Task 2: Alignments

Sequence alignments are a good start to analyse a new protein sequence. Alignment search tools such as Blast, Psiblast or hhblits can be used to find related protein sequences.

Sequence Searches

All searches whre done with an increase number of output lines (10000) in the summary hit list and for the reported alignments in order to have all found hits displayed. The analyses were all conducted using all displayed hits. We set no specific evalue cutoff.

The HFE protein sequence has the Uniprot ID Q30201, NCBI ID 1890180 and PDB ID 1A6Z_A.

All Blast, Psiblast and hhblits output files that where analyses where first parse using the perl script parse_output.pl. For example:

perl /mnt/home/student/kalemanovm/master_practical/Assignment2_Alignments/scripts/task1/parse_output.pl --out_p /mnt/home/student/betza/task2/blast/res_blast.txt  

CATH is a database that hierarchically classifies all domain structures from PDB. This hierarchy consists of four major levels: 1. Class 2. Architecture 3. Topology 4. Homologous superfamily

We compared the first level of the CATH hierarchy (Class) to check whether the hits have the same fold class as the query. The class of a domain is determined by its secondary structure composition (mostly alpha, mostly beta, mixed alpha/beta or few secondary structures). Each domain of a protein is assigned to a CATH class, this means, that one query protein can consist of several fold classes, one for each domain. The python script compareCath.py was written to check the overlap of the query protein's fold classes with those of the hits. The two domains of the HFE protein belong to fold class 2 and 3.

Example call:

python /mnt/home/student/betza/scripts/compareCath.py -i /mnt/home/student/betza/task2/blast/res_blast.txt_results -q 1a6zA > /mnt/home/student/betza/task2/blast
/res_blast.txt/blast_cath

Another database for protein structure clustering is COPS. It consists of different structural groups that contain all proteins with a structural similarity above athreshold. For example, all proteins in the L30 group have a structural similarity of at least 30% percent. This means that if two proteins are not in the same L30 group, they have less than 30% structural similararity.

The L30 groups of the hits were determined to chek if they are in the same group as HFE or not. This analyses were also done using the script parse_output.pl:

perl /mnt/home/student/kalemanovm/master_practical/Assignment2_Alignments/scripts/task1/parse_output.pl --out_p /mnt/home/student/betza/task2/blast/res_blast.txt 
--query 1a6z_A --sot L30 

Blast

A Blast search in big_80 was executed using the standard parameter settings:

blastall -p blastp -i /mnt/home/student/betza/data/hfe.fasta -d /mnt/project/pracstrucfunc13/data/big/big_80 -o /mnt/home/student/betza/task2/blast/res_blast.txt  
-v 10000 -b 10000

Blast finds 1504 hits. There are only a few hits with a very high evalue of 1e-160 but most good hits have an evalue between 1e-40 and 1e-50 (left peak). Most results have a worse evalue above 1e-20. Nearly all hits have an identity of 20% to 40% with the query. The evalue and identity distributions are shown below in the plots.

Evalue distribution of the results of the Blast search against big_80 using standard parameters. The x-axis shows the natural logarithm of the evalue and the y-axis the frequency. The better the evalue the smaller is log(evalue).

Identity distribution of the results of the Blast search against big_80 using standard parameters. The x-axis shows the relative identity between the query and the hits.

The Blast results only contain 13 PDB IDs. Nevertheless, the number of shared CATH classes betwen HFE and the hits were computed as described above. Of 13 pdb hits, only 3 share all domain fild classes with HFE, 6 have one domain fold class in commom and 4 even none.

Histogram of the number of shared CATH classes between HFE and the Blast hits.

number of same fold classes	frequency
0	4
1	6
2	3

Only 6 of the 13 pdb hits are in the same COPS L30 group as the HFE protein.

All results indicate, that some of the Blast hits are not really related to HFE and thus false positives.

