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Crystallization of Macromolecules

David Friedmann1,  Troy Messick2,  Ronen Marmorstein1

1Wistar Institute, Philadelphia, Pennsylvania
2Vironika, Philadelphia, Pennsylvania


Publication Name: 
Current Protocols in Protein Science
Unit Number: 
Unit 17.4
DOI: 
10.1002/0471140864.ps1704s66
Online Posting Date: 
November, 2011
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Abstract

X-ray crystallography has evolved into a very powerful tool to determine the three-dimensional structure of macromolecules and macromolecular complexes. The major bottleneck in structure determination by X-ray crystallography is the preparation of suitable crystalline samples. This unit outlines steps for the crystallization of a macromolecule, starting with a purified, homogeneous sample. The first protocols describe preparation of the macromolecular sample (i.e., proteins, nucleic acids, and macromolecular complexes). The preparation and assessment of crystallization trials is then described, along with a protocol for confirming whether the crystals obtained are composed of macromolecule as opposed to a crystallization reagent. Next, the optimization of crystallization conditions is presented. Finally, protocols that facilitate the growth of larger crystals through seeding are described. Curr. Protoc. Protein Sci. 66:17.4.1-17.4.26. © 2011 by John Wiley & Sons, Inc.

Keywords: macromolecular crystallography; crystal screening; crystal optimization

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Protein for Crystallization
  • Alternate Protocol 1: Preparation of Nucleic Acid Fragments for Crystallization
  • Alternate Protocol 2: Preparation of Macromolecular Complexes for Crystallization
  • Basic Protocol 2: Preparation and Analysis of Crystallization Trials
  • Alternate Protocol 3: Crystallization Screening Using High-Throughput Robotics
  • Support Protocol 1: Establishing Whether Crystals are Macromolecule or Crystallization Reagent Using a Cryoloop
  • Basic Protocol 3: Optimization of Crystallization Parameters
  • Alternate Protocol 4: Optimization of Crystal Equilibration Conditions
  • Alternate Protocol 5: Crystal Optimization Using Under-Oil Microbatch
  • Optimization by Crystal Seeding
  • Alternate Protocol 6: Macroseeding
  • Alternate Protocol 7: Microseeding: Streak Seeding
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Protein for Crystallization

 Materials
  • Soluble protein sample
  • Liquid nitrogen
  • 15 ml to 500 µl and 2 ml to ~50 µl Centriprep and Centricon microconcentrators (Amicon)
  • UV spectrophotometer
  • 1.5-ml screw-cap microcentrifuge tubes
  • Additional reagents and equipment for measurement of dynamic light scattering (unit 17.10)

Alternate Protocol 1: Preparation of Nucleic Acid Fragments for Crystallization

 Materials
  • DNA or RNA oligonucleotides from manufacturer or HPLC purified (e.g., unit 11.6)
  • Desired buffer
  • 1.5 ml screw-cap microcentrifuge tube
  • Beaker
  • Stirring hot plate

Basic Protocol 2: Preparation and Analysis of Crystallization Trials

 Materials
  • Thomas Lubriseal stopcock grease
  • Crystallization screening kit(s) (Hampton Research):
    • For proteins and multiprotein complexes: Crystal Screens 1 (Table 17.4.3) and 2 (latter is optional)
    • For nucleic acids and protein-nucleic acid complexes: Natrix (Table 17.4.4)
  • Concentrated macromolecule sample: protein (see Basic Protocol 1), nucleic acid (see Alternate Protocol 1), or macromolecular complex (see Alternate Protocol 2)
     
