...] [since we have only 2 residues, we *must* include MC and SC :] 0=SC, 1=MC+SC, 2=MC ? ( 1) [...] [since we will use SAVANT, we don't need the O macro file :] O macro and operator file ? (N) [...] SAVANT input file ? (Y) SAVANT input file ? (1fss.savant) [...] LSQMAN input file ? (N) [...] MSEQPRO sequence file ? (N) [...] ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----Now SPASM compares the proteins in its database to your motif and all Trp-Met pairs that are roughly in the same relative spatial positions as those in 1FSS will be listed as "hits" (note that some proteins yield more than one hit). Within half a minute, SPASM thus finds 346 hits in 283 distinct PDB entries:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- [...] ==> HIT : (8FAB) Compound : (FAB FRAGMENT FROM HUMAN IMMUNOGLOBULIN IGG1 (LAMBDA, HIL)) File : (/nfs/pdb/full/8fab.pdb) Residues : ( 428) Resol (A): ( 1.800) MATCH with RMSD 1.28 A for 4 pseudo-atoms TRP A 279 <---> TRP D 36 = 15.0 MET B 33 <---> MET D 81 = 6.0 Total BLOSUM-45 score : ( 21.000) -------------------------------------------------------- ==> HIT : (9LDT) Compound : (LACTATE DEHYDROGENASE (E.C.1.1.1.27) COMPLEX WITH NADH AND OXAMATE) File : (/nfs/pdb/full/9ldt.pdb) Residues : ( 331) Resol (A): ( 2.000) MATCH with RMSD 0.92 A for 4 pseudo-atoms TRP A 279 <---> TRP A 150 = 15.0 MET B 33 <---> MET A 274 = 6.0 Total BLOSUM-45 score : ( 21.000) -------------------------------------------------------- Nr of proteins found : ( 283) Nr of proteins tried : ( 2590) Total number of hits : ( 346) CPU total/user/sys : 36.6 35.8 0.8 Run again ? (Y) n [...] ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Now, if you had produced an O macro to look at the hits in O, you would have noticed that many of the hits are rather poor: the Trp and Met have similar distances, but their interaction is quite different from the one seen in 1FSS. Moreover, it is quite likely that O's database becomes full before the macro has read all 283 proteins. And it becomes difficult to switch 346 O graphics objects on and off. This is where SAVANT comes in ! It enables you to scrutinise the hits in more detail, doing a superpositioning on all (main-chain and/or side-chain) atoms. Here we will only use the side-chain atoms (we are not at all interested in the main chain), which will take about a minute of CPU time. (Note: SAVANT expects that a sub-directory called savant exists; if it does not, it will refuse to run. In that case, create such a directory by typing: mkdir savant)
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- % 715 gerard sarek 21:05:46 spasm/sites > run savant [...] SAVANT library file ? (/home/gerard/lib/savant.lib) [...] SAVANT input file ? (savant.inp) 1fss.savant SAVANT O macro ? (savant.omac) SAVANT ODB file ? (savant.odb) O2D plot file ? (savant.plt) [...] [do the detailed superpositioning using *only* the side-chain atoms:] Select mode: 0 = SC, 1 = MC + SC, 2 = MC Mode (0/1/2) ? ( 1) 0 [...] Hit PDB file : (/nfs/pdb/full/8fab.pdb) Residues in hit : ( 2) Hit will go to PDB file : (savant/1fss_8fab_1.pdb) Atoms in residues : ( 14 8) Atoms in common : ( 14) RMS distance : ( 1.093) Hit PDB file : (/nfs/pdb/full/9ldt.pdb) Residues in hit : ( 2) Hit will go to PDB file : (savant/1fss_9ldt_1.pdb) Atoms in residues : ( 14 8) Atoms in common : ( 14) RMS distance : ( 1.202) [...] CPU-time taken : User - 60.9 Sys - 7.9 Total - 68.8 [...] ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Next we can use DEJANA to select only the best hits for display in O:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- % 716 gerard sarek 21:05:46 spasm/sites > run dejana [...] O macro (DEJAVU/LSQMAN/SPASM/RIGOR/SAVANT) ? (lsqman.omac) savant.omac Reading hits ... # 1 ID 119l1 Nres 14 RMSD 1.43 A # 2 ID 119l2 Nres 14 RMSD 1.35 A [...] # 345 ID 8fab1 Nres 14 RMSD 1.09 A # 346 ID 9ldt1 Nres 14 RMSD 1.20 A Nr of hits (> 0 atoms/residues/SSEs) : ( 346) ------------------------------------------ Min nr of matched atoms/residues/SSEs ? ( 1) Max RMSD of matched atoms/residues/SSEs ? ( 999.990) Sorting hits ... Nr of hits left : ( 346) # 1 ID 1req1 Nres 14 RMSD 0.54 A # 2 ID 1cto1 Nres 14 RMSD 0.57 A # 3 ID 1aro1 Nres 14 RMSD 0.69 A # 4 ID 2hbg1 Nres 14 RMSD 0.69 A # 5 ID 1ppf1 Nres 14 RMSD 0.74 A # 6 ID 4mdh1 Nres 14 RMSD 0.76 A # 7 ID 1pnk1 Nres 14 RMSD 0.77 A # 8 ID 1kmm1 Nres 14 RMSD 0.80 A # 9 ID 1phc1 Nres 14 RMSD 0.80 A # 10 ID 3fru1 Nres 14 RMSD 0.80 A # 11 ID 1bdm1 Nres 14 RMSD 0.81 A # 12 ID 1bak2 Nres 14 RMSD 0.82 A [...] # 344 ID 1yge2 Nres 14 RMSD 2.45 A # 345 ID 1ihp1 Nres 14 RMSD 2.47 A # 346 ID 1gow1 Nres 14 RMSD 2.57 A Select one of the following options: 0 = re-enter criteria and re-sort 1 = write new O macro with current hits 2 = quit program without writing new O macro Option (0, 1, 2) ? ( 0) ------------------------------------------ Min nr of matched atoms/residues/SSEs ? ( 1) 10 Max RMSD of matched atoms/residues/SSEs ? ( 999.990) 0.7 Sorting hits ... Nr of hits left : ( 4) # 1 ID 1req1 Nres 14 RMSD 0.54 A # 2 ID 1cto1 Nres 14 RMSD 0.57 A # 3 ID 1aro1 Nres 14 RMSD 0.69 A # 4 ID 2hbg1 Nres 14 RMSD 0.69 A Select one of the following options: 0 = re-enter criteria and re-sort 1 = write new O macro with current hits 2 = quit program without writing new O macro Option (0, 1, 2) ? ( 0) 1 New O macro file ? (dejana.omac) Writing hits ... Processing PDB code : (1req1) Processing PDB code : (1cto1) Processing PDB code : (1aro1) Processing PDB code : (2hbg1) New O macro written ... [...] ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
The result of running DEJANA is an O macro that will only display the top 4 hits (which all have an RMSD < 0.7 Å for 14 side-chain atoms). Simply start up O, and execute the macro:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- % 717 gerard sarek 21:05:46 spasm/sites > ono [...] As4> File not found in path: on_startup As4> Indirect file does not exist. @dejana.omac As4> Macro in computer file-system. Heap> Created by SAVANT V. 990907/0.3 at Tue Sep 7 21:24:55 1999 for gerard Sam> File type is PDB Sam> Database compressed. [...] Sam> Molecule 2HBG1 contained 2 residues and 22 atoms Sam> PDB is not a visible command. Mol> Object not in list, cannot delete it: 2HBG1 Mol> No connectivity Db for 2HBG1 Mol> Database compressed. mol connectivity is 30 ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----Hint: if you want the hits displayed as ball-and-stick models as well, do the following before executing the macro:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- cat dejana.omac | sed -e 's/! ske/ske/' > q ; mv q dejana.omac ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Hint: don't forget that your savant sub-directory still contains one little PDB file for each of the 346 hits ... You may want to remove some of those.
Does your result look something like this ?
If you have a motif, but you want to allow variations (e.g., Arg could also be Lys, Tyr could also be Phe, etc.), you can do "fuzzy matching" in SPASM. As an example, prepare a PDB file containing Arg 111, Arg 132, and Tyr 134 of PDB entry 1CBS (these residues constitute the fatty-acid-binding motif in cellular retinoic-acid-binding protein).