Psiblast

Four different psiblst runsagaisnt big_80 where done using all four combinaitons of:

• 2 iterations
• 10 iterations
• default E-value cutoff (0.002)
• E-value cutoff 10E-10

Example call for Psiblast with 10 iterations and evalue cutoff of 10E-10:

blastpgp -i /mnt/home/student/betza/data/hfe.fasta -d /mnt/project/pracstrucfunc13/data/big/big_80 -j 10 -h 10E-10 -v 10000 -b 10000 -o /mnt/home/student/betza
/task2/psiblast/new/j1h1/j1hi.txt -Q /mnt/home/student/betza/task2/psiblast/new/j1h1/j1h1.pssm -C /mnt/home/student/betza/task2/psiblast/new/j1h1/j1h1.chk

The perl scrip devide_psiblast_out.pl was then used to divide the different psiblast iterations in order to be able to analyse the results of the last iteration alone.

perl /mnt/home/student/kalemanovm/master_practical/Assignment2_Alignments/scripts/task1/devide_psiblast_out.pl /mnt/home/student/betza/task2/psiblast/new/j1h1
/j1hi.txt

The evalue an identity distributions of the four runs are plotted below.

Histogram of the log(evalue) distribution of the results of the four different Psiblast searches against big_80: 2 iterations with default evalue cutoff (0.002) and 10E-10 and 10 iterations with default evalue cutoff(0.002) and 10E-10. The x-axis shows the natural logarithm of the evalue and the y-axis the frequency. The better the evalue the smaller is log(evalue).

Boxplot of the log(evalue) of the four different Psiblast searches against big_80: 2 iterations with default evalue cutoff (0.002) and 10E-10 and 10 iterations with default evalue cutoff(0.002) and 10E-10. The thick black line inside the box is the median and the upper and lower end of the box the 25% and 75% quantils. The single points above the whiskers are outliers.

Histogram of the identity distribution of the results of the four different Psiblast search against big_80: 2 iterations with default evalue cutoff (0.002) and 10E-10 and 10 iterations with default evalue cutoff(0.002) and 10E-10. The x-axis shows the relative identity between the query and the hits.

Boxplot of the identity of the results of the four different Psiblast search against big_80: 2 iterations with default evalue cutoff (0.002) and 10E-10 and 10 iterations with default evalue cutoff(0.002) and 10E-10. The thick black line inside the box is the median and the upper and lower end of the box the 25% and 75% quantils. The single points above and below the whiskers are outliers.

The results of the CATH and COPS classification analysis are listed below:

Comparison of the results of the four Psiblast searches against big_80.

psiblast parameters	j=2, h=0.002	j=2, h=10E-10	j=10, h=0.002	j=10, h=10E-10
# hits	2564	3163	2601	3343
# PDB hits	66	72	56	66
0 CATH class	21	21	20	21
1 CATH class	42	48	33	42
2 CATH classes	3	3	3	3
same L30 group	6	6	6	6

There are 1095 proteins in the same COPS L30 group as the query HFE. Since the big_80 datatbase does not contain all proteins from pdb, the quality of the program cannot be assesed using the COPS L30 group of HFE. Nevertheless, of the found pdb hits only a few belong to the same L30 and share the same CATH fold classes with HFE.

To get more pdb ids, we also did 4 psiblast searches against /mnt/home/rost/kloppmann/data/blast_db/pdb_seqres using the profiles created with big_80. Those four results were compared as well.

Histogram of the log(evalue) distribution of the results of the four Psiblast searches against pdb_seqres using the checkpoint files from the runs before. (2 iterations with default evalue cutoff (0.002) and 10E-10 and 10 iterations with default evalue cutoff(0.002) and 10E-10). The x-axis shows the natural logarithm of the evalue and the y-axis the frequency. The better the evalue the smaller is log(evalue).

Boxplot of the log(evalue) of the four different Psiblast searches against pdb_seqres using the checkpoint files from the runs before. (2 iterations with default evalue cutoff (0.002) and 10E-10 and 10 iterations with default evalue cutoff(0.002) and 10E-10). The thick black line inside the box is the median and the upper and lower end of the box the 25% and 75% quantils. The single points below the whiskers are outliers.

Histogram of the identity distribution of the results of the four different Psiblast searches against pdb_seqres using the checkpoint files from the runs before. (2 iterations with default evalue cutoff (0.002) and 10E-10 and 10 iterations with default evalue cutoff(0.002) and 10E-10). The x-axis shows the relative identity between the query and the hits.

Boxplot of the identity of the results of the four different Psiblast searches against pdb_seqres using the checkpoint files from the runs before. (2 iterations with default evalue cutoff (0.002) and 10E-10 and 10 iterations with default evalue cutoff(0.002) and 10E-10). The thick black line inside the box is the median and the upper and lower end of the box the 25% and 75% quantils. The single points above and below the whiskers are outliers.

