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Macromolecular Synchrotrons Protocols and Techniques Thayumana “Soma”sundaram Institute of Molecular Biophysics Florida State University.

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Presentation on theme: "Macromolecular Synchrotrons Protocols and Techniques Thayumana “Soma”sundaram Institute of Molecular Biophysics Florida State University."— Presentation transcript:

1 Macromolecular CryoCrystallography @ Synchrotrons Protocols and Techniques Thayumana “Soma”sundaram Institute of Molecular Biophysics Florida State University Tallahassee, FL 32306-4380 soma@sb.fsu.edusoma@sb.fsu.edu | www.sb.fsu.edu/~somawww.sb.fsu.edu/~soma 2007 FLAVS & FMS UCF, Orlando, FL March 12, 2007

2 Thayumanasamy Somasundaram 2 Overview Biological Macromolecules  Proteins, Nucleic Acids, Lipids, Sugars, & Complexes Single Crystal X-Ray Diffraction  Not powder or fiber or solution scattering Synchrotron Data Collection  Techniques CryoCrystallography  Protocols & Advantages Examples of Data

3 March 12, 2007 Thayumanasamy Somasundaram 3 Biological Macromolecules Proteins  20 Naturally Occurring Amino Acids  Anywhere from 20 to ~1500 Amino Acids Nucleic Acids  4 Bases  DNA/RNA Lipids & Sugars Protein + NA Complexes Membrane Proteins

4 March 12, 2007 Thayumanasamy Somasundaram 4 Single Crystal X-Ray Diffraction Provides a Complete Structural Information Needs a Crystal (A Challenge) –Still takes 6-9 months to get a crystallization condition Unit Cell Dimensions:  10s Å (Proteins) | Small Molecules (1s Å)  100s Å (Virus & Complexes) Crystal Dimensions: 0.05-1.0mm Use 1-2Å X-Ray Radiation (Cu: 1.54Å; 8 keV) Bond Lengths: ~1.0 – 2.0Å Structural Repository: www.rcsb.org/pdbwww.rcsb.org/pdb  34,700 (X-Ray), 6000 (NMR), 225 (EM+)

5 March 12, 2007 Thayumanasamy Somasundaram 5 X-Ray Source 1 Home Source  Rotating Anode  Multi-layer Mirror  Wide Usage  Easy Access  Fixed Wavelength Cu (1.54 Å) Cr (2.29 Å) Mo (0.71 Å) Rigku RU-H2R

6 March 12, 2007 Thayumanasamy Somasundaram 6 X-Ray Source 2 Synchrotron  Broad Wavelength Selection  Bending Magnet & Insertion Devices  >1000 Times Intense  Low Divergence (mrad)  Small Beam Size (<0.1 x 0.1 mm 2 )  Access  Travel/Planning APS Aerial View SER-CAT

7 March 12, 2007 Thayumanasamy Somasundaram 7 Brilliance* of X-Ray Sources 1 *Photons/s/mrad/mm 2 /0.1% bandpass 1: JR Helliwell, ITC:F, 155-166 (2001) 1900 1960 1980 1990 2000

8 March 12, 2007 Thayumanasamy Somasundaram 8 Why CryoCrystallography?  A Specialized Field is Now Routine  5% in 1995 and >90% in 2006 But Macromolecular Crystals are  Radiation Sensitive  Contain 40-70% Solvent  Contain Flexible Regions  Low Scattering Cross-section Crystal in Capillary E Garman, COSB 13, 545 (2003)

9 March 12, 2007 Thayumanasamy Somasundaram 9 Buffer in CryoLoop CryoCrystallography  Breakthrough in 1990 T. –Y. Teng (Cornell)  Wire Loop  Viscous Hydrophilic Solvent  Free Standing Crystal  Flash Cooling Crystal in CryoLoop T.-Y. Teng, JAC 23, 387 (1990)

10 March 12, 2007 Thayumanasamy Somasundaram 10 CryoCrystallography  Procedures Flash Cool in Stream (100°K)  For Immediate Collection  Ease of Handling Flash Cool in Liquid (77°K)  For Storage  For Shipping & Transport  Needs Practice Flash Cool in Stream Flash Cool in Liquid

11 March 12, 2007 Thayumanasamy Somasundaram 11 CryoCrystallography  Advantages For Macromolecular Crystals  Reduces Free Radical Diffusion  Reduces Stress on Crystals  Reduces Thermal Motion  Reduces Extra Scattering Crystal in CryoLoop

