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