    Table 17.4.3 Components of Crystal Screen 1 (Hampton Research) for Soluble Proteinsa
    ConditionFormulationb
    130% MPD, 0.1 M sodium acetate, pH 4.6, 0.02 M calcium chloride
    20.4 M potassium/sodium tartrate
    30.4 M ammonium phosphate
    42.0 M ammonium sulfate, 0.1 M Tris×Cl, pH 8.5
    530% MPD, 0.1 M sodium HEPES, pH 7.5, 0.2 M sodium citrate
    630% PEG 4000, 0.1 Tris×Cl, pH 8.5, 0.2 M magnesium chloride
    71.4 M sodium acetate, 0.1 M sodium cacodylate, pH 6.5
    830% 2-propanol, 0.1 M sodium cacodylate, pH 6.5, 0.2 M sodium citrate
    930% PEG 4000, 0.1 M sodium citrate, pH 5.6, 0.2 M ammonium acetate
    1030% PEG 4000, 0.1 M sodium acetate, pH 4.6, 0.2 M ammonium acetate
    111.0 M ammonium phosphate, 0.1 M sodium citrate, pH 5.6
    1230% 2-propanol, 0.1 M sodium HEPES, pH 7.5, 0.2 M magnesium chloride
    1330% PEG 400, 0.1 M Tris×Cl, pH 8.5, 0.2 M sodium citrate
    1428% PEG 400, 0.1 M sodium HEPES, pH 7.5, 0.2 M calcium chloride
    1530% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M ammonium sulfate
    161.5 M lithium sulfate, 0.1 M sodium HEPES, pH 7.5
    1730% PEG 4000, 0.1 M Tris×Cl, pH 8.5, 0.2 M lithium sulfate
    1820% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M magnesium acetate
    1930% 2-propanol, 0.1 M Tris×Cl, pH 8.5, 0.2 M ammonium acetate
    2025% PEG 4000
    2130% MPD, 0.1 M sodium cacodylate, pH 6.5, 0.2 M magnesium acetate
    2230% PEG 4000, 0.1 M Tris×Cl, pH 8.5, 0.2 M sodium acetate
    2330% PEG 400, 0.1 M sodium HEPES, pH 7.5, 0.2 M magnesium chloride
    2420% 2-propanol, 0.1 M sodium acetate, pH 4.6, 0.2 M calcium chloride
    251.0 M sodium acetate, 0.1 M imidazole, pH 6.5
    2630% MPD, 0.1 M sodium citrate, pH 5.6, 0.2 M ammonium acetate
    2720% 2-propanol, 0.1 M sodium HEPES, pH 7.5, 0.2 M sodium citrate
    2830% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M sodium acetate
    290.8 M potassium/sodium tartrate, 0.1 M sodium HEPES, pH 7.5
    3030% PEG 8000, 0.2 M ammonium sulfate
    3130% PEG 4000, 0.2 M ammonium sulfate
    322.0 M ammonium sulfate
    334.0 M sodium formate
    342.0 M sodium formate, 0.1 M sodium acetate, pH 4.6
    351.6 M sodium/potassium phosphate, 0.1 M sodium HEPES, pH 7.5
    368% PEG 8000, 0.1 M Tris×Cl, pH 8.5
    378% PEG 4000, 0.1 M sodium acetate, pH 4.6
    381.4 M sodium citrate, 0.1 M sodium HEPES, pH 7.5
    392% PEG 400, 2.0 M ammonium sulfate, 0.1 M sodium HEPES, pH 7.5
    4020% 2-propanol, 20% PEG 4000, 0.1 M sodium citrate, pH 5.6
    4110% 2-propanol, 20% PEG 4000, 0.1 M sodium HEPES, pH 7.5
    4220% PEG 8000, 0.05 M potassium phosphate
    4330% PEG 1500
    440.2 M magnesium formate
    4518% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M zinc acetate
    4618% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M calcium acetate
    472.0 M ammonium sulfate, 0.1 M sodium acetate, pH 4.6
    482.0 M ammonium phosphate, 0.1 M Tris×Cl, pH 8.5
     aJancarik and Kim (1991).
     bMPD, 2-methyl-2,4-pentanediol; PEG, polyethylene glycol.
     