Start up SPASM and answer the questions, until you get to:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- You may opt to allow substitutions of certain residue types. You have the following options: (1) Do not allow substitutions (2) Only allow D/E, N/Q, L/I, F/Y and R/K (3) Use BLOSUM-45 to decide (4) User-defined substitutions Substitution option ? ( 3) 2 ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
In this case, we only need to allow F/Y and R/K substitutions, so we can select option 2. If you want to, you can also use the BLOSUM-45 substitution matrix instead. It will list the matrix values for those residue types that occur in your motif. You can then decide on a cut-off value, and all residue types whose matrix values is not less than your cut-off value will be allowed:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
You may opt to allow substitutions of certain
residue types. You have the following options:
(1) Do not allow substitutions
(2) Only allow D/E, N/Q, L/I, F/Y and R/K
(3) Use BLOSUM-45 to decide
(4) User-defined substitutions
Substitution option ? ( 3) 3
ALA ARG ASN ASP CYS GLU GLN GLY HIS ILE LEU LYS MET PHE PRO SER THR TRP TYR VAL
ARG -2.0 7.0 0.0 -1.0 -3.0 1.0 0.0 -2.0 0.0 -3.0 -2.0 3.0 -1.0 -2.0 -2.0 -1.0 -1.0 -2.0 -1.0 -2.0
TYR -2.0 -1.0 -2.0 -2.0 -3.0 -1.0 -2.0 -3.0 2.0 0.0 0.0 -1.0 0.0 3.0 -3.0 -2.0 -1.0 3.0 8.0 -1.0
Statistics for entire BLOSUM-45 matrix:
Average value : ( -0.918)
St. dev. : ( 2.467)
Minimum : ( -5.000)
Maximum : ( 15.000)
Matrix value cut-off ? ( 3.000)
Allowed substitutions :
ARG (ARG LYS)
TYR (TYR PHE TRP)
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Alternatively, you can indicate yourself which substitutions will be allowed (and SPASM will allow you to do silly things here, e.g. to allow Ala-Trp substitutions, if you so insist):
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- You may opt to allow substitutions of certain residue types. You have the following options: (1) Do not allow substitutions (2) Only allow D/E, N/Q, L/I, F/Y and R/K (3) Use BLOSUM-45 to decide (4) User-defined substitutions Substitution option ? ( 3) 4 Enter allowed substitutions in 3-letter code: Which types to allow for ARG ? (ARG) arg lys Which types to allow for TYR ? (TYR) tyr phe ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Once you have decided what to do, run SPASM as usual. (Note: you may want to be extra generous with the allowed mismatch distances.)
Run SAVANT (version 1.0 or later) and make sure to feed it a library that contains a description of the atom types in different residue types that you consider matchable (except CB atoms which SAVANT will always consider matchable), e.g.:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- [...] ALT PHE CG TYR CG ALT PHE CD1 TYR CD1 ALT PHE CD1 TYR CD2 ALT PHE CE1 TYR CE1 ALT PHE CE1 TYR CE2 ALT PHE CD2 TYR CD2 ALT PHE CD2 TYR CD1 ALT PHE CE2 TYR CE1 ALT PHE CE2 TYR CE2 ALT PHE CZ TYR CZ ! ALT PHE CG TRP CG ALT PHE CG HIS CG ALT TYR CG TRP CG ALT TYR CG HIS CG ALT TRP CG HIS CG ! ALT ARG CG LYS CG ALT ARG CD LYS CD [...] ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Note that with our motif, any lysine that is matched to one of our arginines will be included in the least-squares superpositioning with not only its main-chain atoms (if you include those, that is), but also with its CB (standard behaviour of SAVANT) and its CG and CD atoms (read from the library file).
Also note that the matching of Tyr and Phe is ambiguous for several atom types (e.g., Phe CD1 can be matched with Tyr CD1 and CD2). SAVANT will use the match that gives the lowest overall RMSD. However ... SAVANT does not try all possible permutations ! This means that the same atom can sometimes be used twice (see the first example in the output below for an example: Tyr CD1 and CD2 are both matched to Phe CD2). Also, sometimes you may get "silly" matches such as CD1-CD1/CD2-CD2 with CE1-CE2/CE2-CE1. However, usually things work fine, and if this is not the case it tends to be for the poorer hits (compare the second example in the output below with the first).
