Crystallization of Macromolecules

David Friedmann1, Troy Messick2, Ronen Marmorstein1

1 Wistar Institute, Philadelphia, Pennsylvania, 2 Vironika, 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

Alternate Protocol 2: Preparation of Macromolecular Complexes for Crystallization

  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 protocol 1), nucleic acid (see protocol 2), or macromolecular complex (see protocol 3)
    Table 7.4.3   Materials   Components of Crystal Screen 1 (Hampton Research) for Soluble Proteins g   Components of Crystal Screen 1 (Hampton Research) for Soluble Proteins   Components of Natrix (Hampton Research) for Nucleic Acid and Protein‐Nucleic Acid Complexes Screen j   Components of Natrix (Hampton Research) for Nucleic Acid and Protein‐Nucleic Acid Complexes Screen

    Condition Formulation h
    1 30% MPD, 0.1 M sodium acetate, pH 4.6, 0.02 M calcium chloride
    2 0.4 M potassium/sodium tartrate
    3 0.4 M ammonium phosphate
    4 2.0 M ammonium sulfate, 0.1 M Tris⋅Cl, pH 8.5
    5 30% MPD, 0.1 M sodium HEPES, pH 7.5, 0.2 M sodium citrate
    6 30% PEG 4000, 0.1 Tris⋅Cl, pH 8.5, 0.2 M magnesium chloride
    7 1.4 M sodium acetate, 0.1 M sodium cacodylate, pH 6.5
    8 30% 2‐propanol, 0.1 M sodium cacodylate, pH 6.5, 0.2 M sodium citrate
    9 30% PEG 4000, 0.1 M sodium citrate, pH 5.6, 0.2 M ammonium acetate
    10 30% PEG 4000, 0.1 M sodium acetate, pH 4.6, 0.2 M ammonium acetate
    11 1.0 M ammonium phosphate, 0.1 M sodium citrate, pH 5.6
    12 30% 2‐propanol, 0.1 M sodium HEPES, pH 7.5, 0.2 M magnesium chloride
    13 30% PEG 400, 0.1 M Tris⋅Cl, pH 8.5, 0.2 M sodium citrate
    14 28% PEG 400, 0.1 M sodium HEPES, pH 7.5, 0.2 M calcium chloride
    15 30% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M ammonium sulfate
    16 1.5 M lithium sulfate, 0.1 M sodium HEPES, pH 7.5
    17 30% PEG 4000, 0.1 M Tris⋅Cl, pH 8.5, 0.2 M lithium sulfate
    18 20% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M magnesium acetate
    19 30% 2‐propanol, 0.1 M Tris⋅Cl, pH 8.5, 0.2 M ammonium acetate
    20 25% PEG 4000
    21 30% MPD, 0.1 M sodium cacodylate, pH 6.5, 0.2 M magnesium acetate
    22 30% PEG 4000, 0.1 M Tris⋅Cl, pH 8.5, 0.2 M sodium acetate
    23 30% PEG 400, 0.1 M sodium HEPES, pH 7.5, 0.2 M magnesium chloride
    24 20% 2‐propanol, 0.1 M sodium acetate, pH 4.6, 0.2 M calcium chloride
    25 1.0 M sodium acetate, 0.1 M imidazole, pH 6.5
    26 30% MPD, 0.1 M sodium citrate, pH 5.6, 0.2 M ammonium acetate
    27 20% 2‐propanol, 0.1 M sodium HEPES, pH 7.5, 0.2 M sodium citrate
    28 30% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M sodium acetate
    29 0.8 M potassium/sodium tartrate, 0.1 M sodium HEPES, pH 7.5
    30 30% PEG 8000, 0.2 M ammonium sulfate
    31 30% PEG 4000, 0.2 M ammonium sulfate
    32 2.0 M ammonium sulfate
    33 4.0 M sodium formate
    34 2.0 M sodium formate, 0.1 M sodium acetate, pH 4.6
    35 1.6 M sodium/potassium phosphate, 0.1 M sodium HEPES, pH 7.5
    36 8% PEG 8000, 0.1 M Tris⋅Cl, pH 8.5
    37 8% PEG 4000, 0.1 M sodium acetate, pH 4.6
    38 1.4 M sodium citrate, 0.1 M sodium HEPES, pH 7.5
    39 2% PEG 400, 2.0 M ammonium sulfate, 0.1 M sodium HEPES, pH 7.5
    40 20% 2‐propanol, 20% PEG 4000, 0.1 M sodium citrate, pH 5.6
    41 10% 2‐propanol, 20% PEG 4000, 0.1 M sodium HEPES, pH 7.5
    42 20% PEG 8000, 0.05 M potassium phosphate
    43 30% PEG 1500
    44 0.2 M magnesium formate
    45 18% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M zinc acetate
    46 18% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M calcium acetate
    47 2.0 M ammonium sulfate, 0.1 M sodium acetate, pH 4.6
    48 2.0 M ammonium phosphate, 0.1 M Tris⋅Cl, pH 8.5
    Condition Formulation k
    1 0.01 M magnesium chloride, 0.05 M MES, pH 5.6, 1.8 M lithium sulfate
    2 0.01 M magnesium acetate, 0.05 M MES, pH 5.6, 2.5 M ammonium sulfate
    3 0.1 M magnesium acetate, 0.05 M MES, pH 5.6, 20% MPD