    Table 17.4.4 Components of Natrix (Hampton Research) for Nucleic Acid and Protein-Nucleic Acid Complexes Screena
    ConditionFormulationb
    10.01 M magnesium chloride, 0.05 M MES, pH 5.6, 1.8 M lithium sulfate
    20.01 M magnesium acetate, 0.05 M MES, pH 5.6, 2.5 M ammonium sulfate
    30.1 M magnesium acetate, 0.05 M MES, pH 5.6, 20% MPD
    40.2 M KCl, 0.01 M magnesium sulfate, 0.05 M MES, pH 5.6, 10% PEG 400
    50.2 M KCl, 0.01 M magnesium chloride, 0.05 M MES, pH 5.6, 5% PEG 8000
    60.1 M ammonium sulfate, 0.01 M magnesium chloride, 0.05 M MES, pH 5.6, 20% PEG 8000
    70.02 M magnesium chloride, 0.05 M MES, pH 6.0, 15% isopropanol
    80.005 M magnesium sulfate, 0.1 M ammonium acetate, 0.05 M MES, pH 6.0, 0.6 M NaCl
    90.1 M KCl, 0.01 M magnesium chloride, 0.05 M MES, pH 6.0, 10% PEG 400
    100.005 M magnesium sulfate, 0.05 M MES, pH 6.0, 5% PEG 4000
    110.01 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.0, 1.0 M lithium sulfate
    120.01 M magnesium sulfate, 0.05 M sodium cacodylate, pH 6.0, 1.8 M lithium sulfate
    130.015 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.0, 1.7 M ammonium sulfate
    140.1 M KCl, 0.025 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.0, 15% isopropanol
    150.04 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.0, 5% MPD
    160.04 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.0, 30% MPD
    170.2 M KCl, 0.01 M calcium chloride, 0.05 M sodium cacodylate, pH 6.0, 10% PEG 4000
    180.01 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 1.3 M lithium sulfate
    190.01 M magnesium sulfate, 0.05 M sodium cacodylate, pH 6.5, 2.0 M ammonium sulfate
    200.1 M ammonium acetate, 0.015 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 10% isopropanol
    210.2 M KCl, 0.005 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.5, 10% 1,6-hexanediol
    220.08 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5
    230.2 M KCl, 0.01 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.5, 10% PEG 4000
    240.2 M ammonium acetate, 0.01 M calcium chloride, 0.05 M sodium cacodylate, pH 6.5, 10% PEG 4000
    250.08 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 30% PEG 4000
    260.2 M KCl, 0.1 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 10% PEG 8000
    270.2 M ammonium acetate, 0.01 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 30% PEG 8000
    280.05 M magnesium sulfate, 0.05 M sodium HEPES, pH 7.0, 1.6 M lithium sulfate
    290.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 4.0 M lithium chloride
    300.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0
    310.005 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 25% PEG monomethyl ether 550
    320.2 M KCl, 0.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 20% 1,6-hexanediol
    330.2 M ammonium chloride, 0.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 30% 1,6-hexanediol
    340.1 M KCl, 0.005 M magnesium sulfate, 0.05 M sodium HEPES, pH 7.0, 15% MPD
    350.1 M KCl, 0.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 5% PEG 400
    360.1 M KCl, 0.01 M calcium chloride, 0.05 M sodium HEPES, pH 7.0, 10% PEG 400
    370.2 M KCl, 0.025 M magnesium sulfate, 0.05 M sodium HEPES, pH 7.0, 20% PEG 200
    380.2 M ammonium acetate, 0.15 M magnesium acetate, 0.05 M sodium HEPES, pH 7.0, 5% PEG 4000
    390.1 M ammonium acetate, 0.02 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 5% PEG 8000
    400.01 M magnesium chloride, 0.05 M Tris×Cl, pH 7.5, 1.6 M ammonium sulfate
    410.1 M KCl, 0.015 M magnesium chloride, 0.05 M Tris×Cl, pH 7.5, 10% PEG monomethyl ether 550
    420.01 M magnesium chloride, 0.05 M Tris×Cl, pH 7.5, 5% isopropanol
    430.01 M magnesium chloride, 0.05 M ammonium acetate, 0.05 M Tris×Cl, pH 7.5, 10% MPD
    440.2 M KCl, 0.05 M magnesium chloride, 0.05 M Tris×Cl, pH 7.5, 10% PEG 4000
    450.025 M magnesium sulfate, 0.05 M Tris×Cl, pH 8.5, 1.8 M ammonium sulfate
    460.005 M magnesium sulfate, 0.05 M Tris×Cl, pH 8.5, 35% 1,6-hexanediol
    470.1 M KCl, 0.01 M magnesium chloride, 0.05 M Tris×Cl, pH 8.5, 30% PEG 400
    480.01 M calcium chloride, 0.2 M ammonium chloride, 0.05 M Tris×Cl, pH 8.5, 30% PEG 4000
     aScott et al. (1995).
     bMES, 2-(N-morpholino)ethanesulfonic acid; MPD, 2-methyl-2,4-pentanediol; PEG, polyethylene glycol.
  • Small aluminum foil weigh dish
  • Hot plate
  • 50-ml Erlenmeyer flask with 2.5-cm diameter opening
  • 24-well crystallization trays (VDX Plate, Hampton Research)
  • 22-mm2 plastic or siliconized coverslips (Hampton Research)
  • Self-closing tweezers
  • Microscope with ³40× magnification, light source, and viewing platform (at least 3 × 4 in.), preferably with polarizer (e.g., Leica MZ16, Zeiss STEMI SV11, Nikon SMZ1000)
  • Tape