Now run SAVANT, and only include the side-chain atoms in the superpositioning:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- SAVANT library file ? (/home/gerard/lib/savant.lib) Nr of AMB lines : ( 11) Nr of ALT lines : ( 37) SAVANT input file ? (savant.inp) cra2.savant SAVANT O macro ? (savant.omac) SAVANT ODB file ? (savant.odb) O2D plot file ? (savant.plt) Select mode: 0 = SC, 1 = MC + SC, 2 = MC Mode (0/1/2) ? ( 1) 0 MODE : ( 0) SC only Pattern PDB file : (cra2.pdb) Residues in pattern : ( 3) Atoms in residues : ( 11 11 12) Total : ( 34) Ambiguous : ( 8) Alternative : ( 10) Hit PDB file : (/nfs/pdb/full/169l.pdb) Residues in hit : ( 3) Hit will go to PDB file : (savant/cra2_169l_1.pdb) Atoms in residues : ( 9 11 11) ==> Match ARG CB <-> LYS CB ==> Alternative ? ARG CG ==> Alternative ? ARG CD ==> Not matchable ARG NE ==> Not matchable ARG CZ ==> Not matchable ARG NH1 ==> Not matchable ARG NH2 ==> Match ARG CB <-> ARG CB ==> Match ARG CG <-> ARG CG ==> Match ARG CD <-> ARG CD ==> Match ARG NE <-> ARG NE ==> Match ARG CZ <-> ARG CZ ==> Match ARG NH1 <-> ARG NH1 ==> Match ARG NH2 <-> ARG NH2 ==> Match TYR CB <-> PHE CB ==> Alternative ? TYR CG ==> Alternative ? TYR CD1 ==> Alternative ? TYR CE1 ==> Alternative ? TYR CD2 ==> Alternative ? TYR CE2 ==> Alternative ? TYR CZ ==> Not matchable TYR OH Atoms in common : ( 9) RMS distance : ( 1.145) Nr ambiguous : ( 2) ARG 132 NH1 <-> NH2 => RMSD (A) : ( 1.404) Lowest RMSD : ( 1.145) Nr alternatives : ( 8) ARG 111 CG <-> LYS A 60 CG => RMSD (A) : ( 1.228) ARG 111 CD <-> LYS A 60 CD => RMSD (A) : ( 1.425) TYR 134 CG <-> PHE A 4 CG => RMSD (A) : ( 1.440) TYR 134 CD1 <-> PHE A 4 CD1 => RMSD (A) : ( 1.526) TYR 134 CD1 <-> PHE A 4 CD2 => RMSD (A) : ( 1.418) TYR 134 CE1 <-> PHE A 4 CE1 => RMSD (A) : ( 1.648) TYR 134 CE1 <-> PHE A 4 CE2 => RMSD (A) : ( 1.452) TYR 134 CD2 <-> PHE A 4 CD1 => RMSD (A) : ( 1.698) TYR 134 CD2 <-> PHE A 4 CD2 => RMSD (A) : ( 1.493) TYR 134 CE2 <-> PHE A 4 CE1 => RMSD (A) : ( 1.884) TYR 134 CE2 <-> PHE A 4 CE2 => RMSD (A) : ( 1.637) TYR 134 CZ <-> PHE A 4 CZ => RMSD (A) : ( 1.791) Final RMSD : ( 1.791) Nr matches : ( 17) [...] Hit PDB file : (/nfs/pdb/full/1waj.pdb) Residues in hit : ( 3) Hit will go to PDB file : (savant/cra2_1waj_1.pdb) Atoms in residues : ( 9 11 11) ==> Match ARG CB <-> LYS CB ==> Alternative ? ARG CG ==> Alternative ? ARG CD ==> Not matchable ARG NE ==> Not matchable ARG CZ ==> Not matchable ARG NH1 ==> Not matchable ARG NH2 ==> Match ARG CB <-> ARG CB ==> Match ARG CG <-> ARG CG ==> Match