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Assignment 2 Due February 20, 2018 Details of assignment begin on p. 6PDB shows us proteins like these→CollagenLamininProteoglycanElectron microscope view of collagen fiber with those two LDL particles attached to itin a heart valve —Frank et al 1994Source: Biosites, David S. Goodsell, ScrippsThe Basement Membrane is a layer of fibers found under the epidermis, also around the capillaries, in kidneys, etc. Goodsell’s painting of it is based microscopic views and on PDB structures like the one belowIntroduction to THE PROTEIN DATA BANK& What X-ray Crystallography Can Show Us About the Structures of Important ProteinsBasement MembraneCLength of dotted line: 8.33 nmPDB ID: 1bkvCollagen IIIPhysics 3750. Dr. Villanueva 1 Winter 2018at crystalX−raysStructures deduced (Fourier analysis, geometry …)HUGE amount of math is done are projectedImages are made crystal layers in different waysX−rays interact withSo urce :R andy Rea de, U nive rsity of Cam brid geWhat is it about X−rays that makes them so useful in crystallography? It is their very short wavelengths. X−rays are between 10 nm and 0.01 nm in length,that is, between 100 and 0.1 Angstroms.The phenomenon of X−rays was discovered in 1895 By 1914 scientists wereusing X−rays to investigatevery simple: halite, or rock salt. atomic structure to be deduced crystals of minerals. The firstHalite crystal (Rock salt)Atoms in Halite crystalDorothy Crowfoot Hodgkins produced the structure of cholesterol. In 1969Thus some X−rays are on the same scale as atoms, 1 or 2 Angstroms,with atoms and molecules, and the interactions can be analyzed as well as bonds between atoms . Being of similar size, X−rays can interactAnalysis of more complex organic compounds came later. In 1937she unravelled the much more complex structure of the insulin molecule.X−ray CrystallographyIntroduction to Assignment 2Physics 3750. Dr. Villanueva 2 Winter 2018An early example ofX-ray Diffraction AnalysisRosalyn Franklin’s “Photo 51”1 nm10 “rungs”on theladder~3.4 nmThis photo ↑ is based on an X-ray diffraction study of DNA.It helped Francis Crick and James Watson show in 1953 that DNA is shaped like the double helix above.For a simple explanation of how the deduction was made, see the PBS slide show, “Anatomy of Photo 51:” X-ray wavelength often used in X-ray crystallography: ~ 0.136 nm (< 1/7 of a nanometer: pretty small)Physics 3750. Dr. Villanueva 3 Winter 2018ANOTHER EXAMPLE OFX-RAY CRYSTALLOGRAPHY The enzyme ElastaseElastin ↓ under an electron microscopeNormal lung tissue Lung damaged by emphysemaElastin is a stretchable protein It is found in arteries, skin, muscles and lungsHow can we learn the structure of elastase? We make a crystal of the protein. We aim X-rays at it.The way the X-rays bounce off it (the diffraction pattern) tells us(after lots of math)that elastase looks rather like thisElastin is broken down by the enzyme elastaseIn the disease of emphysema (maybe because the body is trying to fight bacteria, overdoes it?). elastase damages tissues in the lungsKnowing the structure of the elastase protein may allow us to find therapies to cope with it.Human NeutrophilElastasePDB ID: 1B0FResolution: 3ǺSources: Technion, Israel; Jessica Bon, MD, Virginia Engineering School; Wiki, Bugg; PDB Cregge, JMedChemPhysics 3750. Dr. Villanueva 4 Winter 2018What does “resolution 3 Å” mean? It tells us the greatest fineness of detail that can be distinguished(If two points are just 3 Å apart, we can barely see that they are separate)An example related to X-ray crystallographyLoosely based on material by Bernard Rupp, Crystallography 101IX-ray Pulsar B1507, in 7 kilobytes I 53 kilobyte version of same Resolution in another contextDetail better resolved here ↓Physics 3750. Dr. Villanueva 5 Winter 2018To reach About Molecule of the Month at the Protein Data Bank, you can go right to this: you can search for “about molecule of the month pdb.”The top of the page you should get will look like this ↓ only bigger, and not as fuzzy. If you click on ▲ By Date that should take you to a page like this ↓Assignment 2I. Choose a “Molecule of the Month” [MOTM] from PDB Measure the molecule using the PDB tools. Show us the picture. (see PowerPoint “Measuring Protein Sizes in the PDB” for a quick guide) II. Tell us something about the molecule (details below)The MOTM collection now contains over 200 molecules. Find one that interests youPhysics 3750. Dr. Villanueva 6 Winter 2018Basic steps: I a. Go to About the Molecule of the Month PDB articles arranged by Date or by Category. Choose a protein. If it hasn’t been taken, register it on Blackboard as yours. b. Click on molecule’s name. A discussion of the molecule will come up Inside the discussion will be one or more molecule PDB ID entry codes. Click on one. c. This takes you to a page with a picture of the molecule. Note the molecule’s Molecular Weight and the resolution of X-ray analysis (if available). Express resolution in nanometers (Å). d. Click on 3D View Structure. At the bottom right is a box that says NGL (Web NG) Switch NGL to JSmol. Click. Perform two measurements of molecule. Note them in nanometers and E-notation, in meters e. Note if there are disulfide bonds (sulfur