NOTE: Preparation and crystallization (steps 1 to 9) should be done both at 4°C and room temperature (±3°C).

Alternate Protocol 3: Crystallization Screening Using High-Throughput Robotics

 Materials
  • Crystal Screen 1 and 2 (see Table 17.4.3)
  • Concentrated protein or macromolecular complex (Basic Protocol 1)
  • MASTERBLOCK 96 Deep Well plate (Hampton Research)
  • Microplate adhesive film (USA Scientific or other manufacturer)
  • Intelli-Plate 96 (Art Robbins Instruments)
  • Art Robbins Crystal Phoenix robot
  • Crystal Clear Sealing Tape (Hampton Research)

Support Protocol 1: Establishing Whether Crystals are Macromolecule or Crystallization Reagent Using a Cryoloop

 Materials
  • Crystallization tray(s) containing 0.2-mm3 (or larger) crystal(s) and reservoir solutions (see Basic Protocol 2)
  • Macromolecule storage solution (i.e., solution in which the macromolecule is soluble)
  • Purified macromolecule
  • 2× SDS sample buffer (unit 10.1)
  • Microscope with ³40× magnification, light source, and viewing platform (at least 3 × 4 in.), preferably with polarizer (e.g., Leica MZ16, Zeiss STEMI SV11, Nikon SMZ1000)
  • Cryoloops (Hampton Research)
  • Plastic petri dish
  • Microcentrifuge tubes
  • Vortex
  • Additional reagents and equipment for SDS-PAGE (unit 10.1) and silver staining (unit 10.5)

Basic Protocol 3: Optimization of Crystallization Parameters

 Materials
  • Crystallization screening kit(s) (Hampton Research):
    • For proteins and multiprotein complexes: Crystal Screens 1 (Table 17.4.3) and 2 (latter is optional)
    • For nucleic acids and protein-nucleic acid complexes: Natrix (Table 17.4.4)
  • Concentrated reagents needed to set up crystal screens (for the example described here: 1 M sodium citrate pH 5.6, 4.6, and 6.5; 2M ammonium acetate; 50% PEG 4000)
  • Concentrated macromolecule sample: protein (see Basic Protocol 1), nucleic acid (see Alternate Protocol 1), or macromolecular complex (see Alternate Protocol 2)
  • 1-ml micropipettor
  • Microscope with ³40× magnification, light source, and viewing platform (at least 3 × 4 in.), preferably with polarizer (e.g., Leica MZ16, Zeiss STEMI SV11, Nikon SMZ1000)
  • Additional reagents and equipment for preparation of crystallization trials (see Basic Protocol 2)

Alternate Protocol 5: Crystal Optimization Using Under-Oil Microbatch

 Additional Materials (also see Basic Protocol 3)
  • Paraffin oil (Hampton Research or other manufacturer)
  • Concentrated macromolecule or macromolecular complex (see Basic Protocol 1)
  • Vapor batch 96-well plate (Hampton Research)
  • Microcentrifuge tubes
  • Plastic or siliconized coverslips
  • Temperature-controlled environment

Alternate Protocol 6: Macroseeding

 Additional Materials (also see Basic Protocol 2)
  • Microscope with ³40× magnification, light source, and viewing platform (at least 3 × 4 in.), preferably with polarizer (e.g., Leica MZ16, Zeiss STEMI SV11, Nikon SMZ1000)
  • Tweezers
  • Glass fibers (see recipe) or crystal probe manipulators (Hampton Research)
  • Capillary-tipped syringe (optional; see recipe)

