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Bioinformatics Tutorial

This document summarizes the analysis of a protein sequence (CbThiD) using bioinformatics tools. It was determined that CbThiD belongs to the ribokinase superfamily and is most similar to a sequence from Clostridium Difficile. Secondary structure prediction identified helical regions in CbThiD. A homology model was generated for CbThiD using the structure 2i5b as a template, and no major structural differences were found between the query and template structures.

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Wei Feng Zhen
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110 views

Bioinformatics Tutorial

This document summarizes the analysis of a protein sequence (CbThiD) using bioinformatics tools. It was determined that CbThiD belongs to the ribokinase superfamily and is most similar to a sequence from Clostridium Difficile. Secondary structure prediction identified helical regions in CbThiD. A homology model was generated for CbThiD using the structure 2i5b as a template, and no major structural differences were found between the query and template structures.

Uploaded by

Wei Feng Zhen
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Wei Zhen

Enzymology
Part A:
1) See attached
2) Shown below
Part B
3. To what superfamily does this protein belong?
Ribokinase_pfkb_like superfamily
4. How many significant (e-value < 0.001) matches are there for our
protein?
10
5. List the names of the organisms of the top three significant matches.
Clostridium Difficile (seq1)
Salmoella Typhimurium (seq2)
Bacillus Subtillis
(seq3)
Part C Alignment
Input:
>CB_ThiD
MKKVLTIAGSDSSGGAGIQADIKTMSSLGVYAMSIITAVTAQNSIGVQDVHEVPKNMVEA
QIKSVFDDIDVDAVKIGMLSNSETIKSIKEYLEKYKAKNIVLDPVMVSKSGYFLLKPEAI
EELKKLISITNIVTPNIPEAEVLSKIEINSEDDMKKAAIIIQAIGVKNVLVKGGHRCSDA
NDILLYEDKFITIPGNRIETKNTHGTGCTLSSAIASYLTKGFNIEKSVSLSKEYITKAIE
NSFPIGEGVGPVGHFIELYKKAHLDF
>seq1
MQRINALTIAGTDPSGGAGIQADLKTFSALGAYGCSVITALVAENTCGVQSVYRIEPDFV
AAQLDSVFSDVRIDTTKIGMLAETDIVEAVAERLQRHHVRNVVLDTVMLAKSGDPLLSPS
AIETLRVRLLPQVSLITPNLPEAAALLDAPHARTEQEMLAQGRALLAMGCEAVLMKGGHL
EDAQSPDWLFTREGEQRFSAPRVNTKNTHGTGCTLSAALAALRPRHRSWGETVNEAKAWL
SAALAQADTLEVGKGIGPVHHFHAWW
>seq2
MQRINALTIAGTDPSGGAGIQADLKTFSALGAYGCSVITALVAENTCGVQSVYRIEPDFV
AAQLDSVFSDVRIDTTKIGMLAETDIVEAVAERLQRHHVRNVVLDTVMLAKSGDPLLSPS
AIETLRVRLLPQVSLITPNLPEAAALLDAPHARTEQEMLAQGRALLAMGCEAVLMKGGHL
EDAQSPDWLFTREGEQRFSAPRVNTKNTHGTGCTLSAALAALRPRHRSWGETVNEAKAWL
SAALAQADTLEVGKGIGPVHHFHAWW
>Seq3

MSMHKALTIAGSDSSGGAGIQADLKTFQEKNVYGMTALTVIVAMDPNNSWNHQVFPIDTD
TIRAQLATITDGIGVDAMKTGMLPTVDIIELAAKTIKEKQLKNVVIDPVMVCKGANEVLY
PEHAQALREQLAPLATVITPNLFEASQLSGMDELKTVDDMIEAAKKIHALGAQYVVITGG
GKLKHEKAVDVLYDGETAEVLESEMIDTPYTHGAGCTFSAAVTAELAKGAEVKEAIYAAK
EFITAAIKESFPLNQYVGPTKHSALRLNQQS
ALLIGNMENT:

