Task 4: Homology based structure predictions

Task description

The full description of this task can be found here.

In this task we are going to learn more about several methods of homology modelling. There exists only a small number of known protein structures. Therefore if someone wants to predict the structure of a somehow new protein (newly sequenced, a mutant, etc.). He could use known structures to calculate a model of the unknown structure.

Homology modelling bases on an alignment between the target-sequence and one or more template structures. The aligned coordinates are directly used for the model. That is why it is important to select a good template and alignment.

Calculation of models

Overview of available homologous structures

Search

We used hhsearch with the standard parameter to find homologous structures of our protein. The following command was executed:

• ./hhsearch -i reference_pah_aa.fasta -d pdb70.db -b 500 -o hhsearch.out

We received the following hits:

No.	PDB ID	Description	Prob	E-Value	P-Value	Score	SS	Cols	Query HMM	Template HMM	Residues	Sequence Identity
1	1phz_A	Protein (phenylalanine	1	1	1	1084.4	0	429	1-429	1-429	(429)	92%
2	1j8u_A	Phenylalanine-4-hydroxy	1	1	1	894.5	0	325	103-427	1-325	(325)	100%
3	1toh_A	Tyroh tyrosine hydroxy	1	1	1	890.7	0	342	111-452	2-343	(343)	60%
4	1mlw_A	Tryptophan 5-monooxygen	1	1	1	804.2	0	300	116-415	2-301	(301)	66%
5	1ltz_A	Phenylalanine-4-hydroxy	1	1	1	504.9	0	265	144-414	2-269	(297)	30%
6	2v27_A	Phenylalanine hydroxyla	1	1	1	471.1	0	254	167-424	4-271	(275)	30%
7	2qmx_A	Prephenate dehydratase;	1	1	1	70.0	0	53	33-85	199-251	(283)	40%
8	2qmw_A	PDT prephenate dehydra	1	1	1	66.1	0	51	35-85	190-240	(267)	37%
9	3luy_A	Probable chorismate mut	1	1	1	66.0	0	53	33-85	207-259	(329)	28%
10	1y7p_A	Hypothetical protein AF	1	1	1	19.9	0	38	36-73	6-43	(223)	16%

Template structure selection

We selected the following structures as our template structures:

• > 60% sequence identity: 1phz
• > 40% sequence identity: 1toh
• < 40% sequence identity: 1ltz

Alignment Refinement

We used the reference for a search in PFAM. There were two PFAM-domains detected on the reference sequence: ACT and Biopterin_H Then we used the sequence of the three proteins 1PHZ, 1TOH and 1LTZ to run a search against PFAM and used the alignments with the HMMs of ACT and Biopterin_H and the alignment of the reference sequence with the HMMs of ACT and Biopterin_H as seeds for the improved alignments. The crucial parts of the reference sequence according to the annotation in UniProt was already aligned by the seeds. We predicted the secondary structure of the three sequences of the proteins (TODO ref... seems to be better to predict) and tried to extend the seeds. In PHZ a large gap could be filled by high sequence identity. Afterwards we deleted the unaligned ends of the reference sequence to improve the resulting model. The essential part of the protein, the domains, should now be better modeled.

Seeds by PFAM - ACT:

REFERENCE	SLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKkDEYEFFTHLD-KRSL
1PHZ	SLIFSLKEEVGALAKVLRLFEENDINLTHIESRPSRLNkDEYEFFTYL------

