Assignment #3

Check this page periodically through Wednesday of Finals week for additions and corrections. -- T. Frey

Tuesday, May 19: I will post the 3rd and final question on Thursday morning. I'll be at the San Diego Cell Biology meeting all day tomorrow (Wednesday). -- T. Frey

1. Protein Structure Question:

Download the structure file and the sequence file for Bovine Adenylate Kinase Isozyme-2 from the sites listed in the Protein Structure Classification Page: http://www.sci.sdsu.edu/TFrey/Bio750/ProtStructClass.html

(a) Examine the structure of Adenylate Kinase and describe its classification based upon secondary structure. Include a printout of the the structure to demonstrate your conclusion (the printout does not need to be in color). Use RasMol which is available on the computers in LS 126; RasMol can also be downloaded for computers running the Macintosh, Windows, and UNIX operating systems from the RasMol site: http://www.umass.edu/microbio/rasmol/

(b) Analyze the amino acid sequence of Adenylate Kinase and provide the following plots

What can you conclude about the enzyme structure from these plots? You can use any protein sequence analysis programs you have access to (see the Protein Structure Classification Page) or you can get a copy of the Excel spreadsheet which I demonstrated in class; this will be on the same disk which contains the Myocyte.PICT image file used in the next question. I'll show you how to use the spreadsheet without grade penalty.

(c) Compare in detail the predictions based upon the plots in 1b with the structure. How do the predictions compare with the actual structure?


2. Dr. Paolini's Question:

This exercise requires the use of the NIH Image software package (versions 1.58 or 1.61 are readily available) and an image saved on this website. Examine the provided image "myocyte" of an isolated cardiac myocyte prepared from the heart of an adult rat. Show the intensity histogram from this flat image, then enhance the contrast and display the resultant histogram. Print out the original and enhanced images and histograms side-by-side. Quantify the contrast improvement. Suppose that this cell is known to be 110 micrometers in length. What is the average sarcomere length and its standard deviation? (hint: use the intensity profile feature from Image and a spreadsheet like Excel to analyze your results). Estimate the number of sarcomeres in this cell (one sarcomere = band-to-band spacing along the one micrometer diameter of a cylindrical myofibril.

The image appears below. You have two choices in obtaining the image:

(1) Obtain a copy of the image on floppy disk; either from Christy in LS 104 or from a GA in the Biology Department Computer lab in LS 126. This is probably your best bet because of a file format problem mentioned below.

(2) Place the pointer on the image and hold down the mouse button until you get a popup menu offering you various options for manipulating the image; choose "Save this Image as..." and save it to a disk. This saves the image in .gif format, unfortunately NIH Image does not read .gif formats. If you can convert the .gif to PICT or TIFF you can then read it with NIH Image.

NIH Image runs on Macintosh computers and is available on the computers in LS 126, many computers in labs throughout the Biology Department, and can be downloaded from:

http://rsb.info.nih.gov/nih-image/Default.html


Question 3: Fourier Transforms

The image below is a two-dimensional projection of a cytochrome oxidase crystal reconstructed from the Fourier Transform of an electron micrograph. In answering this question think of this as a two-dimensional protein structure calculated from relatively low resolution structure factors similar to the example shown in IV. D. The Structure Factor, Fhkl of the X-Ray diffraction lecture; you will need to refer to this figure in answering the question.

(a) Outline one unit cell in the image below and justify your choice. Note that there are many possible correct choices of a unit cell (and many possible incorrect choices). Your answers to part (d) will be simplified if you place the corners of the unit cell on a point of symmetry. Draw a second correct choice of a unit cell.

(b) This 2-dimensional projection is centrosymmetric and therefore contains centers of symmetry or inversion centers; it also contains glide planes which is a symmtry operation in which a structure is mirrored across a line (mirror plane) and then translated one half of a unit cell. Indicate the positions of all centers of symmetry and of all glide planes.

(c) Based upon your analysis of the types of symmetry present, what is the asymmetric unit of this crystal? The asymmetric unit is the smallest part of the unit cell from which the entire unit cell can be generated by applying all of the symmetry operations in whatever order necessary. Explain your answer.

(d) Refer to the example of a diffraction pattern in IV.D. mentioned above. List the (1,0), (0,1), (1,1), (1,-1), (2,0), and (0,2) structure factors in order of decreasing intensity (amplitude) and list the phase of each for the first unit cell you've selected above. Note that this is a centrosymmetric projection which means that for the correct choice of unit cell origin the phases of all structure factors are either 0° or 180°. Justify your choices for the phases, perhaps by making drawings similar to the images which make up the figure in IV.D.

(e) Although this projection is centrosymmetric, real protein molecules cannot have centers of symmetry. Describe the three-dimensional symmetry operations present in the actual crystal which appear as centers of symmetry and glide planes when projected onto two dimensions?

Bonus Question (Not for the faint of heart): Why are the phases of the structure factors of centrosymmetric projections 0° or 180° if the proper phase origin is chosen?

This is the end of Assignment #3 (I guess this is enough!). Have a great summer!! --Terry