1. What is your structure/protein? How was this structure determined (ie method – crystallography, NMR, Cryo-EM, other)? What is the resolution? Was this structure published (if so, give the citation)?
2. If your molecule of interest has (hypothetically) different types of structures available (ie NMR, crystal, cryo-EM), How would you determine which to use? List the characteristics you would look for in the structures and explain how they would help you to decide which structure is more well-resolved or a better choice.
3. How many chains are in your structure? Do you have any ligands, ions, nucleotides, etc present? Is this a biological assembly? Is this an asymmetric unit with multiple chains?
4. Are all of the amino acids present in your PDB file? If not, what is missing and postulate why that might be the case.
5. You want to include a representation of your molecule in a figure. Create a rendering of your molecule. Use different types of representations and different colors. If you have multiple chains, please distinguish them. If you have additional non-protein molecules present (ie ligands, nucleotides, ions, etc), highlight those as well. Explain in detail in a figure legend what you have shown and how it is represented. Don’t forget to include the program you used to create your image and cite it properly.
6. Do you have any hydrogen bonding in your protein? Please show a rendering of all hydrogen bonds. （Regarding question 6 and showing the hydrogen bonds, not all of the assigned proteins will have hydrogen bonding. If you are unable to see the hydrogen bonding representation, please confirm whether you have hydrogen atoms in your PDB file. If your protein falls in this category and you cannot create a rendering, offer an explanation regarding the lack of hydrogen bonding.）
7. Where are all of the charged residues on your protein? Are they about equally distributed or do specific surfaces have patches of charges. What about the location of hydrophobic residues? Create a rendering showing (a) the positively and negatively charged residues and (b) the hydrophobic residues. Is this result surprising or expected? Please explain.
8. You would like to covalently link the C-terminus of one chain of your protein to the N-terminus of the next chain. Your lab has some crosslinkers on hand with spacer arm distances of 9Å, 12 Å, 25 Å, and 39 Å. Will any of these crosslinkers work? If so, which would you choose? If not, what length spacer arm would be necessary and why? (Hint – if your protein isn’t completely resolved, use the N and C terminus from the determined structure)
9. You want to specifically label a cysteine residue in your protein with a fluorophore. How many cysteine residues total are in the molecule? Are all of the cysteines resolved in the structure or are some missing? Show a surface rendering of the molecule highlighting cysteines. Does the resolved structure have any surface exposed cysteine residues that may be a good candidate to label? Should you label one of these residues or engineer a new cysteine? If you chose to label an existing cysteine, which residue(s) may be a good candidate and why? If you think we should engineer a new cysteine, suggest a residue to mutate and why.