Seeds by PFAM - Biopterin_H

REFERENCE	PWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPD-EYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCL-SEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQK
1PHZ	PWFPRTIQELDRFANQI------LDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYTEEEKQTWGTVFRTLKALYKTHACYEHNHIFPLLEKYCGFREDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPD-EYIEKLATIYWFTVEFGLCKEGDSIKAYGAGLLSSFGELQYCL-SDKPKLLPLELEKTACQEYSVTEFQPLYYVAESFSDAKEKVRTFAATIPRPFSVRYDPYTQRVEVLDNT-----------------------
1TOH	PWFPRKVSELDKC-----------DLDHPGFSDQVYRQRRKLIAEIAFQYKHGEPIPHVEYTAEEIATWKEVYVTLKGLYATHACREHLEGFQLLERYCGYREDSIPQLEDVSRFLKERTGFQLRPVAGLLSARDFLASLAFRVFQCTQYIRHASSPMHSPEPDCCHELLGHVPMLADRTFAQFSQDIGLASLGASD-EEIEKLSTVYWFTVEFGLCKQNGELKAYGAGLLSSYGELLHSL-SEEPEVRAFDPDTAAVQPYQDQTYQPVYFVSESFNDAKDKLRNYASRIQRPFSVKFDPYTLAIDVLDSPHTIQRSLEGVQDELHTLAHALS-
1LTZ	------------------------------------------------------PQPLDRYSAEDHATWATLYQRQCKLLPGRACDEFLEGLERLEV----DADRVPDFNKLNEKLMAATGWKIVAVPGLIPDDVFFEHLANRRFPVTWWLREPHQLDYLQEPDVFHDLFGHVPLLINPVFADYLEAYGKGGVKAKAlGALPMLARLYWYTVEFGLINTPAGMRIYGAGILSSKSESIYCLdSASPNRVGFDLMRIMNTRYRIDTFQKTYFVIDSFKQ--------------------------------------------------------

unadjusted alignment of 1TOH

REFERENCE	MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK
PSI	CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCHHHHHHHHHHHHCCCCEEEEECCCCCCCCCCEEEEEECCCCCCHHHHHHHHHHCCCCEEEECCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHHHHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHHCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHHCCCCCEEEECCCCCEEEECCCHHHHHHHHHHHHHHHHHHHHHHHHHC
1TOH	-------------K-------------------------------------------------------------------------------------------------------VPWFPRKVSELDKCD---L--------DHPGFSDQVYRQRRKLIAEIAFQYKHGEPIPHVEYTAEEIATWKEVYVTLKGLYATHACREHLEGFQLLERYCGYREDSIPQLEDVSRFLKERTGFQLRPVAGLLSARDFLASLAFRVFQCTQYIRHASSPMHSPEPDCCHELLGHVPMLADRTFAQFSQDIGLASLGASDEEIEKLSTVYWFTVEFGLCKQNGELKAYGAGLLSSYGELLHSLSEEPEVRAFDPDTAAVQPYQDQTYQPVYFVSESFNDAKDKLRNYASRIQRPFSVKFDPYTLAIDVLDSPHTIQRSLEGVQDELHTLAHALSAIS
PSI	-------------C-------------------------------------------------------------------------------------------------------CCCCCCCCCCCCCCC---C--------CCCCCCHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHCCCEEEECCCCCCHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCHHHHHHCCCCCCCCHHHHHHHHHHHHHHCCCCHHHHHHHHCEEEEEEEEEEEEECCCCEECCCCCCCCCCHHCCCCCCCCCCCCCCHHHHHCCCCCCCCCCCEEEEECCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCEECCCCHHHHHHHHHHHHHHHHHHHHHHHHHC

unadjusted alignment of 1PHZ

REFERENCE	MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK
PSI	CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCHHHHHHHHHHHHCCCCEEEEECCCCCCCCCCEEEEEECCCCCCHHHHHHHHHHCCCCEEEECCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHHHHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHHCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHHCCCCCEEEECCCCCEEEECCCHHHHHHHHHHHHHHHHHHHHHHHHHC
1PHZ	------------------GQETSYIEDNSNQNGAISLIFSLKEEVGALAKVLRLFEENDINLTHIESRPSRLNKDEYEFFTYLDKRTKPVLGSIIKSLRNDIGATVHELSRDKEKNTVPWFPRTIQELDRFANQI------LDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYTEEEKQTWGTVFRTLKALYKTHACYEHNHIFPLLEKYCGFREDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKEGDSIKAYGAGLLSSFGELQYCLSDKPKLLPLELEKTACQEYSVTEFQPLYYVAESFSDAKEKVRTFAATIPRPFSVRYDPYTQRVEVLDNT-------------------------
PSI	------------------CCCCCCCCCCCCCCCEEEEEEEECCCCCHHHHHHHHHHHCCCEEEEEECCCCCCCCCEEEEEEEECCCCCCCHHHHHHHHHCCCCCCEEECCCCCCCCCCCCCCCCHHHHHHHHHHH------CCCCCCCCCCHHHHHHHHHHHHHCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHCCCCCCCCCCCHHHHHHHHHCCCCEEEECCCCCCHHHHHHHHHCCCCCEECCCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHHHHHHHHCCCCHHHHHHHHHHEEEEEEEEEEEECCCCEEECCCCCCCCCCCCCCCCCCCCCCCCCHHHHHCCCCCCCCCCCCEEEECCHHHHHHHHHHHHHHCCCCCCCCCCCCCCEEEECCCC-------------------------

