Team:TU Darmstadt/modelling/Structure

All these steps are performed to every template used for the modeling approach. For our project we set the maximum amount of templates to 20. Every derived structure is evaluated using an average per-residue quality Z-scores. At last a hybrid model is built containing the best regions of all predictions. This procedure make prediction’s accurate and thus more realistic. For the evaluation we used the Yasara Z-scores.A Z-score describes how many standard deviations the model quality is away from the average high-resolution X-ray structure. Negative values indicate that the homology model looks worse than a high-resolution X-ray structure. The overall Z-scores for all models have been calculated as the weighted averages of the individual Z-scores using the formula Overall = 0.145*Dihedrals + 0.390*Packing1D + 0.465*Packing3D ^[7].

Parameters
We used the Yasara script hm_build.mcr for the model creation with the following parameters:

- Modeling speed (slow = best): Slow

- Number of PSI-BLAST iterations in template search (PsiBLASTs): 3

- Maximum allowed PSI-BLAST E-value to consider template (EValue Max): 0.5

- Maximum number of templates to be used (Templates Total): 20

- Maximum number of templates with same sequence (Templates SameSeq): 1

- Maximum oligomerization state (OligoState): 4 (tetrameric)

- Maximum number of alignment variations per template: (Alignments): 5

- Maximum number of conformations tried per loop (LoopSamples): 50

- Maximum number of residues added to the termini (TermExtension): 10

Since the target sequence was the only available information, possible templates were identified by running 6 PSI-BLAST iterations to extract a position specific scoring matrix (PSSM) from Uniprot, and then searching the PDB for a match (i.e. hits with an E-value below the homology modeling cutoff 0.5)

We present the best three models from a total of 14 listed in the model ranking and sorted by their overall quality Z-scores.

First model LssmOrange

This model is a monomer, and based on the following sequence alignment:

1. SecStr:
2. CCCCCCCCCCCCCCCCEEEEEEEEEEECCEEEEEEEEEEECCCCCEEEEEEEEEECCCCCCCCCHHHCCCCCCCCCCCCCCCCCHHHHHHCCCCCEEEEEEEEEECCCEEEEEEEEEEECCEEEEEEEEEEECCCCCCCCCCCCCCCCCCEEEEEEEECCEEEEEEEEEEEECCCCEEEEEEEEEEECCCCCCCCCCEEEEEEEEEEEECCCCCEEEEEEEEEEECCCCCCCCCCC
3. Target:
4. MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGFQTVKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALKGEDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK
5. Match:
6. EE:NMAIIKEFMRFK::MEGSVNGHEFEIEGEGEGRPYEG QT:KLKVTKGGPLPFAWDILSPQF SKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMG EA SERMYPEDGALKGE K|RLKLKDGGHY :EVKTTYKAKKPVQLPGAY :: KLDITSHNEDYTIVEQYER EGRHSTGGMDELYK
7. Template:
8. .....EEDNMAIIKEFMRFKTHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQF...SKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEACSERMYPEDGALKGEMKMRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNTNTKLDITSHNEDYTIVEQYERNEGRHSTGGMDELYK
9. SecStr:
10. ....HHHHHHCCCCCEEEEEEEEEEETTEEEEEEEEEEEETTTEEEEEEEEEEECCCCCCGGGGGGGGG...TTTTCECTTTTCHHHHHHCCCEEEEEEEEEETTEEEEEEEEEEEEETTEEEEEEEEEEECCTTTTCTTTTCCEEEECEEEEEEEETTEEEEEEEEEEEETTEEEEEEEEEEEEEECCCCCCCCCEEEEEEEEEEEEETTTEEEEEEEEEEEECCCCTTTTCCC/li>

In the alignment, 228 of 236 target residues (96.6%) are aligned to template residues. Among these aligned residues, the sequence identity is 93.0% and the sequence similarity is 94.7% ('similar' means that the BLOSUM62 score is > 0).

Here, the first hybrid homology model of our protein, LssmOrange is shown in ribbon representation. Furthermore, we illustrate the average quality z-score as a function of residue number. Nevertheless, it was subjected to a final round of simulated annealing minimization in explicit solvent and obtained the following quality Z-scores:

Check Type	Quality Z-score	Comment
Dihedrals	2.363	Optimal
Packing 1D	-0.864	Good
Packing 3D	-1.138	Satisfactory
Overall	-0.524	Good

LssmOrange - second modelling

This model is a monomer, and based on the following sequence alignment:

