Chem120A+Notes+-+Cate+Protein+Crystallography

Chem120A+Notes+-+Cate+Protein+Crystallography - Chem 120A...

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Unformatted text preview: Chem 120A X-ray Crystallography 11/21/08 Jamie Cate X-ray crystallography - Structure to mechanism First Example: Sickle Cell Anemia and Hemoglobin B. J. Strasser (1999) Science 286 , 1488-90. Linus Pauling and Vernon Ingram found that a change in hemoglobin was the cause of Sickle Cell Anemia (E6V). Getting faster - Your Favorite Protein hours-weeks Revolutions in X-ray crystallography Not size limited - ribosome, viruses (> 2.5 MDa) Atomic resolution detail Hemoglobin: 30y! The PDB surpasses >43,000 structures in 2007 total structures ~84% x-ray crystallography ~15% NMR ~1% EM and other Electron density Model building X-ray crystallography can image protein structure Protein Crystal (Source) Structure Data 0. Purify protein 4. Model building minutes/months 5. Model refinement minutes - months 3. Calculate e- density minutes/hours 1. Grow crystals Days - Time table for crystal structure determination 2. Collect data Seconds (hours-months) Principal bottleneck Crystal growth - general requirements Need: Pure macromolecule (>98%) Chemically, conformationally homogeneous sample Add: Precipitating agents (mild organics such as PEG or salts) Buffers, inorganic or organic salts Cofactors, ligands, chemical additives Perturb : Hydration state, temperature, solubility response Get: Random aggregation Amorphous precipitate (common) Ordered phase transition Crystals (hard, rare) Precipitant Protein Free interface diffusion Crystallization methods Precipitant Protein Wait (Microbatch) Dehydrate - wait (Vapor diffusion) Direct mixing Macromolecules are rare/expensive, but crystallography typically requires milligram quantities Screening is laborious - interrogate chemical space by random/systematic guessing (hundreds-thousands of conditions) Process is entirely empirical But at the end of the day Problems Crystals are: Ordered arrays of 10 12-10 14 molecules ~25-80% water - similar to cells Native macromolecule structure/activity retained in crystalline state Crystals 101 0. Purify protein 4. Model building minutes/months 5. Model refinement minutes - months 3. Calculate e- density minutes/hours 1. Grow crystals Days - Time table for crystal structure determination 2. Collect data Seconds (hours-months) Synchrotron CCD Rotating anode Image plate Film Data collection - experimental setup Beam Optics (mirrors) N2 stream (100K) Source Detector Crystal (cryo-preserved!) Diffracted X-rays Why use X-rays?...
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This note was uploaded on 09/28/2009 for the course CHEM 120A taught by Professor Whaley during the Spring '07 term at University of California, Berkeley.

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Chem120A+Notes+-+Cate+Protein+Crystallography - Chem 120A...

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