unadjusted alignment of 1LTZ

REFERENCE	MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPD-EYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCL-SEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK
PSI	CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCHHHHHHHHHHHHCCCCEEEEECCCCCCCCCCEEEEEECCCCCCHHHHHHHHHHCCCCEEEECCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCH HHHHHHHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHH CCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHHCCCCCEEEECCCCCEEEECCCHHHHHHHHHHHHHHHHHHHHHHHHHC
1LTZ	-----------------F--------------------------V-----V-------------------------------------PDITT-----RKNVG-----LSHDANDFTLP------QPLDR-------YSA-----------------------------------------EDHATWATLYQRQCKLLPGRACDEFLEGLERLE----VDADRVPDFNKLNEKLMAATGWKIVAVPGLIPDDVFFEHLANRRFPVTWWLREPHQLDYLQEPDVFHDLFGHVPLLINPVFADYLEAYGKGGVKAKALGALPMLARLYWYTVEFGLINTPAGMRIYGAGILSSKSESIYCLDSASPNRVGFDLMRIMNTRYRIDTFQKTYFVIDSFKQL------FDAD----FAPLY------LQLAD-AQPWG--AGDIAP------DDL--VL
PSI	-----------------C--------------------------C-----C-------------------------------------CCCCC-----CCCCC-----CCCCCCCCCCC------CCCCC-------CCH-----------------------------------------HHHHHHHHHHHHHHHHCCCCCHHHHHHHHHHCC----CCCCCCCCCHHHHHHHHHHHCCEEEECCCCCCHHHHHHHHHCCCCCEEECCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHHHHHHHHCCCCCCCHHHHHHHHHEEEEEEEEEECCCCEEEECCCCCCCCCCCCCCCCCCCCEEECCCHHHHHCCCCCCCCCCCCEEEECCHHHH------HHHC----CHHHH------HHHHH-CCCCC--CCCCCC------CCC--CC

adjusted alignment of 1LTZ

REFERENCE	PIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAAT
PSI	CCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHHHHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHHCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHH
1LTZ	PQPLDRYSAEDHATWATLYQRQCKLLPGRACDEFLEGLERLEV----DADRVPDFNKLNEKLMAATGWKIVAVPGLIPDDVFFEHLANRRFPVTWWLREPHQLDYLQEPDVFHDLFGHVPLLINPVFADYLEAYGKGGVKAKAlGALPMLARLYWYTVEFGLINTPAGMRIYGAGILSSKSESIYCLdSASPNRVGFDLMRIMNTRYRIDTFQKTYFVIDSFKQLFDADFAPL
PSI	CCCCCCCCHHHHHHHHHHHHHHHHHCCCCCHHHHHHHHHHCCCCCCCCCCCHHHHHHHHHHHCCEEEECCCCCCHHHHHHHHHCCCCCEEECCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHHHHHHHHCCCCCCCHHHHHHHHHEEEEEEEEEECCCCEEEECCCCCCCCCCCCCCCCCCCCEEECCCHHHHHCCCCCCCCCCCCEEEECCHHHHHHHCCHHHHHHH

