Announcements Lec 10 Quiz today--ATPase Homework #5 Assigned includes Reading, Chpt 3 ECB Most students like in-class assignments (good!)

Quiz #4 (1) F1 ATPase has three ______________ subunits and three _____________ subunits arranged in a ringlike structure. In the center there is a _______________ shaft. (2) There are three ATP binding sites in the F1 motor, what are the nucleotide states of each site? (3) In Noji et al. Nature 1997, it was shown that F1 has 120 degree steps. However, in the review article it is mentioned that these steps are further broken into one _________ and one _________ degree sub-steps. (4) The whole ATP synthase does / does not rotate. beta alpha ATP ADP+Pi empty 3090 gamma 2/13/08

Resolution: Can’t get better than /2N.A. Accuracy vs. Resolution A single spot will be smeared out, no matter how small the spot is, because of the wavelength of light to ~ /2N.A. (shape = Point Spread Function, PSF). Or can you? We can in-fact, achieve sub- diffraction limited resolution by several ways. Accuracy: Unlimited: limited by # sqrt(photons)

Electron Microscope Images (Usually, but not always, requires staining with metals to get sufficient contrast) [For Transmission E.M. need to make very thin sections.]

Scanning E.M. (Electrons scattered from surface) Specimen is coated with heavy metals. Raster beam across specimen. Good depth of focus. Amount of electrons scattered depends on angle of surface – gives highlights, shadows, 3-d surfaces. D.I.C. (Phase)

sin  = n /d d ~ 2 Å ;  ~ 1-2 Å

Measuring atomic distances X-ray crystallography Another technique: NMR Myoglobin crystal Stryer, pg Å  -helix Atomic Scattering electron density backbone

Optical Scattering Analog

Crystal of protein must tell you something about structure in water. Structure of pltocyanin (protein in photosynthesis) Blue = x-ray crystallography; red = NMR X-ray Crystallography: must crystallize to get reasonable diffraction. (With partially oriented samples, like original DNA, can get some information– see next slide) NMR– in solution. NMR is better, except very limited what range it can do usually <25k Often-times (though not always), get good agreement!

Fluorescence Imaging with One Nanometer Accuracy (1.5 nm, msec)

How are biomolecules moved around within a cell? “Kinesin can carry a packet of neurotransmitter from your spine to the tip of your finger in about two days — a journey that would take a thousand years if left to simple diffusion.” (Molloy and Schmitz, Nature, 2005) Single Molecule Studies of Molecular Motors In vitro & in vivo

Biomolecular Motors: Intra- & Extra- Cellular Motion Actin,  tubules ATP  mechanical work Nature Reviews Microtubule actin Microtubule  polymer (+) direction (- direction) Kinesin Myosin Dynein  Motor ATP-binding heads Cargo binding Actin,  tubules nm scale Move along tracks w ATP intracellular directional movement cell shape & extracellular movement

Kinesin: 0, 16.6 nm or 8.3, 8.3 nm Myosin V: 0, 74 nm or 37, 37 nm Kinesin Myosin V Myosin V (Kinesin): Hand-over-hand or Inchworm?

Class evaluation 1.What was the most interesting thing you learned in class today? 2. What are you confused about? 3. Related to today’s subject, what would you like to know more about? 4. Any helpful comments. Answer, and turn in at the end of class.