Macromolecular Cryo Crystallography Synchrotrons Protocols and Techniques 2007

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

Overview l Biological Macromolecules § Proteins, Nucleic Acids, Lipids, Sugars, & Complexes l Single Crystal X-Ray Diffraction § Not powder or fiber or solution scattering l Synchrotron Data Collection § Techniques l Cryo. Crystallography § Protocols & Advantages l Examples of Data Thayumanasamy Somasundaram March 12, 2007 2

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

Single Crystal X-Ray Diffraction l l Provides a Complete Structural Information Needs a Crystal (A Challenge) – Still takes 6 -9 months to get a crystallization condition l Unit Cell Dimensions: § 10 s Å (Proteins) | Small Molecules (1 s Å) § 100 s Å (Virus & Complexes) l l Crystal Dimensions: 0. 05 -1. 0 mm Use 1 -2Å X-Ray Radiation (Cu: 1. 54Å; 8 ke. V) Bond Lengths: ~1. 0 – 2. 0Å Structural Repository: www. rcsb. org/pdb § 34, 700 (X-Ray), 6000 (NMR), 225 (EM+) Thayumanasamy Somasundaram March 12, 2007 4

X-Ray Source 1 l 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) Thayumanasamy Somasundaram Rigku RU-H 2 R March 12, 2007 5

X-Ray Source 2 l 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 Thayumanasamy Somasundaram SER-CAT APS Aerial View March 12, 2007 6

Brilliance* of X-Ray Sources 1 *Photons/s/mrad/mm 2/0. 1% bandpass 2000 1990 1980 1960 1900 1: JR Helliwell, ITC: F, 155 -166 (2001) Thayumanasamy Somasundaram March 12, 2007 7

Why Cryo. Crystallography? § § l 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) Thayumanasamy Somasundaram March 12, 2007 8

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

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

Cryo. Crystallography § l Advantages For Macromolecular Crystals § § Reduces Free Radical Diffusion Reduces Stress on Crystals Reduces Thermal Motion Reduces Extra Scattering Crystal in Cryo. Loop Thayumanasamy Somasundaram March 12, 2007 11

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

Cryo. Crystallography § l Problems Crystal Damage § Crystal Cracks § Chemical Reaction § Disorder (Mosaicity) l Other § Snow Ice § Embedded Ice § l l l Remedies Annealing Screen Solvents Controlled Humidity Protein ↓ Water ↑ Thorne et al ACD 58 459 (2002) Thayumanasamy Somasundaram March 12, 2007 13

Cryo. Crystallography - 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) • 12 h (Home) • 15 h (No LN 2) Arginine Kinase 100°K | 30 sec • 0 h (NSLS – BM-12 C) • 1. 5 h (NSLS – BM-12 C) G Zhou T Somasundaram E Blanc G P Sarathy WR Ellington and MS Chapman PNAS 95 8449 (1998) Thayumanasamy Somasundaram March 12, 2007 14

Cryo. Crystallography - Example § • Example: Virus AAV 2 293°K | Ambient § 24 h (Home | Capillary) • AAV 2 277°K | 4°C • • • 30 s (CHESS | F 1 | Capillary) Survived 3 exposures AAV 2 100°K | Cryo • • 70 s (CHESS | F 1 | Cryo. Loop) Survived >180 exposures Q, Xie W Bu S Bhatia J Hare T Somasundaram A Azzi, and MS Chapman PNAS 99 10405 (2002) Thayumanasamy Somasundaram March 12, 2007 15

Cryo. Crystallography - 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) Thayumanasamy Somasundaram March 12, 2007 16

Cryo. Crystallography - 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) Thayumanasamy Somasundaram March 12, 2007 17

Cryo. Tools Cryo. Cap Cryo. Loop Cryo. Tong Cryo. Cane Thayumanasamy Somasundaram Hampton Research Cryo. Vial Cryo. Puck Cryo. Shipper Cryo. Puck March 12, 2007 18

SER-CAT Beamline @ APS Cryo Sample CCD System Thayumanasamy Somasundaram March 12, 2007 19

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

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