adjusted alignment of 1PHZ

REFERENCE	GQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQ
PSI	CCCCCCCCCCCCCCCCEEEEEECCCCCHHHHHHHHHHHHCCCCEEEEECCCCCCCCCCEEEEEECCCCCCHHHHHHHHHHCCCCEEEECCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHHHHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHHCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHHCCCCCEEEECCCCCEEEECCCHHH
1PHZ	GQETSYIEDNSNQNGAISLIFSLKEEVGALAKVLRLFEENDINLTHIESRPSRLNkDEYEFFTYLDKRTKPVLGSIIKSLRNDIGATVHELSRDKEKNTVPWFPRTIQELDRFANQI------LDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYTEEEKQTWGTVFRTLKALYKTHACYEHNHIFPLLEKYCGFREDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPD-EYIEKLATIYWFTVEFGLCKEGDSIKAYGAGLLSSFGELQYCL-SDKPKLLPLELEKTACQEYSVTEFQPLYYVAESFSDAKEKVRTFAATIPRPFSVRYDPYTQRVEVLDNT
PSI	CCCCCCCCCCCCCCCEEEEEEEECCCCCHHHHHHHHHHHCCCEEEEEECCCCCCCCCEEEEEEEECCCCCCCHHHHHHHHHCCCCCCEEECCCCCCCCCCCCCCCCHHHHHHHHHHH------CCCCCCCCCCHHHHHHHHHHHHHCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHCCCCCCCCCCCHHHHHHHHHCCCCEEEECCCCCCHHHHHHHHHCCCCCEECCCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHHHHHHHHCCCCH-HHHHHHHHHEEEEEEEEEEEECCCCEEECCCCCCCCCCCCCCC-CCCCCCCCCCHHHHHCCCCCCCCCCCCEEEECCHHHHHHHHHHHHHHCCCCCCCCCCCCCCEEEECCCC

adjusted alignment of 1TOH

REFERENCE	VPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK
PSI	CCCCCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHHCCEEEECCCCCCHHHHHHHCCCCEECCCEEEECCCCCCCCCCCCHHHHHHCCCCCCCCCHHHHHHHHHHHHCCCCCHHHHHHHHHHEEEEEEEEEECCCCCEEEECCCCCCCHHHHHHHHCCCCCCCCCCHHHHHCCCCCCCCCCEEEEEECCHHHHHHHHHHHHHHCCCCCEEEECCCCCEEEECCCHHHHHHHHHHHHHHHHHHHHHHHHHC
1TOH	VPWFPRKVSELDKC-----------DLDHPGFSDQVYRQRRKLIAEIAFQYKHGEPIPHVEYTAEEIATWKEVYVTLKGLYATHACREHLEGFQLLERYCGYREDSIPQLEDVSRFLKERTGFQLRPVAGLLSARDFLASLAFRVFQCTQYIRHASSPMHSPEPDCCHELLGHVPMLADRTFAQFSQDIGLASLGASD-EEIEKLSTVYWFTVEFGLCKQNGELKAYGAGLLSSYGELLHSL-SEEPEVRAFDPDTAAVQPYQDQTYQPVYFVSESFNDAKDKLRNYASRIQRPFSVKFDPYTLAIDVLDSPHTIQRSLEGVQDELHTLAHALSA
PSI	CCCCCCCCCCCCCC CCCCCCCCHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHCCCHHHHHHHHHHHHHCCCCCCCCCCHHHHHHHHHHHCCCEEEECCCCCCHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCHHHHHHCCCCCCCCHHHHHHHHHHHHHHCCCCHHHHHHHHCEEEEEEEEEEEEECCCCEECCCCCCCCCCHHCCCCCCCCCCCCCCHHHHHCCCCCCCCCCCEEEEECCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCEECCCCHHHHHHHHHHHHHHHHHHHHHHHHHC

Homology modelling with Modeller

Modeller uses two types of files to be run. The first one contains the used alignment in the PIR-format (see PIR FORMAT), the second one is a python script, which tells Modeller which steps it has to perform. There are some examples in /apps/modeller9.9/examples/automodel. But python seems to be falsely configured on the virtual machines (at least the linux virtual machine).