bonds, SS bonds) that help shape the protein. For details, see p,18 &19 in PowerPoint Measuring Protein Sizes. f. Save a PNG image of your molecule for posting (Right-click on page, then click on FILE, then Export PNG Image). Make sure the image shows the measurements you have made. II Describe the molecule in approximately 300 words, using at least two references (with URLs). Summary: On Blackboard : I Tell us a) your name and the name of your molecule and its PDB ID, (b) its molecular weight, c) the resolution of the X-rays, (d) some measurements of your molecule, e) if there are disulfide bonds in the molecule. (f) Post an image, showing measurementsII Provide a 300 word discussion of whatever you find interesting about the molecule, based on D.Goodsell’s essay (which you may have to read a few times) and another source.Detailed Guide to doing all this. Please, start a little early. The PDB site is sometimes down, and it can take a little time to get used to the measuring technique. The essay will take some time too.NOTE: I’ve also provided a PowerPoint Introduction to the site and to Measuring Protein SizesI a. First, go to “About Molecule of the Month PDB” with this link: Note that below, the collection contains around 200 molecules Choose one. Check Blackboard to see if it has not been taken. If it has, choose another. There are plenty.For example, I chose “Zinc Fingers,” Next, I go to Blackboard to see if anyone else has taken Zinc Fingers. No one has. So I register the molecule: “Zinc Fingers, Dr. V.”b. Click on ►the molecule’s name (not PDF). This takes you to the discussion page. Read it to gather enough information for a brief essay about the protein molecule. You’ll want to use two other sources as well. Then, somewhere in the discussion, part way down the page, you’ll find a reference to a picture of a protein, and a link to where in the Protein Data Bank it is analyzed. It says “PDB entry θabc ” ] Click the entry code, Click to connect with an example of a structurePhysics 3750. Dr. Villanueva 7 Winter 2018c. Now go to the structure of the molecule. For Zinc Fingers structure 1TF6, I got a page from which this is part (except that I had to shrink it):The Resolution of this structure, 3.1 Å, is under Experimental Data Snapshot. 3.1 Å = 0.31 nm Molecular Weight, or Total Structure Weight, is further down, on the left, under Macromolecule Content →d. Click on 3D View: Structure A picture will appear. Go to Select a different viewer (right, below picture), and select JSMol. Now another ribbon-shaped version of the molecule will appear. —————–► Move your mouse to any point on the molecule, res
t on it and double-click. Move the mouse somewhere else. Double-click. An instant later, a dashed line will appear, with distance in nanometers [nm], Ǻngstroms, Å, or picometers [pm] written nearby. 1.736 nm = 17.36 Å = 1736 pm Getting the measurements may take practice. (Thing may rotate instead measuring, or do nothing) If no numbers appear, you might try right-clicking. Then a set of options appears. Make sure the Show measurement box is clicked.• There’s extra help with measuring & manipulating the picture on pages 12 and 13. Also the PowerPoint Introduction →to Measuring Proteins gives step-by-step picturesDNA ►Zinc Fingers→←Enlarged version of part of ↑ the above image, but with measurements (& different background)Physics 3750. Dr. Villanueva 8 Winter 2018e. Note if there are any SS bonds, or sulfur-sulfur bonds in this molecule. You find these by clicking on the Sulfur Bonds box If any tiny gold lines appear in the molecule image, they are sulfur bonds between two non-adjacent cysteine molecules These bonds help to shape the molecule. Finally: a little background Structure 1TF6 was produced in 1998 from a protein found in a frog, Xenopus laevis. This protein consists of six “fingers” coordinated by zinc atoms (Zn→). (For more, see the Sample of Completed Assignment) No need to provide pictures of the source of your molecule. But Xenopus laevis is also called the African Clawed Frog → It’s aquatic, as you might have guessed. Image from AskNature.orgOrange arrows point to some of the zinc atoms, which help to structure this molecule. If you want to see more, go to 1TF6 and give it a spinAbout the essay part—please don’t let it intimidate you. You just have to gather enough about the molecule to be able to say something coherent about it. The good part is that David Goodsell doesn’t choose any old molecule to be The Molecule of the Month. These molecules are quite interesting. Sometimes it’s because of their historical importance. Sometimes it’s because of their biological importance, because scientists want to make drugs that imitate what these molecules do—or prevent what they do! So even though the molecule may look difficult at first, just read what Goodsell has to say, maybe a couple of times. Almost always, it is reasonably clear. Look up some of the words, if you don’t know them. Then read something by another author on the molecule. Then write something with two references (with URLs). Please don’t use Wikipedia alone, though. It’s a good first start, especially for quick definitions, but it’s not always peer-reviewed, and there have been problems with that. But you can read what it says, and consult the references.All in all, this essay assignment is a bit like the story I heard once about what to do if you are appointed ambassador to a country that you’ve never heard of before. The usual trick, the story goes, is to findseveral books about that country, and read them quickly. You probably wouldn’t remember everything that you read. But you would recall the main points. That, and a friendly smile, would get you throughthe dinner parties. Then you worked hard.