Comparison of the results of the four Psiblast searches against pdb_seqres.

psiblast checkfiles	j=2, h=0.002	j=2, h=10E-10	j=10, h=0.002	j=10, h=10E-10
# hits	4307	4814	3874	4058
0 CATH class	561	586	529	539
1 CATH class	2798	3280	2397	2571
2 CATH classes	948	948	948	948
same L30 group	1047	1053	1045	1052

1095 proteins are in the same COPS L30 group as HFE. The differnent psiblast searches found nearly all proteins that are in the same L30 group as HFE. Therefore,the decision which of the parameter settings leads to the best results cannot be based ont he pdb hits, since the differences are minimal.Nevertheless, j=10, h=0.002 had the least hits, but the highest percentage of hits in the same L30 group as the query.

HHblits

hhblits -i /mnt/home/student/betza/data/hfe.fasta -d /mnt/project/rost_db/data/hhblits/uniprot20_02Sep11 -o /mnt/home/student/betza/task2/hhblits/hfe.hhr -oa3m /mnt/home/student/betza/task2/hhblits/hfe.a3m -oalis /mnt/home/student/betza/task2/hhblits/hfe -ohhm /mnt/home/student/betza/task2/hfe.hhm -Z 10000 -B 10000

The output files were analysed using the script parse_output.pl. example: perl /mnt/home/student/kalemanovm/master_practical/Assignment2_Alignments/scripts/task1/parse_output.pl --out_p /mnt/home/student/betza/task2/blast/res_blast.txt --query 1a6z_A --sot L30 --out_h /mnt/home/student/betza/task2/hhblits/

Evalue distribution of the results of the HHblits search with default parameters using the using the uniprot20_02Sep11 database. The x-axis shows the natural logarithm of the evalue and the y-axis the frequency. The better the evalue the smaller is log(evalue).

Identity distribution of the results of the HHblits search in the uniprot20_02Sep11 database using default parameters. The x-axis shows the relative identity between the query and the hits.

It is remarkable that HHblits finds so many hits with a very low evalue. This can maybe explained by the fact, that hhblits found several clusters with a good evalue. Since used all proteins in the clusters for the analyses and not only the cluster representative the number of hits is clearly higher than the number of hits from the Blast and Psiblast searches.

Comparison

TODO evalue

TODO idenity

TODO: Venn diagramms of hits overlap.

Multiple sequence alignments

The following data sets were chosen for the Multiple Sequence Alignments:

Below 30% SeqID:

AC Number	Sequence Identity
F6JYA9	0.29
D9J389	0.28
D5MSB3	0.27
B3FRK2	0.20
3ov6_A	0.22
1p7k_L	0.21
H0Y1D0	0.20
B3FRK3	0.18
Q8HWL2	0.17

Above 60% SeqID:

G5BQE5 0.67

AC Number	Sequence Identity
B4DDZ1	0.83
Q5EEZ1	0.76
G3THV5	0.75
G1MBW1	0.73
H0VAR7	0.72
F1PX48	0.71
G1T7D7	0.70
O35799	0.68

Whole range of SeqID:

AC Number	Sequence Identity
B4DDZ1	0.83
Q5EEZ1	0.76
G1MBW1	0.73
H0VAR7	0.72
F1PX48	0.71
G1T7D7	0.70
G5BQE5	0.67
Q95IT9	0.38
2qrt-A	0.38
Q8HX83	0.36
F6WCX4	0.34
F6JYA9	0.29
D9J389	0.28
D5MSB3	0.27
2zok_E	0.24
H0Y1D0	0.20
Q8SNJ4	0.22
B3FRK3	0.18
Q8HWL2	0.17

pdb: 1p7k_L 0.21
3ov6_A 0.22

2qrt_A 0.38
2zok_E 0.24

ClustalW

Below 30% SeqID: ClustalW_Below_30
Above 60% SeqID: ClustalW_Above_60
Whole range of SeqID: ClustalW_whole

MAFFT

Below 30%:MAFFT_30
Above 60%: MAFFT_60
Whole range: MAFFT_whole

T-Coffee

Below 30%: Tcoffee_30
Above 60%:Tcoffee_60
Whole range: Tcoffee_whole