• The used fix of the python installation is described at the software section.
• The Modeller modules were still not importable by Python, that is why it was necessary to reinstall Modeller. The steps for this are described in software section.

After all this stuff we tried to write a alignment-file with the hhsearch alignments and a python-file (according to the example /apps/modeller9.9/examples/automodel/model-default.py. But modeller seems to be very sensible due to missing acids in the coordinate section of the pdb, which are of course mentioned in the sequence used in the alignment.

To avoid this problem there are at least two possibilities:

• Repair the pdb by the script repairPDB. Map the sequence in the alignment on the sequence in the coordinate section of the used pdb (a lot of work - need some kind of alignment and a pdb-parser... both is not trivial)
• The other possibility is to let Modeller create the alignment on the basis of the repaired PDB (with repairPDB).

We have chosen the second option, which is really nice described in the Modeller tutorial (see basic modeller tutorial).

At the example of 1PHZ. We prepared three files.

• The alignment file, which contains only the reference sequence: pah.ali
  >P1;PAH
sequence:reference::::::::
MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEEN
DVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKD
TVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPI
PRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQ
FLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGH
VPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLL
SSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATI
PRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK*

• The python script, which tells Modeller to create an alignment
  from modeller import *

env = environ()
aln = alignment(env)
mdl = model(env, file='../structures/1phz.pdb', model_segment=('FIRST:A','LAST:A'))
aln.append_model(mdl, align_codes='1phz', atom_files='../structures/1phz.pdb')
aln.append(file='pah.ali', align_codes='PAH')
aln.align2d()
aln.write(file='phz.ali', alignment_format='PIR')


• Then we tried to optimize the alignment. (for the different criteria see below)

• The python script, which tells Modeller to create a model
  from modeller.automodel import *

a = automodel(env, alnfile='phz.ali',
knowns='1phz', sequence='PAH',
assess_methods=(assess.DOPE, assess.GA341))
a.starting_model = 1
a.ending_model = 1
a.make()


As already mentioned above, we took a closer look at the sequence above.

• We removed gaps at the beginning and at the end of the alignment
• We tried to shift the alignment, such that the dssp secondary structures of the reference (used an alignment to 2PAH from task 3) and the template (new dssp run) have a higher coverage
• We tried to match the corresponding annotated functional sites (PDB).

Homology modelling with Swissmodel

Standard workflow

The standard workflow of Swissmodel is the automated mode. For this mode only the UniProt accession number or the amino acid sequence of the target protein is required. As an optional parameter it is possible to enter the template structure as well. However, if this field is left blank Swissmodel will search automatically for a suitable template.

The input for all three models is as follows:

Category	Template	Target	Image
> 60% sequence identity	1PHZ Chain: A	P00439 (Phenylalanine-4-hydroxylase)
> 40% sequence identity	1TOH Chain: A	P00439 (Phenylalanine-4-hydroxylase)
< 40% sequence identity	1LTZ Chain: A	P00439 (Phenylalanine-4-hydroxylase)

Homology modelling with iTasser

ITasser is a prediction server, which participated in several CASP competitions. It claims of itself to be the best one. The interface of this server can be seen in Figure 1. The runtime of the jobs is approximately 24 to 48 hours. The server seems to receive a lot of jobs, that is why it is not allowed to add more than one job at a time from the same ip-address. Therefore it is probably not possible to run all six variants described in the task.

Evaluation of the calculated models

Selection of the reference structures

We had the following choice of reference structures for PAH:

Entry	Method	Resolution (A)	Chain	Positions
1DMW	X-Ray	2.00	A	118-424
1J8T	X-Ray	1.70	A	103-427
1J8U	X-Ray	1.50	A	103-427
1KW0	X-Ray	2.50	A	103-427
1LRM	X-Ray	2.10	A	103-427
1MMK	X-Ray	2.00	A	103-427
1MMT	X-Ray	2.00	A	103-427
1PAH	X-Ray	2.00	A	117-424
1TDW	X-Ray	2.10	A	117-424
1TG2	X-Ray	2.20	A	117-424
2PAH	X-Ray	3.10	A/B	118-452
3PAH	X-Ray	2.00	A	117-424
4PAH	X-Ray	2.00	A	117-424
5PAH	X-Ray	2.10	A	117-424
6PAH	X-Ray	2.15	A	117-424

