Sandbox 143

This Sandbox is Reserved from October 27, 2010, through December 21, 2010 for use by the Biochemistry 642 class at the University of Massachusetts at Amherst taught by Wayne Decatur. This reservation includes Sandbox Reserved 101 through Sandbox Reserved 142.

To get started: Click the edit this page tab at the top. Save the page after each step, then edit it again. Click the 3D button (when editing, above the wikitext box) to insert a 3D applet Jmol scene window. show the Scene authoring tools, create a molecular scene, and save it. Copy the green link into the page. Add a description of your scene. Use the buttons above the wikitext box for bold, italics, links, headlines, etc. More help: Help:Editing

Molecular Visualization Problem #3Molecular Visualization Problem #3

by: Karen Plevock, Chris Meaden, Alyssa Gable

IMAGESIMAGES

spinqualitylabelsall models 2KTQ Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

spinqualitylabelsall models 3KTQ distance between oxygen atom of residue 671 and incoming nucleotide Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

spinqualitylabelsall models 2KTQ distance between oxygen atom of residue 671 and incoming nucleotide Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Part IPart I

Page 1:Page 1:

-How many polypeptide and polynucleotide chains are on the screen? 3
-Are there nucleic acid, protein, or both? Both
-If nucleic acid is present, what type of nucleic acid? DNA
-How many chains of protein make up this enzyme? 1
-How many total residues of each chain are actually visible on the screen?
Chain A=538
Chain B=12
Chain D=13

Page 2:Page 2:

-What is the approximate molecular weight of the polypeptide chain visible on the screen?
(538 residues)x(110 daltons/residue)=59.2kD

-What is the pdb identification code of this structure? 2KTQ
-What does the pdb file associtaed with this structure say the structure is? Open ternary complex of large fragment of DNA polymerase I from Thermus aquaticus.
-Was this structure solved with NMR or X-Ray crystallography? X-Ray crystallography
-What is the resolution of this structure? 2.3 Angstroms
-What organism is the native biological source of the enzyme in the structure? Thermus Aquaticus.
-What very general term describes that ranges of temperatures that native organism prefers? high temperatures
-What scientific term is used to broadly classify organisms showing a preference for this temperature range? thermophilic
-In what popular lab procedure is this enzyme most commonly used? What is one reason this enzyme is a good choice for this common lab procedure? PCR. DNA polI of other organisms would denature at the repeated high temperatures required for the melting apart of DNA strands during PCR reactions.
-At what pH does the polypeptide chain visible on the screen have a net charge of zero?

-Which chain is the template? D
-Which chain is the primer? B

-In order to get a homogenous population of a paused complex of a functional polymerase, one approach is to leave out at least one of the four nucleotides so that it will stop when it gets to the point that nucleotide is needed. Was that done for this structure? No

Page 3: Page 3:

-The primer chain ends in a nucleotide not defined as G,A,T or C because it is unusual. What is unusual about it? There is no reactive OH group to incorporate another nucleotide at the 3' end.
- What is a more general, chemically descriptive name for such types of nucleotides? dideoxyribonucleotide
-Noting the unusual feature, why has the enzyme not added the incoming nucleotide to the chain? Th OH group is needed to provide energy to form a phosphodiester bond.
-Name a common lab procedure that relies on the use of such a type of nucleotide. Sequencing
-Can you tell what base is on the 3' end of the primer chain? C
-What is the sequence of each strand of nucleic acid shown?
Chain B 5' GACCACGGCGC*DOC 3'
Chain D 5' GGGCGCCGTGGTC 3'
-Where is the oncoming nucleotide relative to the primer strand? It will be incorporated at the 3' end of the primer strand. It will be a C base pairing with a G on the template strand
-What base is the incoming nucleotide? C
-How does the incoming nucleotide differ from the last nucleotide of the primer? The incoming nucleotide still has 3 phosphate groups, whereas the incorporated nucleotide only has one phosphate group.
-One ion is near the site of chemistry. What is it? Mg²⁺
-Would you expect this to be significant for catalysis? Yes. It promotes deprotonation of the 3' OH of the primer strand and assists that leaving of the pyrophosphate.

Page 4: SCHEMATIC INTERACTIONS OF TWO CHAINSPage 4: SCHEMATIC INTERACTIONS OF TWO CHAINS

-Look at the nucleotide of the template that is 5' of the one base-pared to the last nucleotide ofthe primer. Could the first nucleotide of the template potentially base-pair to the incoming nucleotide? Yes
- Are they base-paired in the structure? Yes
-Is the incoming base paired to any nucleic acid? No
-What type of regular secondary structure element dominates in the protein? Alpha-helices
-What do you need to make an abundant amount of double stranded product of a particular size using this enzyme? PCR Machine, dNTP's, primers, template.

Part IIPart II

Page 5:Page 5:

-Does the new structure include essentially the same protein as in the original structure? Yes
-What is the pbd identification code of the new structure? 3KTQ
-What is the resolution of this new structure? 2.3 Angstrom
-Which chain is the template in the new structure? Chain C
-What is the distance between the oxygen atom of the sidechain of residue 671 in Chain A and the oxygen atom of the base of the incoming nucleotide in 3ktq?

Page 6:Page 6:

-In 2KTQ, what is the distance between the oxygen atom of the sidechain of residue 671 in Chain A and the oxygen atom of the base of the incoming nucleotide? 9.78 Angstrom
-Assuming the end of the primer and the incoming nucleotide sit in essentially that same place in the active site of both 2KTQ and 3KTQ, has 671 undergone a substantial shift between the two structures?
-In 3KTQ, look at the nucleotide of the template that is 5' of the one base-paired to the last nucleotide of the primer. Could this nucleotide of the template potentially base-pair to the incoming nucleotide?
-Are they base-paired in 3KTQ?

spinqualitylabelsall models 3ktq Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

spinqualitylabelsall models 2ktq Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image