(Finally, don’t worry; Assignment 3, the last one, will be shorter.)ZnSource of structure: Nolte, Conlin, Harrison, Brown PNAS 1998, “Roles of Zinc Fingers in DNA Recognition”[Xenopus? I expect that it means “foreign foot” in Greek, presumably because frogs’ feet don’t usually look like that.](□ SS Bonds).SS bondFree Cysteine▲ Sulfur ►Physics 3750. Dr. Villanueva 9 Winter 2018SAMPLE of a completed assignmentThe Protein Data Bank Assignment: Zinc Fingers Dr. VillanuevaI choose zinc fingers simply because they were the last molecule in the alphabetical list on the “Molecule of the Month” page. However, I am learning that they are quite interesting. From the Protein Data Bank1 we are told that proteins are large molecules composed of hundreds of amino acids. The reason for so many amino acids is that the forces used to hold protein structures together are rather weak. They include hydrogen bonds, charge-charge interactions (aka salt bridges2), and hydrophobic (water repelling) forces. So you need a lot of hydrogen bonds and salt bridges to hold the structure together. Zinc fingers take another approach. The protein uses a zinc ion to stabilize its structure. The zinc ion has a charge of +2 and can attract the negative ends of the amino acids histidine and cysteine3 far more effectively than a hydrogen bond or salt bridge. Instead of a chain of hundreds of amino acids a short chain of 20-30 amino acids can form a stable structure. Zinc fingers are able to recognize three letters of DNA. This means that several fingers can be linked together to recognize a sequence unique to a particular site on the DNA. This means that zinc fingers could be very useful in gene therapy. A chain of zinc fingers could be used to direct DNA-cutting enzymes to a precise spot on the DNA. This means a bad genetic sequence can be cut away and replaced by a new sequence4 All in all zinc fingers look like fascinating and potentially very useful little proteins!1.1 The Protein Data Bank, Molecule of the Month. 2 Wikipedia article on salt bridge. 3 Shows a zinc ion surrounded by two histidines and two cytsteines. 4 The New York Times. Dec 28, 2009Physics 3750. Dr. Villanueva 10 Winter 20181. a) The data requested from the Molecule of the Month site for Zinc Fingers PDB entry 1tf6: b) Molecular wt = 83174.56 Daltons = 83.17 kDa. c) Resolution = 3.1 Å = 310 pm = 3.1 E-10 md) Measurements: Size: Length of one zinc finger, ~ 19.94 Angstroms = 1.99 E-9 m (or ~2 nm) Width, 5.63 Å = 5.6 E-10 m (or ~ 0.6 nm) e) There are no disulfide bonds in this molecule (but there are some hydrogen bonds) From the PDB Abstract: PubMed Abstract: The crystal structure of the six NH2-terminal zinc fingers of Xenopus laevis transcription factor IIIA (TFIIIA) bound with 31 bp of the 5S rRNA gene promoter has been determined at 3.1 A resolution. Individual zinc fingers are positioned differently in the major groove and across the minor groove of DNA to span the entire length of the duplex. These results show how TFIIIA can recognize several separated DNA sequences by using fewer fingers than necessary for continuous winding in the major groove.You can see the six zinc fingers in blue. From the two measurements each chain is about 20Å (1994 pm ) long and about 6 Å (563 pm) wide. (Some lines and measurements were superimposed by me as they didn’t come out so well in the original)← 19.94 Å5.63 ÅPhysics 3750. Dr. Villanueva 11 Winter 2018More from d. EXAMPLE OF MEASUREMENTS OF Zinc Fingers 1TF6On my computer this Measurement List ↑appeared in a larger box. (It was just shorted to make it fit.)What if you can’t read the distance numbers? One solution is to use the cursor to rotate the molecule until the numbers are not obscured.The other solution is to right-click to pull up a column of options. Click on Measurements and then List of measurements A box will come up, providing the distances that you measured, in nm, Å, or pm← List of MeasurementsBox ↓Physics 3750. Dr. Villanueva 12 Winter 2018More on measurements, centering the molecule, etc.Trouble getting measure- ments to appear?? The system may work better if you right-click on the picture. Then the left column appears. Move the cursor to Measurements ► and the right column appears. Then click on “Double-Click begins…”The you double-click at one place on the image, then at another place. May have to do this for each measurement About centering, zooming in or out, etc:If you type in RCSB PDB Help, it offers ▼a chart that gives a little adviceMyeloperoxidase is an enzyme (as you may recall) that is secreted by neutrophils and macrophages. It is used to produce ← HOCl (bleach) to kill bacteriaPhysics 3750. Dr. Villanueva 13 W
inter 2018


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