All these structures have in common that they did not solve the structure of the whole PAH protein. In addition, there is no complete true apo structure available either. All structures have at least a Fe2+ atom bound. So we defined these structures as our apo structure. Finally, we decided to select 1J8T (apo) and 1J8U (complexed). As mentioned before our apo structure has complexed Fe2+ and our complexed structure is complexed with Fe2+ and BH4 (5,6,7,8-TETRAHYDROBIOPTERIN). The reason for our decision was that both structures are solved from the same group which somehow guaranties a more consistent methodology as if we had selected structures from two different groups. Another reason is the resolution, both structures are the two with the best resolved resolution which is 1.5 Angstrom and 1.7 Angstrom for 1J8U and 1J8T respectively. Finally for more easy comparison, both structures include the same range of amino acids which is from 103 to 427.

Numeric evaluation of the calculated models

Modeller

We have chosen three scores to be calculated by Modeller: molpdf, DOPE and GA341

model	modlpdf	DOPE	GA341
1phz	2568.91309	-53912.11328	1.00000
1toh	20481.25977	-37609.82031	1.00000
1ltz	6824.36182	-37422.13672	0.98868
1phz with adjusted alignment	2567.52441	-49139.31250	1.00000
1loh with adjusted alignment	1790.78723	-38113.64453	1.00000
1ltz with adjusted alignment	1197.55518	-26429.44531	1.00000
multiple alignment	61561.77344	-29682.17578	0.00012

Swissmodel

Automated Mode: Modelling with template structure 1phz_A (>60%)

QMEAN Z-Score: -0.828

QMEAN4 global scores:

QMEANscore4	Estimated absolute model quality	Score components
0.715

Local scores:

Coloring by residue error	Residue error plot

Global scores: QMEAN4:

Scoring function term	Raw score	Z-score
C_beta interaction energy	-157.98	-0.16
All-atom pairwise energy	-12503.57	-0.1
Solvation energy	-50.66	1.01
Torsion angle energy	-78.77	-1.48
QMEAN4 score	0.715	-0.83

Local Model Quality Estimation: Anolea / QMEAN / Gromos:

Automated Mode: Modelling with template structure 1toh_A (>40%)

QMEAN Z-Score: -2.745

QMEAN4 global scores:

QMEANscore4	Estimated absolute model quality	Score components
0.604

Local scores:

Coloring by residue error	Residue error plot

Global scores: QMEAN4:

Scoring function term	Raw score	Z-score
C_beta interaction energy	-78.67	-1.16
All-atom pairwise energy	-7899.47	-0.89
Solvation energy	-20.56	-1.3
Torsion angle energy	-47.34	-2.22
QMEAN4 score	0.604	-2.74

Local Model Quality Estimation: Anolea / QMEAN / Gromos:

Automated Mode: Modelling with template structure 1ltz_A (<40%)

QMEAN Z-Score: -4.282

QMEAN4 global scores:

QMEANscore4	Estimated absolute model quality	Score components
0.47

Local scores:

Coloring by residue error	Residue error plot

Global scores: QMEAN4:

Scoring function term	Raw score	Z-score
C_beta interaction energy	-40.25	-2.1
All-atom pairwise energy	-3528.81	-2.29
Solvation energy	-15.22	-1.18
Torsion angle energy	-4.99	-3.78
QMEAN4 score	0.47	-4.28

Local Model Quality Estimation: Anolea / QMEAN / Gromos:

Comparison to experimental structure

To calculate the C-alpha RMSD we used DaliLite.

To calculate the TM-Score we used the TM-score webservice from the University of Michigan.