CLUSTALO(1.2.1)multiplesequencealignment
CB_ThiDMKKVLTIAGSDSSGGAGIQADIKTMSSLGVYAMSIITAVTAQNSIGVQDVHEVPKN
seq1MQRINALTIAGTDPSGGAGIQADLKTFSALGAYGCSVITALVAENTCGVQSVYRIEPD
seq2MQRINALTIAGTDPSGGAGIQADLKTFSALGAYGCSVITALVAENTCGVQSVYRIEPD
Seq3MSMHKALTIAGSDSSGGAGIQADLKTFQEKNVYGMTALTVIVAMDPNNSWNHQVFPIDTD
:.*****:**********:**:..*.::*.:.*::.*.::
CB_ThiDMVEAQIKSVFDDIDVDAVKIGMLSNSETIKSIKEYLEKYKAKNIVLDPVMVSKSGYFLLK
seq1FVAAQLDSVFSDVRIDTTKIGMLAETDIVEAVAERLQRHHVRNVVLDTVMLAKSGDPLLS
seq2FVAAQLDSVFSDVRIDTTKIGMLAETDIVEAVAERLQRHHVRNVVLDTVMLAKSGDPLLS
Seq3TIRAQLATITDGIGVDAMKTGMLPTVDIIELAAKTIKEKQLKNVVIDPVMVCKGANEVLY
:**:::.::*:****::::::.::*:*:***:.*..:*
CB_ThiDPEAIEELKKLISITNIVTPNIPEAEVLSKIEINSEDDMKKAAIIIQAIGVKNVLVKGG
seq1PSAIETLRVRLLPQVSLITPNLPEAAALLDAPHARTEQEMLAQGRALLAMGCEAVLMKGG
seq2PSAIETLRVRLLPQVSLITPNLPEAAALLDAPHARTEQEMLAQGRALLAMGCEAVLMKGG
Seq3PEHAQALREQLAPLATVITPNLFEASQLSGMDELKTVDDMIEAAKKIHALGAQYVVITGG
*.:*:*..::***:***..:::*.:*:*:*::.**
CB_ThiDHRCSDANDILLYEDKFITIPGNRIETKNTHGTGCTLSSAIASYLTKGFNIEKSVSLSK
seq1HLEDAQSPDWLFTREGEQRFSAPRVNTKNTHGTGCTLSAALAALRPRHRSWGETVNEAK
seq2HLEDAQSPDWLFTREGEQRFSAPRVNTKNTHGTGCTLSAALAALRPRHRSWGETVNEAK
Seq3GKLKHEKAVDVLYDGETAEVLESEMIDTPYTHGAGCTFSAAVTAELAKGAEVKEAIYAAK
**::.::****:***:*:*::::.::::*
CB_ThiDEYITKAIENSFPIGEGVGPVGHFIELYKKAHLDF
seq1AWLSAALAQADTLEVGKGIGPVHHFHAWW
seq2AWLSAALAQADTLEVGKGIGPVHHFHAWW
Seq3EFITAAIKESFPLNQYVGPTKHSALRLNQQS
:::*:::::::**.*

6. Based on the alignment scores, which sequence is the most similar


to CbThiD (give the PDB code)?
Seq1 has the most similarity compared to CbThiD (PDB code =
4JJP_A
7. Print out the alignment (use view alignment file and print this
page) and on that page highlight the longest continuous stretch of
completely conserved (identical) amino acids. There is a 19 residue
stretch that possesses approximately 70% identity. Put a box around
this region.

Part D. Secondary Structure Prediction


8. Highlight all helical regions of the amino acid sequence (as predicted
by Jpred) on the amino acid sequence that you printed out for question
two (hint: use the simple HTML viewing option).
Red = Helices
Green = Sheets

MKKVLTIAGSDSSGGAGIQADIKTMSSLGVYAMSIITAVTAQNSIGVQDVHEVPKNMVEA
QIKSVFDDIDVDAVKIGMLSNSETIKSIKEYLEKYKAKNIVLDPVMVSKSGYFLLKPEAI
EELKKLISITNIVTPNIPEAEVLSKIEINSEDDMKKAAIIIQAIGVKNVLVKGGHRCSDA
NDILLYEDKFITIPGNRIETKNTHGTGCTLSSAIASYLTKGFNIEKSVSLSKEYITKAIE
NSFPIGEGVGPVGHFIELYKKAHLDF
Part E: Homology modeling
9. Open the structure with Chimera (see directions on our Blackboard
site for a refresher on how to use Chimera) and examine the overall
fold. Show your structure as cartoon, color by secondary structure,
then print out an image of the 3D structure (use File, save image as,
select TIFF, and then insert the .TIFF picture into Word). You should
probably set the background color to white to make printing easier.

10. What structure (provide PDB code) was used as a template for the
homology model?
2i5b
11. Download the PDB file for the structure you identified in question
10 from the protein data bank (from the PDB website, type in the PDB
code and when the entry comes up, go to the right side where

download files is displayed and click there to select PDB file (text) or
fetch in the main Chimera window. Using Chimera, print out a similar
figure as in question 9 for this protein.

(pentemer)
12. You can align two structures in Chimera by going to Tools "
Structure comparison " Match Maker. Are there any major
structural differences between the query (CbThiD) and the template?

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