Bi1_2009_Lecture3_full

Bi1_2009_Lecture3_full - Student/faculty conference Monday...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: Student/faculty conference Monday April 6 http://arc.caltech.edu/sfc2009.php 8:00 - 8:25 - Opening 8:30 - 9:25 - Core Curriculum Task Force 9:30 - 10:25 - Undergraduate Research 10:30 - 11:55 - Student Experience 12:00 - 12:55 - Lunch 1:00 - 1:55 - Honor Code 2:00 - 2:55 - Humanities and Social Sciences 3:15 - 4:25 - Options We will have class as usual on Apr 6, but will post a podcast of the lecture on the course website so you can attend the SFC. Richard Feynman “An Invitation to Enter a New Field of Physics” 12/29/59 talk at the annual meeting of the American Physical Society It is very easy to answer many of these fundamental biological questions; you just look at the thing! You will see the order of bases in the chain; you will see the structure of the microsome. Unfortunately, the present microscope sees at a scale which is just a bit too crude. Make the microscope one hundred times more powerful, and many problems of biology would be made very much easier. I exaggerate, of course, but the biologists would surely be very thankful to you---and they would prefer that to the criticism that they should use more mathematics. The electron microscope is not quite good enough, with the greatest care and effort, it can only resolve about 10 Ångstroms. I would like to try and impress upon you while I am talking about all of these things on a small scale, the importance of improving the electron microscope by a hundred times. It is not impossible; it is not against the laws of diffraction of the electron. The wave length of the electron in such a microscope is only 1/20 of an Ångstrom. So it should be possible to see the individual atoms. Clicker question Viruses can be seen with: 1) 2) 3) 4) 5) 6) The naked eye A light microscope A fluorescent microscope An electron microscope A telescope An oscilloscope Size comparison • • • • Typical eucaryotic cell: 10s of microns ( m) Typical bacteria: one or a few microns Protozoa (e.g., Cryptosporidium or Giardia): 3-6 m Viruses: less than 1 m ( 100 nm) • Water filters: 0.1 to 1 m pore size. Will NOT filter out most viruses. From macroscopic to microscopic Light or fluorescence microscopy (can be done on live cells) Electron microscopy (cannot be done on live cells) or X-ray crystallography 0.1 nm = 1 Ångstrom Transmission Electron Microscopy A 2-D projection Can magnify up to 1x106 times. This is also a 2-D projection image Computed tomography (CT) -a medical imaging method Tomography: 3D reconstruction of an object from a series of projections Set of 2D projections recorded while tilting the object. Calculate the backprojection for each projection. The sum of all the backprojections represents the density of the original object. Baumeister et al., 1999, Electron tomography of molecules and cells. Trends Cell Biol. 9: 81-85. Electron tomogram Model of tomogram ~0.5 m 2D images of mitochondria from textbooks suggest an incorrect 3D model Tomogram of mitochondria http://www.sci.sdsu.edu/TFrey/MitoMovie.htm Segmented model derived from tomogram of mitochondria http://www.sci.sdsu.edu/TFrey/MitoMovie.htm 3D reconstruction of Golgi 6-8 nm resolution Colors indicate different Golgi cisternae ER: blue-gray Ribosomes: purple spheres ER-Golgi intermediate compartment: yellow Golgi cisternae: C1: green C2: purple C3: rose C4: olive C5: pink C6: bronze C7: red Ladinsky et al. (1999) J. Cell Biol. 144: 1135-1149 High resolution 3D electron tomography of pancreatic beta cell line Boulder Laboratory for 3D Structure of Cells, University of Colorado ER: yellow membrane-bound ribosomes: blue free ribosomes: orange microtubules: bright green mitochondria: dark green dense core vesicles: blue clathrin-negative vesicles: white clathrin-positive compartments and vesicles: red clathrin-negative compartments and vesicles: purple 500 nm Marsh et al. (2001) PNAS 98: 2399-2406 High resolution 3D electron tomography of pancreatic beta cell line Boulder Laboratory for 3D Structure of Cells, University of Colorado ER: yellow free ribosomes: orange microtubules: bright green mitochondria: dark green dense core vesicles: blue clathrin-negative vesicles: white clathrin-positive compartments and vesicles: red clathrin-negative compartments and vesicles: purple 500 nm ~0.5 m Marsh et al. (2001) PNAS 98: 2399-2406 From macroscopic to microscopic Light or fluorescence microscopy (can be done on live cells) Electron microscopy (cannot be done on live cells) or X-ray crystallography 0.1 nm = 1 Ångstrom Clicker question A fluorescent molecule that absorbs a photon of light at one wavelength 1) always emits a photon at a shorter wavelength 2) always emits a photon at a longer wavelength 3) can emit a photon at a shorter or a longer wavelength depending on the type of fluorophore Fluorophores • component of a molecule that causes it to be fluorescent • Fluorescein isothiocynate (FITC) Absorb: 495 nm (blue) Emit: 521 nm (yellow green) FITC – -N=C=S is reactive group allowing coupling to proteins Rhodamine • Rhodamine Absorb: 515 nm (yellow green) Emit: 546 nm (red) Alexa Fluor