The main goal of this assignment is two-fold:
1. Enable you to critically read and understand a structure paper and apply what you have learnt so far.
2. To acquire expertise in using the various structural tools/resources you have learnt in the course to a protein-ligand complex of your choice. This will also serve as building blocks for your final project. If you choose your molecule wisely, you can get a head-start on your final project.

(Total: 30 points, will account for 15% of your final grade)

Please upload your assignment directly to Blackboard. DO NOT e-mail the assignment. Please submit by the due date/time. LATE ASSIGNEMENTS AND ASSIGNMNETS E-MAILED TO ME WILL BE SUBJECT TO A PENALTY.

Some rules to follow:

You must choose your own protein-ligand complex No duplicates.
Make sure your answers are brief and to the point
You are welcome to discuss your doubts with me at anytime
HOWEVER, you MUST COMPOSE AND TYPE IN your final answers yourself
AND you are expected to be able to explain the basis of your answer Please DO NOT copy sentences from the article. Rephrase it in your
own words.
DO NOT COPY from your friends. YOUR ASSIGNMENT WILL NOT BE GRADED.

Keep in mind that presentation is an important aspect of communication in Science. Bonus points are possible.
SECTION 1:

The main purpose of this section is to read and understand the differences between X-ray and NMR methodologies. Read the following paper and answer the questions that follow:

Structure of the interleukin-2 tyrosine kinase Src homology 2 domain; comparison between X-ray and NMR-derived structures.
Joseph RE et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Feb 1;68(Pt 2):145-53. Epub 2012 Jan 25.PMID:22297986. The paper has been uploaded to Blackboard.
1. Summarize in not more than four to five sentences the main goal and the rationale for undertaking structural studies of the interleukin-2 tyrosine kinase Src homology 2 domains. What are the PDB-Ids of the structures described in this paper? (2 point)
2. Answer the following questions based on the crystal structure: (4 points)
a. What methodology (for phasing) was used to solve the structure? Experimentally what changes had to be made to the wild-type protein that enabled the authors to crystallize and eventually solve the structure by the described methodology and why did the authors have to make this change? In other words, couldn’t they have solved the wild-type structure without this change?
b. Summarize in the form of a table the following: (DO NOT CUT &
PASTE the Table from the paper)
(i). Resolution of the final refined structure
(ii). R-factor and R-free
(iii). Data Completeness
(iv). Average Temperature Factors (this reflects the dynamics and not the temperature at which the experiment was conducted)
(v). Using one of the tools you have learnt in class provide a snapshot of the Ramachandran plot of the crystal structure. What % of the residues is in the disallowed region of the plot if any?
(vi). Based on all the crystallographic parameters above, is the structure reliable? Does it support the author’s claims? Describe your answer in not more than 3-4 sentences. You can use bullet points.
3. What kind of NMR spectra was used to solve the structure? Provide the experimental details used for the NMR experiment including the instrumentation used. Provide a snap-shot of the NMR spectra of WT and mutant SH2 (you are welcome to take this from the paper by providing a
reference). (2 point)

4. Summarize in 4-5 sentences the major differences if any, between the Xray and the NMR structures. Summarize in 2-3 sentences what major conclusions were made from each of the two structures? Was there agreement between the two structures? Which methodology is yielding more information to the authors and why? (You may tie this to the rationale behind this paper). (4 points)

5. What were the major unresolved questions that the NMR and X-ray structures described in this paper help to address? In one or two sentences, comment if you think these structures are going to further our understanding of the role of cis-trans isomerization in proteins. (3 points)

SECTION 2:

Using Heteroatom navigator
(http://hetpdbnavi.nagahama-i-bio.ac.jp/index.php?mode=0) or

PDB web-site (http://www.rcsb.org/pdb/home/home.do) identify a structure of a complex determined either by X-ray crystallography or NMR with your favorite ligand bound (small molecule). Make sure that the ligand is a biologically relevant one! If you have questions about this, please let me know. Your assignment will not be graded if you choose a ligand that is not physiologically/biologically relevant. For example acetate, glycerol and azide are not biologically relevant in crystal structures. Examples of biologically relevant molecules are ATP, GTP, GDP, NAD, NADP, NADH, PLP, FMN, FAD, SAM etc. This list by no means is exhaustive.
Before you proceed make sure that your structure has electron density data in the EDS server and is biologically relevant.

1) Write out the PDB-ID and the PMID associated with the structure you have chosen. Write a few sentences about the biological function of the protein-ligand complex structure you have chosen. Not more than 4-5 sentences please! DO NOT COPY sentences from the paper. This is considered plagiarism (3 points)

2) Tabulate (present as a table) the H-bond interactions that the ligand makes with protein using PDBSum and Ligand Explorer (provided within the structure record in PDB) tool/resource. Please include both the ligand atoms and the protein residues involved in the interaction.

Is there any bridged water molecules involved in stabilizing the complex? (you can get this information using the Ligand Explorer tool provided within the PDB record). If yes, what residues is it bridging? If no, just mention that there are no bridged water molecules.
Do not just provide a snap-shot of the interactions! (6 points).

Example:
GDP
(Ligand) Atoms Amino Residue Acid Hydrogen Bonds Avg. Distance (Å)
N7 Asn 116 A 1 3.10

3) Write-out any interesting structural features seen using ProMotif (for example, does it have any 310 helices or gamma turns etc.). If none of this exist, list the ones you see.

Provide a snapshot of the Ramachandran plot for your chosen structure.
Comment about the stereochemistry based on the plot in one sentence.
(3 points). For example, is the structure reliable?

4) Classify your protein-complex using the SCOP database. Provide fold, superfamily, family and class information. If SCOP does not contain the information for your structure, please use the Superfamily database.

Provide the topology of your complex. If you see more than one topology, pick the domain that contains your ligand. Snap-shot of the topology will be fine but make sure you label the topology. Make sure your labels are legible. (for example is it 243217 or 3214576 etc…) (3 points).