ARG CD <-> ARG CD ==> Match ARG NE <-> ARG NE ==> Match ARG CZ <-> ARG CZ ==> Match ARG NH1 <-> ARG NH1 ==> Match ARG NH2 <-> ARG NH2 ==> Match TYR CB <-> PHE CB ==> Alternative ? TYR CG ==> Alternative ? TYR CD1 ==> Alternative ? TYR CE1 ==> Alternative ? TYR CD2 ==> Alternative ? TYR CE2 ==> Alternative ? TYR CZ ==> Not matchable TYR OH Atoms in common : ( 9) RMS distance : ( 0.703) Nr ambiguous : ( 2) ARG 132 NH1 <-> NH2 => RMSD (A) : ( 1.239) Lowest RMSD : ( 0.703) Nr alternatives : ( 8) ARG 111 CG <-> LYS 240 CG => RMSD (A) : ( 0.738) ARG 111 CD <-> LYS 240 CD => RMSD (A) : ( 0.774) TYR 134 CG <-> PHE 266 CG => RMSD (A) : ( 0.761) TYR 134 CD1 <-> PHE 266 CD1 => RMSD (A) : ( 0.907) TYR 134 CD1 <-> PHE 266 CD2 => RMSD (A) : ( 0.984) TYR 134 CE1 <-> PHE 266 CE1 => RMSD (A) : ( 1.120) TYR 134 CE1 <-> PHE 266 CE2 => RMSD (A) : ( 1.163) TYR 134 CD2 <-> PHE 266 CD1 => RMSD (A) : ( 1.173) TYR 134 CD2 <-> PHE 266 CD2 => RMSD (A) : ( 1.094) TYR 134 CE2 <-> PHE 266 CE1 => RMSD (A) : ( 1.190) TYR 134 CE2 <-> PHE 266 CE2 => RMSD (A) : ( 1.066) TYR 134 CZ <-> PHE 266 CZ => RMSD (A) : ( 1.128) Final RMSD : ( 1.128) Nr matches : ( 17) [...] Hit PDB file : (/nfs/pdb/full/7aat.pdb) Residues in hit : ( 3) Hit will go to PDB file : (savant/cra2_7aat_1.pdb) Atoms in residues : ( 11 9 12) ==> Match ARG CB <-> ARG CB ==> Match ARG CG <-> ARG CG ==> Match ARG CD <-> ARG CD ==> Match ARG NE <-> ARG NE ==> Match ARG CZ <-> ARG CZ ==> Match ARG NH1 <-> ARG NH1 ==> Match ARG NH2 <-> ARG NH2 ==> Match ARG CB <-> LYS CB ==> Alternative ? ARG CG ==> Alternative ? ARG CD ==> Not matchable ARG NE ==> Not matchable ARG CZ ==> Not matchable ARG NH1 ==> Not matchable ARG NH2 ==> Match TYR CB <-> TYR CB ==> Match TYR CG <-> TYR CG ==> Match TYR CD1 <-> TYR CD1 ==> Match TYR CE1 <-> TYR CE1 ==> Match TYR CD2 <-> TYR CD2 ==> Match TYR CE2 <-> TYR CE2 ==> Match TYR CZ <-> TYR CZ ==> Match TYR OH <-> TYR OH Atoms in common : ( 16) RMS distance : ( 2.209) Nr ambiguous : ( 6) ARG 111 NH1 <-> NH2 => RMSD (A) : ( 2.212) TYR 134 CD1 <-> CD2 => RMSD (A) : ( 2.123) TYR 134 CE1 <-> CE2 => RMSD (A) : ( 2.020) Lowest RMSD : ( 2.020) Nr alternatives : ( 2) ARG 132 CG <-> LYS A 258 CG => RMSD (A) : ( 2.037) ARG 132 CD <-> LYS A 258 CD => RMSD (A) : ( 2.036) Final RMSD : ( 2.036) Nr matches : ( 18) ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Use DEJANA as usual to select the best hits (you may want to sort on RMSD first, and number of matched atoms second; this can be done using option 3 in DEJANA version 1.4 or later).
