Post on 14-Jan-2016
Macromolecular CryoCrystallography Macromolecular CryoCrystallography @ Synchrotrons@ Synchrotrons
Protocols and Techniques
Thayumana “Soma”sundaram
Institute of Molecular Biophysics
Florida State University
Tallahassee, FL 32306-4380
soma@sb.fsu.edu | www.sb.fsu.edu/~soma
2007 FLAVS & FMS
UCF, Orlando, FL
March 12, 2007
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Overview 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
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Biological MacromoleculesBiological 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
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Single Crystal X-Ray DiffractionSingle 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/pdb
34,700 (X-Ray), 6000 (NMR), 225 (EM+)
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X-Ray Source 1X-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
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X-Ray Source 2X-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 mm2) Access Travel/Planning APS Aerial View
SER-CAT
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Brilliance* of X-Ray SourcesBrilliance* of X-Ray Sources11
1.0E+07
1.0E+09
1.0E+11
1.0E+13
1.0E+15
1.0E+17
1.0E+19
SealedTube
RotAnode
BendMag
IDWiggler
ID Undul
Brilliance
*Photons/s/mrad/mm2/0.1% bandpass
1: JR Helliwell, ITC:F, 155-166 (2001)
1900
1960
1980
1990
2000
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Why CryoCrystallography?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 CapillaryE Garman, COSB 13, 545 (2003)
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Buffer in CryoLoop
CryoCrystallographyCryoCrystallography
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)
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CryoCrystallographyCryoCrystallography
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
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CryoCrystallographyCryoCrystallography
Advantages
For Macromolecular Crystals Reduces Free Radical Diffusion Reduces Stress on Crystals Reduces Thermal Motion Reduces Extra Scattering
Crystal in CryoLoop
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CryoCrystallographyCryoCrystallography
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 (Tg: 140°K) Prevent Water-Water
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CryoCrystallographyCryoCrystallography
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)
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CryoCrystallography - ExampleCryoCrystallography - 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 LN2)• 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)
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CryoCrystallography - ExampleCryoCrystallography - 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)
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CryoCrystallography - ExampleCryoCrystallography - 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)
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CryoCrystallography - ProblemsCryoCrystallography - 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)
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CryoToolsCryoTools Hampton Research
CryoCap CryoLoop CryoVial
CryoTong CryoCane
CryoPuck
CryoPuckCryoShipper
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SER-CAT Beamline @ APSSER-CAT Beamline @ APS
CCD System
Cryo Sample
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AcknowledgementsAcknowledgements
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
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Contact InformationContact Information
Thayumanasamy “Soma”sundaram414 Institute of Molecular Biophysics
Florida State UniversityTallahassee, FL 32306-4380
Phone: 850-644-6448 | Fax: 850-644-7244E-mail: soma@sb.fsu.edu
Web: www.sb.fsu.edu/~somaWeb: www.sb.fsu.edu/~xray