In this lab we'll investigate the structure of a S-Adenosyl Methionine Synthetase, sams-1 (NP_510002). sams-1 has not been studied biochemically and its structure has not been determined experimentally. We'll investigate the structure of sams-1 using secondary structure prediction and 3D structure prediction.
The protein structure prediction servers can take a long time to respond (from an hour to several days), so I have submitted prediction requests to several 2° and 3° structure prediction servers ahead of time.
The results can be found in the links above and are in the formats returned by the prediction servers.
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sams-1 function
- 1. Investigating what is known about sams-1.
1a. What is this protein? What organism is it from?
1b. Is sams-1 an enzyme, and if so, what reaction does it catalyse? sams-1 has not been studied extensively, so looking at the function of homologs in human or mouse may be helpful.
Secondary structure prediction
- 2. The secondary structure prediction results are linked above from JPRED, PSIpred, and SAM-T08. PHYRE also makes secondary structure predictions but we'll not use them for question 1. The SAM_T08 FAQ question 6 explains some of the secondary structure prediction alphabets used.
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a. Examine the PSIpred secondary structure predictions. How are different secondary structures represented by PSIpred? Answer briefly.
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b. Is this protein predicted to have primarily one class of secondary structure or is it mixed? Indicate mixed or which type predominates.
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c. Compare the PSIpred secondary structure predictions with those from JPRED and SAM-T08. To do this line them up in a txt file or word precessor. Estimate the agreement between the three prediction programs (70%, 99%, etc). Notice that SAM-T08 uses STR2 for secondary structure predictions and a different prediction alphabet (http://compbio.soe.ucsc.edu/SAM_T08/faq.html#secondary-meaning).
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d. Find a region of 10-15 aa where the predictions disagree. For you answer provide an alignment of the aa sequence with the predictions from the three programs. Include the aa numbering of this region.
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e. The secondary structure preograms give confidence values along with the predictions. For the region of the protein in your answer for part d., was this a region where the programs were confident of the prediction?
3D structure prediction
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3. Now examine tertiary structure predictions for this protein made by PHYRE.
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a. Does PHYRE find potential 3D structures for this protein?
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b. What structure does PHYRE indicate as the best structure model?
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c. Examine the best PHYRE 3D structure model. The Jmol protein model links seem to display best. Paste a screen capture of this structure displayed in 'cartoon' format from an angle that allows the three-fold symmetry to be seen.
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d. View the alignment of the best library sequence to sams-1 (in the View Alignments column). Typeically a 3D structure is predicted for only part of a submitted sequence. What part of the protein is aligned to the library sequence and thus included in the structure model?
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e. Examining the 3D model, what type of secondary structures form the modeled region of the protein? What type of beta sheets are in the structure?
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f. Looking at the PHYRE results, the 'View Alignments' column shows the query sequence secondary structure predictions aligned with the library seqeunce. How well does the predicted secondary structure of the library sequences (the best two models) generally agree with the predicted secondary structure of sams-1?
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g. Examine the predictions ESyPred3D makes for the structure of sams-1. (Use RasMol to view it, select the program to open the file. RasMol is at 'Local Disk'->'Program Files'->'Instructional Software'->'Ras Win'. Then in RasMol select the Display->Strands or Ribbons for easy viewing. Does the ESyPred3D structure prediction agree with the prediction made by PHYRE? To compare the structures, orient them both simlarly and then examine the secondary structure elements. You may find it helpful to break down this question as 1) are the overall folds the same? 2) where are the structures most similar, and 3) where are the structures show the greatest divergence?
h. The ESyPred3D structure is built using PDB ID 1XRA as a template. On what PDB structure is the top PHYRE structure modeled? Are these structures from the same organism?
BIO520