dyes -- more photostable, more colors http://www.invitrogen.com/site/us/en/home/References/ Molecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlights/The-Alexa-Fluor-Dye-Series.html Confocal microscopy Insect embryo Conventional fluorescent microscope Confocal fluorescent microscope Conventional fluorescence microscopy results in a blurry image because there are fluorescent structures above and below the plane of focus. A laser scanning confocal microscope uses a pinhole aperature in the detector that allows only fluorescence emitted from the plane of focus to be included in the image. A laser beam is scanned across the specimen to give a sharp 2-D image in each plane of focus. A series of xy planes can be taken at different depths to yield a 3-D image. Confocal microscopy can be used to derive 3D reconstructions of cells Rice et al., 2009, J. Mol. Biol. 386; 717-732 5m http://www.conncoll.edu/ccacad/zimmer/GFP-ww/ Live cell imaging An endosomal protein was fused to GFP Intracellular vesicles can move 1-2 m/sec Rice et al., 2009, J. Mol. Biol. 386; 717-732 Live imaging of HIV spread http://www.telegraph.co.uk/scienceandtechnology/science/sciencenews/5058131/ Scientists-film-HIV-spreading-for-first-time.html http://www.sciencemag.org/cgi/content/full/323/5922/1743 From macroscopic to microscopic Light or fluorescence microscopy (can be done on live cells) Electron microscopy (cannot be done on live cells) or X-ray crystallography 0.1 nm = 1 Ångstrom An experimental method to determine macromolecular structures: X-ray Crystallography Crystal Growth X-ray Data Electron Density Protein Model The final output of X-ray crystallography is an electron density map -- a map showing the positions of the atoms in a macromolecular structure How do we get protein structures? Each shows one plane of a 3-D electron density map X-ray crystallography • Why X-rays? Right wavelength (Å) to resolve atoms • Why crystals? Immobilize protein, enhance weak signal from scattering • What is a protein crystal? Large solvent channels (20-80% solvent) Same density as cytoplasm Enzymes active in crystals A crystal is a 3D repeating lattice Overview of imaging No lens to refocus X-rays, so must understand reciprocal space and diffraction Diffraction: Scattering followed by interference Bragg’s law Consider simultaneous reflection of a large number of X-rays. See diffraction maximum in direction phase. only if diffracted waves are in Path difference (2dsin ) must represent an integral number of wavelengths to get constructive interference. Learned two things from Bragg’s Law • sin = n /2 x 1/d Low angle: large interplanar spacing High angle: small interplanar spacing – Since sin is proportional to 1/d, structures with large interplanar spacings (d) will have diffraction patterns with small spacings and vice-versa. • Repeating unit in real space (crystal) --> diffraction maxima and minima Resolution An inverse FT including all of the high angle information gives back the original image. An inverse FT including only the low angle information gives back a low resolution view of Mickey. From Harburn, Taylor, Wellbery, An Atlas of Optical Transforms The electron density equation and the phase problem (xyz) is what we want: the electron density at every point x,y,z 1 (xyz ) = V | F( hkl ) | exp[( 2 i(hk + ky + lz ) + i h k l hkl Can measure amplitude of scattered wave: |F(hkl)|=I1/2 Can’t measure the phase • There are experimental methods for determining the phase for each reflection hkl. Clicker question: What would you get if you performed a Fourier transform using the amplitudes from an image of a duck combined with the phase information from an image of a cat? 1) A funny looking duck 2) A funny looking cat 3) A ducat 4) A caduck 5) A Bi 1 student 6) A Bi 1 TA ? Clicker question: What would you get if you performed a Fourier transform using the amplitudes from an image of a duck combined with the phase information from an image of a cat? 1) A funny looking duck 2) A funny looking cat 3) A ducat 4) A caduck 5) A Bi 1 student 6) A Bi 1 TA For more info see http:// www.ysbl.york.ac.uk/~cowtan/ fourier/fourier.html You can use the electron density equation to calculate an electron density map once you have amplitudes and phases for every hkl Coordinates are deposited in the Protein Data Base (PDB) X-ray detection • Film (relic of the past) • Diffractometers (almost relic of past, but used for small molecules) • Multiwire detectors (almost relic of past) • Phosphorimager detectors (R-AXIS, MAR) • CCD detectors Synchrotron X-ray sources • High-intensity X-ray emitted by charged particles accelerated in a curved path • X-ray wavelength in range of 0.5 - 2 Å (from E=h =hc/ ) • Is tunable!! • Caltech’s “Molecular Observatory” includes a high intensity synchrotron radiation beam line at the Stanford Synchrotron Radiation Laboratory (SSRL). http://www.br.caltech.edu/mobservatory e- Radiation emitted by accelerating charged particle tangent to path of circle ...
View Full Document

This note was uploaded on 09/25/2010 for the course BIO 1 taught by Professor Bakorman during the Spring '09 term at Caltech.

Ask a homework question - tutors are online