Does your result look something like this ?
Just to show that SPASM rocks, check out this (big) picture to see the hits in a different way ...
Let's see how SPASM works when we are only interested in a chunk of main-chain (e.g., an unusual or otherwise interesting loop). For this purpose, first generate a 7-residue bit of left-handed helix with MOLEMAN2:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- MOLEMAN2 > auto spink alpha 7 [...] MOLEMAN2 > xyz mirror x [...] MOLEMAN2 > write lal7.pdb ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Now run SPASM:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- SPASM database file ? (/home/gerard/lib/spasm.lib) [...] Which PDB file ? (0xyz.pdb) lal7.pdb [...] Four-character ID for this run ? (LAL7) [...] Max superpositioning RMSD ? ( 1.500) 1.0 [...] Max CA-CA distance mismatch ? ( 2.000) Max SC-SC distance mismatch ? ( 2.000) [...] Resolution cut-off (A) ? ( 999.900) [...] Max nr of residues ? ( 9999) [...] Substitution option ? ( 3) 1 [...] Conserve sequence directionality ? (N) y [...] Conserve neighbouring residues ? (N) y [...] Conserve sequence gaps ? (N) [...] Print distance matrices ? (N) [...] Print operators ? (N) [...] Extensive output ? (N) [...] 0=SC, 1=MC+SC, 2=MC ? ( 1) 2 [...] O macro and operator file ? (N) y O macro file ? (lal7.omac) O operator file ? (lal7.odb) [...] SAVANT input file ? (Y) SAVANT input file ? (lal7.savant) [...] LSQMAN input file ? (N) [...] MSEQPRO sequence file ? (N) [...] ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Note that, in general, main-chain searches are quite a bit slower than searches for small motifs.
The results may look as follows:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- ... Searching ... -------------------------------------------------------- ==> HIT : (1ACP) Compound : (ACYL CARRIER PROTEIN (NMR, 2 STRUCTURES)) File : (/nfs/pdb/full/1acp.pdb) Residues : ( 77) Resol (A): ( 99.990) MATCH with RMSD 0.85 A for 7 pseudo-atoms ALA Z 1 <---> LYS 9 -1.0 ALA Z 2 <---> ILE 10 -1.0 ALA Z 3 <---> ILE 11 -1.0 ALA Z 4 <---> GLY 12 0.0 ALA Z 5 <---> GLU 13 -1.0 ALA Z 6 <---> GLN 14 -1.0 ALA Z 7 <---> LEU 15 -1.0 Total BLOSUM-45 score : ( -6.000) -------------------------------------------------------- ==> HIT : (1AK0) Compound : (MOL_ID: 1; MOLECULE: P1 NUCLEASE; CHAIN: NULL; EC: 3.1.30.1) File : (/nfs/pdb/full/1ak0.pdb) Residues : ( 264) Resol (A): ( 1.800) MATCH with RMSD 0.62 A for 7 pseudo-atoms ALA Z 1 <---> ALA 129 = 5.0 ALA Z 2 <---> TYR 130 -2.0 ALA Z 3 <---> ALA 131 = 5.0 ALA Z 4 <---> VAL 132 0.0 ALA Z 5 <---> GLY 133 0.0 ALA Z 6 <---> GLY 134 0.0 ALA Z 7 <---> ASN 135 -1.0 Total BLOSUM-45 score : ( 7.000) -------------------------------------------------------- ==> HIT : (1BD0) Compound : (MOL_ID: 1; MOLECULE: ALANINE RACEMASE; CHAIN: A, B; EC: 5.1.1.1; BIOLOGICA) File : (/nfs/pdb/full/1bd0.pdb) Residues : ( 381) Resol (A): ( 1.600) MATCH with RMSD 0.40 A for 7 pseudo-atoms ALA Z 1 <---> LYS A 39 -1.0 ALA Z 2 <---> ALA A 40 = 5.0 ALA Z 3 <---> ASN A 41 -1.0 ALA Z 4 <---> ALA A 42 = 5.0 ALA Z 5 <---> TYR A 43 -2.0 ALA Z 6 <---> GLY A 44 0.0 ALA Z 7 <---> HIS A 45 -2.0 Total BLOSUM-45 score : ( 4.000) -------------------------------------------------------- ==> HIT : (2OMF) Compound : (MOL_ID: 1; MOLECULE: MATRIX PORIN OUTER MEMBRANE PROTEIN F; CHAIN: NULL; S) File : (/nfs/pdb/full/2omf.pdb) Residues : ( 340) Resol (A): ( 2.400) MATCH with RMSD 0.77 A for 7 pseudo-atoms ALA Z 1 <---> ASN 141 -1.0 ALA Z 2 <---> SER 142 + 1.0 ALA Z 3 <---> ASN 143 -1.0 ALA Z 4 <---> PHE 144 -2.0 ALA Z 5 <---> PHE 145 -2.0 ALA Z 6 <---> GLY 146 0.0 ALA Z 7 <---> LEU 147 -1.0 Total BLOSUM-45 score : ( -6.000) -------------------------------------------------------- ==> HIT : (2SXL) Compound : (MOL_ID: 1; MOLECULE: SEX-LETHAL PROTEIN; CHAIN: NULL; FRAGMENT: RNA-BINDIN) File : (/nfs/pdb/full/2sxl.pdb) Residues : ( 88) Resol (A): ( 99.990) MATCH with RMSD 0.92 A for 7 pseudo-atoms ALA Z 1 <---> PRO 82 -1.0 ALA Z 2 <---> GLY 83 0.0 ALA Z 3 <---> GLY 84 0.0 ALA Z 4 <---> GLU 85 -1.0 ALA Z 5 <---> SER 86 + 1.0 ALA Z 6 <---> ILE 87 -1.0 ALA Z 7 <---> LYS 88 -1.0 Total BLOSUM-45 score : ( -3.000) -------------------------------------------------------- Skipped (resolution) : ( 0) Skipped (nr residues) : ( 0) Nr of proteins tried : ( 2590) Nr of proteins found : ( 5) Total number of hits : ( 5) CPU total/user/sys : 373.7 372.8 0.9 ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Now, you can look at the hits by executing the lal7.omac macro in O. Doesn't look so bad, aye ?
If you want, you can also run SAVANT, of course. Let's see how good the matches are if we include all main-chain atoms and the CB atoms:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- SAVANT library file ? (/home/gerard/lib/savant.lib) Nr of AMB lines : ( 11) Nr of ALT lines : ( 37) SAVANT input file ? (savant.inp) lal7.savant SAVANT O macro ? (savant.omac) SAVANT ODB file ? (savant.odb) O2D plot file ? (savant.plt) Select mode: 0 = SC, 1 = MC + SC, 2 = MC Mode (0/1/2) ? ( 2) 1 MODE : ( 1) MC + SC ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Run DEJANA:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- Nr of hits left : ( 5) # 1 ID 1bd01 Nmatch 34 RMSD 1.18 A # 2 ID 1ak01 Nmatch 33 RMSD 1.25 A # 3 ID 2omf1 Nmatch 34 RMSD 1.49 A # 4 ID 1acp1 Nmatch 34 RMSD 1.61 A # 5 ID 2sxl1 Nmatch 33 RMSD 1.64 A ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Now look at the hits again in O. In particular, check the match of 1BD0. It looks pretty good - except the CBs are pointing in different directions compared to our motif ! Explain why this is so (remember how the motif was generated ...) !
