Student level introduction to autoBUSTER tutorial:  Refinement and model building of a molecular replacement from a homology model of SULT1A3  Question sheet.
See: https://www.globalphasing.com/buster/wiki/index.cgi?AutoBusterExampleIntro
Your name: Your name
Part A: calculating a map for the initial MR model.
	Question A1: what do the following options do in autobuster refine?: 
		-p 		*
		-m 		*
		-M MapOnly	*
		-d		*
Question A2: what are the R and Rfree values reported by autoBUSTER refine?
	R value:  	*
	Rfree value:	*
Note from these values you cannot tell whether the MR has been successful or not.

For questions A3, A4, A5. You should be using coot to look at the autobuster map of the initial MR model around residue TRP A 155: 
Question A3:.Residue 155 is a TRP in sult1a3 but is a PHE in estrogen sulfotransferase. Are these amino acids similar? How do they differ.
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Question A4: Is residue A155 complete in the MR model?
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Question A5: What do the autobuster maps show about A155?
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For question A6 to A9 look at residue A 272 GLN that is adjacent to TRP A155. This residue is a GLN in sult1a3 but is a LEU in estrogen sulfotransferase.
Question A6:.Is the sidechain of A272 complete? Is it in density?
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Question A7: How are LEU and GLN sidechains similar and how do they differ.
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Question A8: looking at the autoBUSTER maps around the backbone of A272, is there indication that the model needs correction? 
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Question A9: What is the backbone secondary structure  around residue A272? 
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For question A10 to A14  you should be using the "Validate" "Unmodelled Blobs" feature to look for unmodelled blobs in the difference map.
Question A10:.look for arginine  and lysine residues that are close to the blobs identified. What residues are close to the blobs found?
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Question A11: Are arginine and lysine amino acid side chains normally charged at physiological pH?.
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Question A12: Looking at the unmodelled blob in the A chain are the lysine and arginine residues close to the protein surface..
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Question A13. Display just the difference map then use the + and – keys .to change the contour level to 4.0 sigma. What do you see? At what sigma level do the blobs disappear?
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Question A14. What kind of atom would be expected to produce large difference map peaks? Re-read background – what do you think the difference density is for?
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For questions A15 and A16 you should be have superposed 1aqu.pdb onto the A chain of the MR model.
Question A15:.What molecule is found in 1aqu.pdb into the unmodelled blob next to A130 ARG? Are the phosphate atoms in this ligand bound where you expect (see question A14)?
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Question A16:.Produce as pretty a picture of the PAP molecule and difference density and attach it here
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Part B: Using autoBUSTER to do a quick refinement from the SULT1A3 MR model

For questions B1 to B4 you should be looking at partB.log for initial warning messages
Question B1:.How many very bad bond lengths, very bad bond angles and very bad ideal distance constants are there in the initial structure? Are there any chiral atoms that are inverted?
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Question B2:. What are the initial Rfact, Rfree, rms bond length and rms bond angle? (Hint look for the Ncyc=0 line in the table).
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Question B3:. Scroll to the WARNINGS at the start of BIG cycle 2. How many warning are there now? What residue is mentioned in the warnings.
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Question B4:. Now scroll to the end of table reporting cycle 2 How have the Rfact and Rfree changed. What about the rms bond and rms bond angle
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Conclusion refinement allows the model to adjust the models atoms positions and B factors to better fit the X-ray data and have good geometry.

For question B5 and B6 you should be looking at looking at a graph of Rwork and Rfree produced by the graph_autobuster_R command. Rwork is the Rfactor measuring the agreement between calculated and observed structure factors that are used in the refinement. Rfree is the Rfactor for the validation set that are not used in the refinement. In this case Rfree starts below Rwork by chance.
Question B5:How Rwork and Rfree vary during this refinement? 
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Question B6:Do you think the refinement has converged.
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Part C: Using autoBUSTER to do a long refinement from the SULT1A3 MR model

For questions C1  you should be looking at the graph of Rwork and Rfree in the run
Question C1: What does the graph show? Has Rwork dropped below Rfree? What is the approximate %Rfree and % gap between Rwork and Rfree.
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For questions C2 & C3  look at partC.log
Question C2: Are there any geometry warnings at the start of the last big cycle? 
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Note that geometry WARNING are only for really bad geometry problems just because they are no warnings does not mean the geometry is OK (see below).

Question C3: What is the final rms bond and angle deviation in run partC? 
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Note that by default autoBUSTER refine adjusts the weight on the X-ray function to achieve an rms bond of 0.010 Angs.

For questions C4 & C5 you should be using coot to look at the initial and final structures of the partC refinement for an area with big movements.
Question C4: What is the biggest atomic displacement you can find? (Hint look around residue A 27). 
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Question C5: Now load the partA/refine.mtz and partC/refine.mtz to get the maps and look at the area with the big displacement. What has the refinement done? Does the 2Fo-Fc map look the same at the start and finish. If you wish illustrate your answer with snapshot pictures.
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For questions C6 you should be using coot to look at the difference density at the expected PAP binding site after refinement.
Question C6: What has happened to the difference density at the expected PAP binding site compared to that in the initial MR map. 
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Part D:  Manually building three residues using coot

For questions D1  you should be running coot looking at the model and map from partC looking at residue A 30
Question D1: What amino acid is at residue number 30 in the A chain? Is the amino acid complete? Does the density indicate how the model should corrected?
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Now follow the instructions on the wiki page to correct this residue.
Question D2: What happens when click on an atom and drag it in coot? What happens when you ctrl-click and drag an atom? What do the traffic lights indicate?
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For questions D3 you should be running coot and have rebuilt residues 30, 155 and be considering the placement of residue 272
Question D3 How should the sidechain of GLN 272 be placed and what interactions can it make?
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Question D4 Is there any difference around the changes you made?

Part E: Using coot automated tools to rebuild and refit all the side chains

For question E1 you should have used coot to fill partial residues in  the partC model/map
Question E1: Has the automated sidechain fixer made the same changes to residue 30, 155 and 272 that you did.
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For question E2 you should have refined the structure with the coot fixed up side chains.
Question E2: What is the Rfree at the end of partE refinement? How does this compare to the Rfree at the end of partC?.
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Only for advanced users with enough time. 
Question E3: Has improving the side chains of the model improved the density at the expected PAP binding site? What about at the expected dopamine binding site? Hint dopamine could be expected to bind somewhere here the phenol like end of estradiol in 1aqu.pdb For question E2 you should have refined the structure with the coot fixed up side chains.
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Part F: ****
To be written