1. SecStr:
2. CCCCCCCCCCCCCCCCEEEEEEEEEEECCEEEEEEEEEEECCCCCEEEEEEEEEECCCCCCCCCHHHCCCCCCCCCCCCCCCCCHHHHHHCCCCCEEEEEEEEEECCCEEEEEEEEEEECCEEEEEEEEEEECCCCCCCCCCCCCCCCCCEEEEEEEECCEEEEEEEEEEEECCCCEEEEEEEEEEECCCCCCCCCCEEEEEEEEEEEECCCCCEEEEEEEEEEECCCCCCCCCCC
3. Target:
4. MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGFQTVKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALKGEDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK
   Match: AIIKEFMRFKV:MEGSVNGH FEIEGEGEGRPYEG QT:KLKVTKGGPLPF:WDILSPQF S:AYVKHPADIPDY:KLSFPEGFKWERVM:FEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMG EA SERMYPEDGALKGE K R:KLKDGGHY :EVKTTYKAKKPVQLPGAY V: KLDITSHNEDYTIVEQYERAEGRHS
   
5. Template:
6. ..........AIIKEFMRFKVHMEGSVNGHVFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFTWDILSPQF...SNAYVKHPADIPDYFKLSFPEGFKWERVMKFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEALSERMYPEDGALKGEVKPRVKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNRKLDITSHNEDYTIVEQYERAEGRHS.........
   
   
7. SecStr:
8. ..........CCCCCCEEEEEEEEEEETTEEEEEEEEEEEETTTEEEEEEEEEEECCCCCCCCCCGGGGC...TTTTCECTTTTCHHHHHHCCCEEEEEEEEEETTEEEEEEEEEEEEETTEEEEEEEEEEECCTTTTCTTTTCCEEEECEEEEEEEETTEEEEEEEEEEEETTEEEEEEEEEEEEEECCCCCCCCCEEEEEEEEEEEEETTTEEEEEEEEEEEECC.........

In the alignment, 214 of 236 target residues (90.7%) are aligned to template residues. Among these aligned residues, the sequence identity is 91.6% and the sequence similarity is 93.0% ('similar' means that the BLOSUM62 score is > 0).

Here, the second hybrid homology model of our protein, LssmOrange is shown in ribbon representation. Again, we illustrate the average quality z-score as a function of residue number and obtained the following quality Z-scores:

Check Type	Quality Z-score	Comment
Dihedrals	2.125	Optimal
Packing 1D	-0.936	Good
Packing 3D	-1.259	Satisfactory
Overall	-0.642	Good

Third model LssmOrange (dimer)

This model is a dimer, add based on the following sequence alignment:

1. SecStr:
2. CCCCCCCCCCCCCCCCEEEEEEEEEEECCEEEEEEEEEEECCCCCEEEEEEEEEECCCCCCCCCHHHCCCCCCCCCCCCCCCCCHHHHHHCCCCCEEEEEEEEEECCCEEEEEEEEEEECCEEEEEEEEEEECCCCCCCCCCCCCCCCCCEEEEEEEECCEEEEEEEEEEEECCCCEEEEEEEEEEECCCCCCCCCCEEEEEEEEEEEECCCCCEEEEEEEEEEECCCCCCCCCCC
   Target: MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGFQTVKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALKGEDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK
   Match: |IKE M::K: MEG:VN:H:F: :EGEG|PYEG QT ||KV::GGPLPFA:DIL: SK:||:H IPD| K SFPEGF WERV :|EDGGV:T:TQD:SLQDG :IY:VK|RG:NFPS:GPVMQKKT|G EA::E MYP DG:L:G:: | LKL GGH ::KTTY|:KKP |PG:Y VD |L| :: | T VEQ|E A :R|
   Template:...........LIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTQRIKVVEGGPLPFAFDILA......SKTFINHTQGIPDFWKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMMYPADGGLEGRNYMALKLVGGGHLICSLKTTYRSKKP..MPGVYYVDRRLERIKEADKETYVEQHEVAVARY.......... SecStr: ...........CCCCCEEEEEEEEEEETTEEEEEEEEEEEETTTEEEEEEEEEEECCCCCCGGGGGG......TTTTCEEETTTCCCTTTTCCCEEEEEEEEEETTEEEEEEEEEEEEETTEEEEEEEEEEECCTTTTCTTTTCCEEEECEEEEEEEETTEEEEEEEEEEEETTEEEEEEEEEEEEEECCG..CCCCEEEEEEEEEEEEETTTEEEEEEEEEEEEC..........

In the alignment, 207 of 236 target residues (87.7%) are aligned to template residues. Among these aligned residues, the sequence identity is 58.0% and the sequence similarity is 73.9% ('similar' means that the BLOSUM62 score is > 0)

Here, the third hybrid homology model of our protein, LssmOrange is shown in ribbon representation. Again, we illustrate the average quality z-score as a function of residue number and obtained the following quality Z-scores:

Check Type	Quality Z-score	Comment
Dihedrals	1.237	Optimal
Packing 1D	-2.083	Poor
Packing 3D	-1.651	Satisfactory
Overall	-1.400	Satisfactory

References