    4 0.2 M KCl, 0.01 M magnesium sulfate, 0.05 M MES, pH 5.6, 10% PEG 400
    5 0.2 M KCl, 0.01 M magnesium chloride, 0.05 M MES, pH 5.6, 5% PEG 8000
    6 0.1 M ammonium sulfate, 0.01 M magnesium chloride, 0.05 M MES, pH 5.6, 20% PEG 8000
    7 0.02 M magnesium chloride, 0.05 M MES, pH 6.0, 15% isopropanol
    8 0.005 M magnesium sulfate, 0.1 M ammonium acetate, 0.05 M MES, pH 6.0, 0.6 M NaCl
    9 0.1 M KCl, 0.01 M magnesium chloride, 0.05 M MES, pH 6.0, 10% PEG 400
    10 0.005 M magnesium sulfate, 0.05 M MES, pH 6.0, 5% PEG 4000
    11 0.01 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.0, 1.0 M lithium sulfate
    12 0.01 M magnesium sulfate, 0.05 M sodium cacodylate, pH 6.0, 1.8 M lithium sulfate
    13 0.015 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.0, 1.7 M ammonium sulfate
    14 0.1 M KCl, 0.025 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.0, 15% isopropanol
    15 0.04 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.0, 5% MPD
    16 0.04 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.0, 30% MPD
    17 0.2 M KCl, 0.01 M calcium chloride, 0.05 M sodium cacodylate, pH 6.0, 10% PEG 4000
    18 0.01 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 1.3 M lithium sulfate
    19 0.01 M magnesium sulfate, 0.05 M sodium cacodylate, pH 6.5, 2.0 M ammonium sulfate
    20 0.1 M ammonium acetate, 0.015 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 10% isopropanol
    21 0.2 M KCl, 0.005 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.5, 10% 1,6‐hexanediol
    22 0.08 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5
    23 0.2 M KCl, 0.01 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.5, 10% PEG 4000
    24 0.2 M ammonium acetate, 0.01 M calcium chloride, 0.05 M sodium cacodylate, pH 6.5, 10% PEG 4000
    25 0.08 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 30% PEG 4000
    26 0.2 M KCl, 0.1 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 10% PEG 8000
    27 0.2 M ammonium acetate, 0.01 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 30% PEG 8000
    28 0.05 M magnesium sulfate, 0.05 M sodium HEPES, pH 7.0, 1.6 M lithium sulfate
    29 0.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 4.0 M lithium chloride
    30 0.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0
    31 0.005 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 25% PEG monomethyl ether 550
    32 0.2 M KCl, 0.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 20% 1,6‐hexanediol
    33 0.2 M ammonium chloride, 0.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 30% 1,6‐hexanediol
    34 0.1 M KCl, 0.005 M magnesium sulfate, 0.05 M sodium HEPES, pH 7.0, 15% MPD
    35 0.1 M KCl, 0.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 5% PEG 400
    36 0.1 M KCl, 0.01 M calcium chloride, 0.05 M sodium HEPES, pH 7.0, 10% PEG 400
    37 0.2 M KCl, 0.025 M magnesium sulfate, 0.05 M sodium HEPES, pH 7.0, 20% PEG 200
    38 0.2 M ammonium acetate, 0.15 M magnesium acetate, 0.05 M sodium HEPES, pH 7.0, 5% PEG 4000
    39 0.1 M ammonium acetate, 0.02 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 5% PEG 8000
    40 0.01 M magnesium chloride, 0.05 M Tris⋅Cl, pH 7.5, 1.6 M ammonium sulfate
    41 0.1 M KCl, 0.015 M magnesium chloride, 0.05 M Tris⋅Cl, pH 7.5, 10% PEG monomethyl ether 550
    42 0.01 M magnesium chloride, 0.05 M Tris⋅Cl, pH 7.5, 5% isopropanol
    43 0.01 M magnesium chloride, 0.05 M ammonium acetate, 0.05 M Tris⋅Cl, pH 7.5, 10% MPD
    44 0.2 M KCl, 0.05 M magnesium chloride, 0.05 M Tris⋅Cl, pH 7.5, 10% PEG 4000
    45 0.025 M magnesium sulfate, 0.05 M Tris⋅Cl, pH 8.5, 1.8 M ammonium sulfate
    46 0.005 M magnesium sulfate, 0.05 M Tris⋅Cl, pH 8.5, 35% 1,6‐hexanediol
    47 0.1 M KCl, 0.01 M magnesium chloride, 0.05 M Tris⋅Cl, pH 8.5, 30% PEG 400
    48 0.01 M calcium chloride, 0.2 M ammonium chloride, 0.05 M Tris⋅Cl, pH 8.5, 30% PEG 4000