To calculate the RMSD of the 6A radius of the catalytic center we had to first identify the catalytic center. We defined the center position of the catalytic side as the position where our Fe2+ atom is. With the position in hand we now have to extract the residues in a 6A radius around this Fe2+ atom. In order to do so we executed the following steps:

• We opened the complexed or apo structure and one of the modeled structures with Pymol.
• Then we aligned both structures to each other
• Then we selected the Fe2 atom of the apo/complexed structure and expanded this selection by 6A, residue
• Then we extracted the selected residues into two objects each object contains only the residues of either the apo/complexed structure or the modeled structure
• Then we saved both objects in seperate PDB structures
• Now we used the rms.pl script to calculate the all atom RMSD with the following command "./rms.pl -out all first.pdb second.pdb"

Modeller

Standard Workflow

Template Structure	Compared To	Apo/Complexed	C-alpha RMSD	TM score	All Atoms RMSD, 6A
1PHZ Chain: A	1J8T	Apo	0.9	0.9711	4.2082
1PHZ Chain: A	1J8U	Complexed	0.9	0.9718	-
1TOH Chain: A	1J8T	Apo	1.9	0.8844	5.9302
1TOH Chain: A	1J8U	Complexed	1.9	0.8850	4.3264
1LTZ Chain: A	1J8T	Apo	2.3	0.6986	1.0011
1LTZ Chain: A	1J8U	Complexed	2.3	0.6985	-
Multiple	1J8T	Apo	2.1	0.1886	-
Multiple	1J8U	Complexed	2.1	0.1879	-

Adjusted Alignment Workflow

Template Structure	Compared To	Apo/Complexed	C-alpha RMSD	TM score	All Atoms RMSD, 6A
1PHZ Chain: A	1J8T	Apo	1.0	0.1957	-
1PHZ Chain: A	1J8U	Complexed	1.0	0.1954	-
1TOH Chain: A	1J8T	Apo	1,1	0.1592	-
1TOH Chain: A	1J8U	Complexed	1.1	0.1592	-
1LTZ Chain: A	1J8T	Apo	1.7	0.1021	-
1LTZ Chain: A	1J8U	Complexed	1.7	0.1020	-

Swissmodel

Standard Workflow

Template Structure	Compared To	Apo/Complexed	C-alpha RMSD	TM score	All Atoms RMSD, 6A
1PHZ Chain: A	1J8T	Apo	0.9	0.7400	0.5162
1PHZ Chain: A	1J8U	Complexed	0.9	0.7408	0.5154
1TOH Chain: A	1J8T	Apo	1.3	0.8889	0.4616
1TOH Chain: A	1J8U	Complexed	1.2	0.8894	0.3361
1LTZ Chain: A	1J8T	Apo	2.3	0.8816	0.9225
1LTZ Chain: A	1J8U	Complexed	2.3	0.8814	0.9208

Adjusted Alignment Workflow

Template Structure	Compared To	Apo/Complexed	C-alpha RMSD	TM score	All Atoms RMSD, 6A
1PHZ Chain: A	1J8T	Apo
1PHZ Chain: A	1J8U	Complexed
1TOH Chain: A	1J8T	Apo
1TOH Chain: A	1J8U	Complexed
1LTZ Chain: A	1J8T	Apo
1LTZ Chain: A	1J8U	Complexed

iTasser

Standard Workflow

Template Structure	Compared To	Apo/Complexed	C-alpha RMSD	TM score	All Atoms RMSD, 6A
1PHZ Chain: A	1J8T	Apo
1PHZ Chain: A	1J8U	Complexed
1TOH Chain: A	1J8T	Apo
1TOH Chain: A	1J8U	Complexed
1LTZ Chain: A	1J8T	Apo
1LTZ Chain: A	1J8U	Complexed

Adjusted Alignment Workflow

Template Structure	Compared To	Apo/Complexed	C-alpha RMSD	TM score	All Atoms RMSD, 6A
1PHZ Chain: A	1J8T	Apo
1PHZ Chain: A	1J8U	Complexed
1TOH Chain: A	1J8T	Apo
1TOH Chain: A	1J8U	Complexed
1LTZ Chain: A	1J8T	Apo
1LTZ Chain: A	1J8U	Complexed