If you only use the main-chain atoms, the results are as follows:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- Nr of hits left : ( 5) # 1 ID 1bd01 Nmatch 28 RMSD 0.81 A # 2 ID 1ak01 Nmatch 28 RMSD 1.05 A # 3 ID 2omf1 Nmatch 28 RMSD 1.21 A # 4 ID 1acp1 Nmatch 28 RMSD 1.40 A # 5 ID 2sxl1 Nmatch 28 RMSD 1.56 A ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
If you include the CB atoms, the fit of 1BD0 and the motif should look something like this:
If you only use the main-chain atoms, the fit should look something like this:
If you want to play some more, here are some suggestions:
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- Nr of hits left : ( 17) # 1 ID 8tln2 Nmatch 17 RMSD 0.19 A # 2 ID 1ezm2 Nmatch 17 RMSD 0.20 A # 3 ID 1npc2 Nmatch 17 RMSD 0.27 A # 4 ID 1prc2 Nmatch 17 RMSD 0.62 A # 5 ID 1toh2 Nmatch 17 RMSD 0.69 A # 6 ID 1prc3 Nmatch 17 RMSD 0.76 A # 7 ID 1prc4 Nmatch 17 RMSD 0.83 A # 8 ID 1aij2 Nmatch 17 RMSD 0.84 A # 9 ID 1prc7 Nmatch 17 RMSD 0.85 A # 10 ID 1prc10 Nmatch 17 RMSD 0.85 A # 11 ID 1fua5 Nmatch 17 RMSD 0.89 A # 12 ID 1guq6 Nmatch 17 RMSD 0.91 A # 13 ID 2mhr10 Nmatch 17 RMSD 0.91 A # 14 ID 1fua7 Nmatch 17 RMSD 0.93 A # 15 ID 1prc6 Nmatch 17 RMSD 0.93 A # 16 ID 1guq4 Nmatch 17 RMSD 0.94 A # 17 ID 2hmz9 Nmatch 17 RMSD 0.95 A ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- Nr of hits left : ( 7) # 1 ID 3ovw1 Nmatch 14 RMSD 0.30 A # 2 ID 2ayh1 Nmatch 14 RMSD 0.34 A # 3 ID 1gbg1 Nmatch 14 RMSD 0.41 A # 4 ID 3ovw2 Nmatch 14 RMSD 0.95 A # 5 ID 2ayh2 Nmatch 14 RMSD 1.02 A # 6 ID 1gbg2 Nmatch 14 RMSD 1.05 A # 7 ID 1dhk1 Nmatch 14 RMSD 1.19 A ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- Nr of hits left : ( 30) # 1 ID 1as81 Nmatch 18 RMSD 0.15 A # 2 ID 1nif1 Nmatch 18 RMSD 0.18 A # 3 ID 2cbp1 Nmatch 18 RMSD 0.29 A # 4 ID 1adw1 Nmatch 18 RMSD 0.33 A # 5 ID 1pmy1 Nmatch 18 RMSD 0.35 A # 6 ID 1ndr1 Nmatch 18 RMSD 0.40 A # 7 ID 1paz1 Nmatch 18 RMSD 0.40 A # 8 ID 1iuz1 Nmatch 18 RMSD 0.44 A # 9 ID 1ndt1 Nmatch 18 RMSD 0.44 A # 10 ID 1plc1 Nmatch 18 RMSD 0.45 A # 11 ID 1zia1 Nmatch 18 RMSD 0.46 A # 12 ID 7pcy1 Nmatch 18 RMSD 0.47 A # 13 ID 1pcs1 Nmatch 18 RMSD 0.48 A # 14 ID 1a8z1 Nmatch 18 RMSD 0.49 A # 15 ID 1rcy1 Nmatch 18 RMSD 0.49 A # 16 ID 1ag61 Nmatch 18 RMSD 0.50 A # 17 ID 2plt1 Nmatch 18 RMSD 0.53 A # 18 ID 1aoz1 Nmatch 18 RMSD 0.63 A # 19 ID 2azu1 Nmatch 18 RMSD 0.63 A # 20 ID 9pcy1 Nmatch 18 RMSD 0.63 A # 21 ID 1joi1 Nmatch 18 RMSD 0.64 A # 22 ID 1nwp1 Nmatch 18 RMSD 0.65 A # 23 ID 2aza1 Nmatch 18 RMSD 0.65 A # 24 ID 1arn1 Nmatch 18 RMSD 0.67 A # 25 ID 1plb1 Nmatch 18 RMSD 0.68 A # 26 ID 1rkr1 Nmatch 18 RMSD 0.73 A # 27 ID 1bxa1 Nmatch 18 RMSD 0.77 A # 28 ID 1nin1 Nmatch 18 RMSD 0.91 A # 29 ID 1kcw2 Nmatch 18 RMSD 0.98 A # 30 ID 1kcw1 Nmatch 18 RMSD 1.06 A ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Latest update at 18 November, 1999.