     gJancarik and Kim ( ).
     hMPD, 2‐methyl‐2,4‐pentanediol; PEG, polyethylene glycol.
    Table 7.4.4   Materials   Components of Crystal Screen 1 (Hampton Research) for Soluble Proteins g   Components of Crystal Screen 1 (Hampton Research) for Soluble Proteins   Components of Natrix (Hampton Research) for Nucleic Acid and Protein‐Nucleic Acid Complexes Screen j   Components of Natrix (Hampton Research) for Nucleic Acid and Protein‐Nucleic Acid Complexes Screen

    Condition Formulation h
    1 30% MPD, 0.1 M sodium acetate, pH 4.6, 0.02 M calcium chloride
    2 0.4 M potassium/sodium tartrate
    3 0.4 M ammonium phosphate
    4 2.0 M ammonium sulfate, 0.1 M Tris⋅Cl, pH 8.5
    5 30% MPD, 0.1 M sodium HEPES, pH 7.5, 0.2 M sodium citrate
    6 30% PEG 4000, 0.1 Tris⋅Cl, pH 8.5, 0.2 M magnesium chloride
    7 1.4 M sodium acetate, 0.1 M sodium cacodylate, pH 6.5
    8 30% 2‐propanol, 0.1 M sodium cacodylate, pH 6.5, 0.2 M sodium citrate
    9 30% PEG 4000, 0.1 M sodium citrate, pH 5.6, 0.2 M ammonium acetate
    10 30% PEG 4000, 0.1 M sodium acetate, pH 4.6, 0.2 M ammonium acetate
    11 1.0 M ammonium phosphate, 0.1 M sodium citrate, pH 5.6
    12 30% 2‐propanol, 0.1 M sodium HEPES, pH 7.5, 0.2 M magnesium chloride
    13 30% PEG 400, 0.1 M Tris⋅Cl, pH 8.5, 0.2 M sodium citrate
    14 28% PEG 400, 0.1 M sodium HEPES, pH 7.5, 0.2 M calcium chloride
    15 30% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M ammonium sulfate
    16 1.5 M lithium sulfate, 0.1 M sodium HEPES, pH 7.5
    17 30% PEG 4000, 0.1 M Tris⋅Cl, pH 8.5, 0.2 M lithium sulfate
    18 20% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M magnesium acetate
    19 30% 2‐propanol, 0.1 M Tris⋅Cl, pH 8.5, 0.2 M ammonium acetate
    20 25% PEG 4000
    21 30% MPD, 0.1 M sodium cacodylate, pH 6.5, 0.2 M magnesium acetate
    22 30% PEG 4000, 0.1 M Tris⋅Cl, pH 8.5, 0.2 M sodium acetate
    23 30% PEG 400, 0.1 M sodium HEPES, pH 7.5, 0.2 M magnesium chloride
    24 20% 2‐propanol, 0.1 M sodium acetate, pH 4.6, 0.2 M calcium chloride
    25 1.0 M sodium acetate, 0.1 M imidazole, pH 6.5
    26 30% MPD, 0.1 M sodium citrate, pH 5.6, 0.2 M ammonium acetate
    27 20% 2‐propanol, 0.1 M sodium HEPES, pH 7.5, 0.2 M sodium citrate
    28 30% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M sodium acetate
    29 0.8 M potassium/sodium tartrate, 0.1 M sodium HEPES, pH 7.5
    30 30% PEG 8000, 0.2 M ammonium sulfate
    31 30% PEG 4000, 0.2 M ammonium sulfate
    32 2.0 M ammonium sulfate
    33 4.0 M sodium formate
    34 2.0 M sodium formate, 0.1 M sodium acetate, pH 4.6
    35 1.6 M sodium/potassium phosphate, 0.1 M sodium HEPES, pH 7.5
    36 8% PEG 8000, 0.1 M Tris⋅Cl, pH 8.5
    37 8% PEG 4000, 0.1 M sodium acetate, pH 4.6
    38 1.4 M sodium citrate, 0.1 M sodium HEPES, pH 7.5
    39 2% PEG 400, 2.0 M ammonium sulfate, 0.1 M sodium HEPES, pH 7.5
    40 20% 2‐propanol, 20% PEG 4000, 0.1 M sodium citrate, pH 5.6
    41 10% 2‐propanol, 20% PEG 4000, 0.1 M sodium HEPES, pH 7.5
    42 20% PEG 8000, 0.05 M potassium phosphate
    43 30% PEG 1500
    44 0.2 M magnesium formate
    45 18% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M zinc acetate
    46 18% PEG 8000, 0.1 M sodium cacodylate, pH 6.5, 0.2 M calcium acetate
    47 2.0 M ammonium sulfate, 0.1 M sodium acetate, pH 4.6
    48 2.0 M ammonium phosphate, 0.1 M Tris⋅Cl, pH 8.5
    Condition Formulation k
    1 0.01 M magnesium chloride, 0.05 M MES, pH 5.6, 1.8 M lithium sulfate
    2 0.01 M magnesium acetate, 0.05 M MES, pH 5.6, 2.5 M ammonium sulfate
    3 0.1 M magnesium acetate, 0.05 M MES, pH 5.6, 20% MPD
    4 0.2 M KCl, 0.01 M magnesium sulfate, 0.05 M MES, pH 5.6, 10% PEG 400
    5 0.2 M KCl, 0.01 M magnesium chloride, 0.05 M MES, pH 5.6, 5% PEG 8000
