name: midterm chem 645, fall 2013 part i. (4 questions, 10 ... · midterm chem 645, fall 2013 chem...

Midterm CHEM 645, Fall 2013

CHEM 645 midterm, page 1 of 9

Part I. (4 questions, 10 pts. each) A. Short Answers 1. Rob Townley from the Shapiro lab used this technique to identify where to make truncations of his constructs for crystallization. ____________________________________ 2. Name of phasing technique used to solve the phase for ______________________________ the majority of new structures by crystallography. 3. The theoretical lower limit of resolution in X-ray crystallography using X-rays of 1.0 Å is: ____________________________________ 4. The 2013 Nobel Prize in Chemistry was awarded for:

____________________________________

5. In space to right, draw a picture of a peptide plane. ____________________________________ 6. 2D NMR method used to assess the homogeneity of a protein sample. ___________________________________ 7. and 8. Name the two types of data collected in protein NMR that are analogous to intensities of reflections in crystallography._________________________ and ________________________ 9. Protein crystallization technique used to screen and grow protein crystals: ______________________________ 10. Name of the parameter used to evaluate how good a homology model is. ______________________________

Name: _______________________________



B. Resolution of Experimental Models 1) What does the resolution of a structure mean when you are referring to a protein structure solved by NMR? 2) What does the resolution of a structure mean when you are referring to a protein structure solved by X-ray crystallography? Use what you know about Miller indices, parallel planes, and Bragg’s law to help explain your answer. Also, what does higher resolution give you?



C. The expression for the crystallographic R-factor is shown below. R-factor = |Fo|-|Fc| / |Fo|

This equation does not have the phase explicitly shown. However, the R-factor is without a doubt the best criteria to evaluate when a crystal structure refinement has converged on the best phases possible. Explain this apparent discrepancy and at the same time demonstrate that you understand how the R-factor, and specifically the R-free value (Rfree or Rtest), is used to guide the completion of a crystal structure of a protein.



D. The equations that describe the refinement of an NMR or an X-ray crystal structure of a protein are shown to the right. In each method of solving protein structures, the refinement incorporates weighting factors (wNMR, wFhkl and wideal). 1) What would be the result to your structural model if the weighting factor-wideal used in the

refinement was too high?

2) What would be the result to your structural model if the weighting factor-wideal used in the refinement was too low?



Part II. Structural Genomics and High-Throughput Approaches (15 pts. each). Suppose you are working for a pharmaceutical company and are given the responsibility of obtaining structural models for 300 specific proteins of interest. All of these 300 genes have been cloned and are in your lab in a plasmid form. In your answers below, think about coming up with a reasonable model as fast as possible for each of the proteins and then following up with obtaining the best possible structural models. A) Describe briefly how you would initially use homology modeling to obtain a working model

for some of the 300 targets. In your description, explain what you would do, the limitations of the method and finally how you could use this approach to give at least a partial model for some of the more challenging targets.



B) For crystallography and NMR you need to have a source of pure and homogeneous protein. Describe how you would setup a high-throughput expression and purification of the targets. In your description be sure to answer the following two questions: (1) How would you evaluate the quality of each expressed target? (2) How would the approach differ here depending on whether you were going to attempt an NMR or X-ray crystallography structure solution?



C) Describe how you would decide which of the 300 targets you would attempt to solve a crystal structure for. In your description, answer the following questions: (1) What are the experimental limitations of crystallography? (2) What would be your approach to obtain crystals in a high-throughput manner? (3) What would be your approach to obtain phase? (4) How could you use structural and biochemical characterization information obtained in parts-A or B to help with the optimization of crystallization and with the crystal structure solution?



D) How would you decide which of the 300 targets you would attempt to solve an NMR structure for? In your description, answer the following questions: (1) What are the experimental limitations and/or technical challenges of NMR? (2) What types of proteins are particularly good NMR targets due to difficulties from the other two methods? (3) How could you use information obtained in parts-A, B or C to help with the NMR structure solution?



Extra space:

Question Points Your

score I-A 10 I-B 10 I-C 10 I-D 10 II-A 15 II-B 15 II-C 15 II-D 15 Total 100

name: midterm chem 645, fall 2013 part i. (4 questions, 10 ... · midterm chem 645, fall 2013 chem...

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