Alternate Protocol 7: Microseeding: Streak Seeding

 Materials (also see Basic Protocol 2)
  • Stabilization solution (e.g., reservoir solution with 20% higher precipitating reagent)
  • Glass fibers or crystal probe manipulators (Hampton Research)
  • Capillary-tipped syringe (see recipe)
  • Glass minipestle (see recipe)
  • Rabbit hair streaker (see recipe)
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Figures

  • Figure 17.4.1
    Typical crystallization setups. See text for descriptions. (A) Described in Basic Protocol 2. (B) Described in Alternate Protocol 3. (C) Described in Alternate Protocol 5.

    View Image
  • Figure 17.4.2
    Crystal tray greasing setup.

    View Image
  • Figure 17.4.3
    Optimization of crystallization parameters (see Basic Protocol 3 for discussion). A, B, C, and D represent four different trays that could be set up during the process of crystal optimization through rounds of varying the different crystallization condition reagents. Shaded wells represent conditions yielding the best crystals, and upon which the next round of optimization was based. Abbreviations: O, clear drop; C, crystal; P, precipitate; C/P, crystals and precipitate.

    View Image
  • Figure 17.4.4
    Optimization of crystal equilibration parameters (see Alternate Protocol 4 for discussion). A, B, and C represent three trays that could be set up to optimize crystal equilibration conditions. Shaded squares indicate conditions that yielded the best crystals in the example. Abbreviations: O, clear drop; CS, small-sized crystal; CM, medium-sized crystal; CL, large-sized crystal.

    View Image
  • Figure 17.4.5
    Typical home-made crystal manipulation tools.

    View Image

Literature Cited

Literature Cited
    Aggarwal, A.K. 1990. Crystallization of DNA binding proteins with oligonucleotides. Methods Companion 1:83-90.
    Chayen, N.E. 1998. Comparative studies of protein crystallization by vapor-diffusion and microbatch techniques. Acta. Cryst. D54:8-15.
    D'Arcy, A. 1994. Crystallizing proteins: A rational approach Acta Cryst. D50:469-471.
    Ferre-D'Amare, A. and Burley, S. 1994. Use of dynamic light scattering to assess crystallizability of macromolecules and macromolecular assemblies. Structure 2:357-359.
    Jancarik, J. and Kim, S.-H. 1991. Sparse matrix sampling: A screening method for crystallization of proteins. J. Appl. Cryst. 24:409-411.
    McPherson, A. 1982. The Preparation and Analysis of Protein Crystals. John Wiley & Sons, New York.
    McPherson, A. 1999. Crystalization of Biological Macromolecules. Cold Spring Harbor Press, Cold Spring Harbor, New York.
    Muecke, M., Samuels, M., Davey, M., and Jeruzalmi, D. 2008. Preparation of multimilligram quantities of large, linear DNA molecules for structural studies. Structure 16:837-841.
    Rupp, B. 2010. Biomolecular Crystallography: Principles, Practice, and Application to Structural Biology. Garland Science, New York.
    Scott, W.G., Finch, J.T., Grenfell, R., Fogg, J., Smith, T., Gait, M.J., and Klug, A. 1995. Rapid crystallization of chemically synthesized hammerhead RNAs using a double screening procedure. J. Mol. Biol. 250:327-332.
    Stura, E.A. and Wilson, I.A. 1990. Analytical and production seeding techniques. In Methods: A Companion to Methods in Enzymology. 1:38-49.
    Stura, E.A. and Wilson, I.A. 1991. "Seeding techniques". In Crystallization of Nucleic Acids and Proteins: A Practical Approach ( A. Ducruix and G. Giegé, eds.) pp. 241-254. IRL Press, Oxford.
    Stura, E.A., Satterthwait, A.C., Calvo, J.C., Kaslow, D.C., and Wilson, I.A. 1994. Reverse screening. Acta Cryst. 50:448-455.
    Weber, P. 1997. Overview of protein crystallization methods. Methods Enzymol. 276:13-22.
     
 
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