    6 0.1 M ammonium sulfate, 0.01 M magnesium chloride, 0.05 M MES, pH 5.6, 20% PEG 8000
    7 0.02 M magnesium chloride, 0.05 M MES, pH 6.0, 15% isopropanol
    8 0.005 M magnesium sulfate, 0.1 M ammonium acetate, 0.05 M MES, pH 6.0, 0.6 M NaCl
    9 0.1 M KCl, 0.01 M magnesium chloride, 0.05 M MES, pH 6.0, 10% PEG 400
    10 0.005 M magnesium sulfate, 0.05 M MES, pH 6.0, 5% PEG 4000
    11 0.01 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.0, 1.0 M lithium sulfate
    12 0.01 M magnesium sulfate, 0.05 M sodium cacodylate, pH 6.0, 1.8 M lithium sulfate
    13 0.015 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.0, 1.7 M ammonium sulfate
    14 0.1 M KCl, 0.025 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.0, 15% isopropanol
    15 0.04 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.0, 5% MPD
    16 0.04 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.0, 30% MPD
    17 0.2 M KCl, 0.01 M calcium chloride, 0.05 M sodium cacodylate, pH 6.0, 10% PEG 4000
    18 0.01 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 1.3 M lithium sulfate
    19 0.01 M magnesium sulfate, 0.05 M sodium cacodylate, pH 6.5, 2.0 M ammonium sulfate
    20 0.1 M ammonium acetate, 0.015 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 10% isopropanol
    21 0.2 M KCl, 0.005 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.5, 10% 1,6‐hexanediol
    22 0.08 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5
    23 0.2 M KCl, 0.01 M magnesium chloride, 0.05 M sodium cacodylate, pH 6.5, 10% PEG 4000
    24 0.2 M ammonium acetate, 0.01 M calcium chloride, 0.05 M sodium cacodylate, pH 6.5, 10% PEG 4000
    25 0.08 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 30% PEG 4000
    26 0.2 M KCl, 0.1 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 10% PEG 8000
    27 0.2 M ammonium acetate, 0.01 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.5, 30% PEG 8000
    28 0.05 M magnesium sulfate, 0.05 M sodium HEPES, pH 7.0, 1.6 M lithium sulfate
    29 0.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 4.0 M lithium chloride
    30 0.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0
    31 0.005 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 25% PEG monomethyl ether 550
    32 0.2 M KCl, 0.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 20% 1,6‐hexanediol
    33 0.2 M ammonium chloride, 0.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 30% 1,6‐hexanediol
    34 0.1 M KCl, 0.005 M magnesium sulfate, 0.05 M sodium HEPES, pH 7.0, 15% MPD
    35 0.1 M KCl, 0.01 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 5% PEG 400
    36 0.1 M KCl, 0.01 M calcium chloride, 0.05 M sodium HEPES, pH 7.0, 10% PEG 400
    37 0.2 M KCl, 0.025 M magnesium sulfate, 0.05 M sodium HEPES, pH 7.0, 20% PEG 200
    38 0.2 M ammonium acetate, 0.15 M magnesium acetate, 0.05 M sodium HEPES, pH 7.0, 5% PEG 4000
    39 0.1 M ammonium acetate, 0.02 M magnesium chloride, 0.05 M sodium HEPES, pH 7.0, 5% PEG 8000
    40 0.01 M magnesium chloride, 0.05 M Tris⋅Cl, pH 7.5, 1.6 M ammonium sulfate
    41 0.1 M KCl, 0.015 M magnesium chloride, 0.05 M Tris⋅Cl, pH 7.5, 10% PEG monomethyl ether 550
    42 0.01 M magnesium chloride, 0.05 M Tris⋅Cl, pH 7.5, 5% isopropanol
    43 0.01 M magnesium chloride, 0.05 M ammonium acetate, 0.05 M Tris⋅Cl, pH 7.5, 10% MPD
    44 0.2 M KCl, 0.05 M magnesium chloride, 0.05 M Tris⋅Cl, pH 7.5, 10% PEG 4000
    45 0.025 M magnesium sulfate, 0.05 M Tris⋅Cl, pH 8.5, 1.8 M ammonium sulfate
    46 0.005 M magnesium sulfate, 0.05 M Tris⋅Cl, pH 8.5, 35% 1,6‐hexanediol
    47 0.1 M KCl, 0.01 M magnesium chloride, 0.05 M Tris⋅Cl, pH 8.5, 30% PEG 400
    48 0.01 M calcium chloride, 0.2 M ammonium chloride, 0.05 M Tris⋅Cl, pH 8.5, 30% PEG 4000