12 March 12, 2007 Thayumanasamy Somasundaram 12 CryoCrystallography  Cryoprotectants Alcohols  Glycerol (20%)  Poly Ethylene Glycols (20-30%)  2-Methyl-2,4-pentanediol (30%) Salts  Sodium Formate (4M)  Lithium Sulfate (2M)  Mechanism Grow In or Add Solute Glassy Ice (Non- Crystalline) 100°K (T g : 140°K) Prevent Water-Water

13 March 12, 2007 Thayumanasamy Somasundaram 13 CryoCrystallography  Problems Crystal Damage  Crystal Cracks  Chemical Reaction  Disorder (Mosaicity) Other  Snow Ice  Embedded Ice  Remedies Annealing Screen Solvents Controlled Humidity Protein ↓ Water ↑ Thorne et al ACD58 459 (2002)

14 March 12, 2007 Thayumanasamy Somasundaram 14 CryoCrystallography - Example  Example: Enzyme Arginine Kinase 293°K | 12 min  0 h (Home)  12 h (Home); More needed Arginine Kinase 100°K | 15 min 0 h (Home) 12h (Home) 15h (No LN 2 ) Arginine Kinase 100°K | 30 sec 0 h (NSLS – BM-12C) 1.5 h (NSLS – BM-12C) G Zhou T Somasundaram E Blanc G P Sarathy WR Ellington and MS Chapman PNAS 95 8449 (1998)

15 March 12, 2007 Thayumanasamy Somasundaram 15 CryoCrystallography - Example  Example: Virus AAV2 293°K | Ambient  24 h (Home | Capillary) AAV2 277°K | 4°C 30 s (CHESS | F1 | Capillary) Survived 3 exposures AAV2 100°K | Cryo 70 s (CHESS | F1 | CryoLoop) Survived >180 exposures Q, Xie W Bu S Bhatia J Hare T Somasundaram A Azzi, and MS Chapman PNAS 99 10405 (2002)

16 March 12, 2007 Thayumanasamy Somasundaram 16 CryoCrystallography - Example  Example: Protein Fibroblast Growth Factor 100°K |  40 min (Home) Fibroblast Growth Factor 100°K | 20 s (APS 14-BM-C) Offset Detector 1.1 -1.2Å Diffraction MJ Bernett T Somasundaram and M Blaber Proteins 57 626 (2004)

17 March 12, 2007 Thayumanasamy Somasundaram 17 CryoCrystallography - Problems  Problems Embedded Ice  Hard to Remove  Anneal & Flash Cool  Problem w/ Lattice Snow Ice Nuisance Doesn’t Affect Lattice Problem w/ Processing M Yousef SA Clark PK Pruett T Somasundaram WR Ellingtion and MS Chapman Prot Sci 12 103 (2003)

18 March 12, 2007 Thayumanasamy Somasundaram 18 CryoTools Hampton Research CryoCapCryoLoopCryoVial CryoTongCryoCane CryoPuck CryoShipper

19 March 12, 2007 Thayumanasamy Somasundaram 19 SER-CAT Beamline @ APS CCDSystem CryoSample

20 March 12, 2007 Thayumanasamy Somasundaram 20 Automounter | BCSB bcsb.lbl.gov/autoMounter.htm

21 March 12, 2007 Thayumanasamy Somasundaram 21 Remote Access | SSRL

22 March 12, 2007 Thayumanasamy Somasundaram 22 Acknowledgements Michael Chapman, OHSU, Portland, OR  Genfa Zhou, Qing Xie, & Jeff Bush Michael Blaber, CoM, FSU  Matthew Bernett, Jihun Lee, & Sumit Khurana Hong Li, FSU  Song Xue and Sri Vidya SER-CAT, APS, Argonne, IL Inst Molecular Biophysics Florida State University

23 March 12, 2007 Thayumanasamy Somasundaram 23 Contact Information Thayumanasamy “Soma”sundaram 414 Institute of Molecular Biophysics Florida State University Tallahassee, FL 32306-4380 Phone: 850-644-6448 | Fax: 850-644-7244 E-mail: soma@sb.fsu.edusoma@sb.fsu.edu Web: www.sb.fsu.edu/~somawww.sb.fsu.edu/~soma Web: www.sb.fsu.edu/~xraywww.sb.fsu.edu/~xray

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