MOLEC STRU DETER XRAY
MOLEC STRU DETER XRAY BCH 6744
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This 13 page Class Notes was uploaded by Kavon Huel on Friday September 18, 2015. The Class Notes belongs to BCH 6744 at University of Florida taught by Robert McKenna in Fall. Since its upload, it has received 26 views. For similar materials see /class/206958/bch-6744-university-of-florida in Biochemistry and Molecular Biology at University of Florida.
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Date Created: 09/18/15
L4 Crystal preparation So you have crystals how do you get them in the Xray beam Crystals are fragile Mechanical movement Lecture 4 l Before you do anything consider 1 Which crystal do I use 2 What stabilization buffer should I use 3 What method of sample preparation should I use roomcryo temperature data collection When Viewingmanipulating a crystal try to keep it in the conditions that it grew from Lecture 4 Crystal selection Size Size of beam Shape Regular Surface Clear Separated Single Lecture 4 Microseeding Protein Crystals Prepare micro seeding solution Place small crystals in stabilizing solution Crush crystals use needle or microtools n Pipette and Spin 30 sec to remove macroscopic crystals Either Serially dilute solution in stabilizing solution dilutions of 1 10 100 and 1000 and add to preequilibrated drops 1413 the drop s volume of one of the serially diluted microcrystal solutions Or Pass tip of cat whisker or similar small ber through microcrystal solution and then streak tip of whisker successively through 3 or 4 pre equilibrated drops Note Successive streaking with a ber is effectively a serial dilution Preequilibrated drops contain protein and precipitant that is at a concentration just below what is required for precipitation or Lecture 4 4 crystallization of the protein Crystal manipulation Crystals are fragile 1 Don t touch them directly unless you have to 2 If you have to touch them be very gentle acupuncture needles cat hairs current ows 3 Don t let them dry out stabilization buffer reservoir solution Lecture 4 Stabilization buffer Why do you need it 1 Might want to soak crystals eg a substrate inhibitor 2 Don t have much droplet solution batch method 3 Evaporation temperature drying out of drop What should you use Precipitant solution should be a higher concentration of precipitant than crystal Stop crystals dissolving What about pH soaking experiments Remember concentration should be higher Lecture 4 Methods of crystal preparation Three choices two for protein work 1 Coat crystals cover with a protective layer against evaporation epoxy resin oil 2 Seal crystals in a quartz capillary in equilibrium with precipitant solution WET MOUNT 3 Cryo freeze crystals cool to 100K Lecture 4 To wet mount crystals in capillary tubes you need Quartz capillaries size 0220 mm Diamond cutter Syringe Paper wicks or small glass capillm Marker pen Beeswax Lecture 4 Wet mount method Advantages Room temp data collection Disadvantages Takes a steady hand Radiation damage uumhmm my 0 mum mm mhu mm liqum g MW a mmAs mm Ml Hy pm 1d Lecture 4 To cryo mount crystals you need Loops size 01 10mm Pin length 10 20mm Copper cap Magnetic base Lecture 4 Cryo mount method Pick up crystal in a nylon loop Advantages asy Prevent radiation damage m m nl Disadvantages Need cryo protectant Lecture 4 1 1 Commonly used cryo protectants Cryo protectant Concentration in precipitant Glycerol 1030 VV Vacuum pump oils 100 V Ethylene glycol 2040 VV polyethylene glycol 400 1030 VV Lecture 4 12 Now you are ready to put the crystal on the Xray machine Interface is a Goniometer head XYZ translations goniometer Spindle axis 2 Lecture 4 Beam stop