     JScott et al. ( ).
     kMES, 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 ≥40× 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).

Basic Protocol 2: Preparation and Analysis of Crystallization Trials

  Materials
  • Crystal Screen 1 and 2 (see Table 17.4.3)
  • Concentrated protein or macromolecular complex ( 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)

Alternate Protocol 3: Crystallization Screening Using High‐Throughput Robotics

  Materials
  • Crystallization tray(s) containing 0.2‐mm3 (or larger) crystal(s) and reservoir solutions (see protocol 4)
  • Macromolecule storage solution (i.e., solution in which the macromolecule is soluble)
  • Purified macromolecule
  • 2× SDS sample buffer (unit 10.1)
  • Microscope with ≥40× 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)

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

  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 protocol 1), nucleic acid (see protocol 2), or macromolecular complex (see protocol 3)
  • 1‐ml micropipettor
  • Microscope with ≥40× 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 protocol 4)

Basic Protocol 3: Optimization of Crystallization Parameters

  • Paraffin oil (Hampton Research or other manufacturer)
  • Concentrated macromolecule or macromolecular complex (see protocol 1)
  • Vapor batch 96‐well plate (Hampton Research)
  • Microcentrifuge tubes
  • Plastic or siliconized coverslips
  • Temperature‐controlled environment

Alternate Protocol 4: Optimization of Crystal Equilibration Conditions

  • Microscope with ≥40× 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 5: Crystal Optimization Using Under‐Oil Microbatch

  • 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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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.
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