BSTR521 02 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
BSTR521: BioCrystallography
Protein Crystallography
Protein Crystallization, Crystal CryoProtection and Crystal Annealing
January 2009
Wim G.J. Hol
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Figure Courtesy of Focco van den Akker
PROTEIN CRYSTAL GROWTH - Literature
Literature
A major text on protein crystallization is: Crystallization of Biological Macromolecules
Alex Mcpherson
Cold Spring Harbor Laboratory Press Chpt 4 : Some physical and Energetic Principles Chpt 5 : Practical Procedures for Macromolecular Crystallization Chpt 6 : Important Considerations in Macromolecular Crystallization Chpt 7 : Strategies and Special Approaches in Growing Crystals
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Some older information can be found in; Methods in Enzymology, Vol 114 "Diffraction Methods of Biological Macromolecules Eds. H. W. Wyckoff, C. H. W. Hirs, and S. N. Timasheff, Academic Press, Orlando FL, 1985. In particular the following chapters: Chapter 3: "Theory of Protein Solubility" T. Arakawa and S. N. Timasheff, pp. 49-76. Chapter 4: "Nucleation and Growth of Protein Crystals: General Principles and Assays" G. Feher and Z. Kam, pp. 77-111. Chapter 5: "Crystallization of Macromolecules: General Principles" A. McPherson, pp. 112-119. Chapter 6: "Use of Polyethylene Glycol in the Crystallization of Macromolecules" A. McPherson, pp. 120-124. Chapter 7: "Crystallization of Protein by Variation of pH or Temperature" A. McPherson, pp. 125-127. Chapter 8: "Crystallization in Capillary Tubes" G. N. Phillips, Jr., pp. 128-131. Chapter 9: "Seed Enlargement and repeated Seeding" C. Thaller, G. Eichele, L. H. Weaver, E. Wilson, R. Karlsson, and J. N. Jansonius, pp. 132-135.
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PROTEIN CRYSTAL GROWTH (II) - Literature Methods in Enzymology Vol 276 Section II"Crystals"-Carter and Sweet(1997) Macromolecular Crystallography Part A Chapter 2: "Overview of Protein Crystallization Methods Patricia C. Weber, pp. 13-22. Chapter 3: "Inferences Drawn from Physicochemical Studies of Crystallogenesis and Precrystalline States" Madeleine Ries-Kautt and Arnaud Ducruix., pp. 23-59. Chapter 4: "Membrane Protein Crystallization: Application of Sparse Matrices to the -Hemolysin Heptamer" Langzhou Song and J. Eric Gouaux, pp. 60-73. Chapter 5: "Response Surface Methods for Optimizing and Improving Reproducibility of Crystal Growth" Charles W. Carter, Jr. , pp. 74-99. Chapter 6: "Second Virial Coefficient as Predictor in Protein Crystal Growth" A. George et al., pp. 100-109. Chapter 7: "Kinetic Aspects of Macromolecular Crystallization" Joseph R. Luft and George T. Detitta , pp. 110-130. Chapter 8: "Using Cosolvents to Stabilize Protein Conformation for Crystallization" Rui Sousa , pp. 131-142. Chapter 9: "Preparation and Crystallization of RNA: A Sparse Matrix Approach" Craig E. Kundrot, pp. 143-156. Chapter 10: "Dynamic Light Scattering in Evaluating Crystallizability of Macromolecules" Adrian R. Ferre-D'amare and Stephen K. Burley , pp. 157-165 Chapter 11: "Two-Dimensional Protein Crystals in Aid of Three-Dimensional Protein Crystal Growth" Aled M. Edwards et al , pp. 166-170. Chapter 12: 5 "Reductive Alkylation of Lysine Residues to Alter Crystallization Properties of Proteins Ivan Rayment , pp. 171-182.
How to make the most of your precious protein solution?
Protect your protein from damage
Use Flash freezing for Long-term protein storage Deng et al, Acta D (2004) Use Protease Inhibitors Use DTT, TCEP against oxidation Find Ligands in the Literature Find Ligands by gel shifts
ALWAYS check pH of every solution you add to your protein solution
Use your protein solution efficiently
Use small volumes in your crystallization micro-experiments Use minute amounts of protein to explore precipitation properties of your protein (in a so-called pre-screen) Explore the effect of temperature on solubility
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02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
How to make the most of your precious protein solution?
Optimize your protein buffer Dynamic Light Scattering (DLS)
Low polydispersity in DLS is correlated with crystal growth. Low polydispersity is an indication that your protein is present as a welldefined assembly and not a mixture of different aggregation states. So, testing different buffers (increasing salt, glycerol concentration, changing pH, adding additives, etc) makes sense.
How to make the most of your precious protein solution?
HEAVY ATOM COMPOUND NATIVE GELS (HAC GELS) Follow-ups: HAC as Additive in Crystallization HAC for Soaks in Crystal Mounting
Monitor positional shifts as a result of (presumed) HAC binding
Optimize your protein concentration
In particular when your protein is forming multimers it might be good to try as high a protein concentration as possible so that your solution contains multimers only and is not a mixture of monomers and multimers (watch also your size exclusion chromatogram for hints)
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How to make the most of your precious protein solution?
Limited Proteolysis Provides information about: Speed by which protein gets broken down At different pH values At different temperatures Protective effects Of general additives like NaCl, glycerol, phosphate, etc Of specific additives like inhibitors, substrates,ligands,metals Of protein partners, antibodies Of DNA, RNA Useful chunks For crystallization after or without prurification For creation of truncated constructs
Limited Proteolysis
Tbru018012AAA, R5679 Tcru023995AAA, W8424
Key: Time for proteolysis 0 = 0 hour 1 = Proteolysis in 1 hour 24= Proteolysis in 24 hours
4 Proteases: T = Trypsin Th = Thermolysin S = Subtilisin G = Glu-C
Proteins differ greatly in Protease-sensitivity.
You might like to use more than one protease trypsin, GluC, thermolysin, 9 subtilisin, chymotrypsin, elastase, ...
The more stable the protein (or protein-ligand complex) the greater the probability of crystal growth.
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Worth serious consideration
FOR A NEW STRUCTURE ONLY PREPARE SEMET PROTEIN...
Reason: there are few things as sad as having a nice diffraction data set from a native (i.e. no heavy-atomcontaining sulfur-Met) crystal and having great troubles obtaining such a crystal again. With a SeMet crystal you might as well have had a perfect structure at once!! Caveat: sometimes SeMet protein is difficult to express, is hard to purify, does not wish to crystallize, does not diffract as 11 well
Principle of Protein Crystallization
The general idea is: induce supersaturation, form a limited number of nuclei increase the size of these nuclei However, nucleation is a very difficult process to control
Usually, one aims for a few to a few dozen nuclei per crystallization microexperiment. But quite frequently a significant precipitate is formed quickly, Which means one needs to lower either protein or precipitate concentration. Sometimes crystals do form from the initial precipitate but this is usually a slow process.
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02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
Factors Affecting Protein Crystal Growth
1. 2. 3. 4. 5. 6. 7. 8. 9. pH Ionic Strength Temperature Concentration of Precipitant Concentration of Macromolecule Purity of Macromolecules Additives, Effectors and Ligands Organism source of Macromolecule Substrates, Coenzymes, Inhibitors 12. Rate of Equilibration 13. Surfactants or Detergents 14. Gravity 15. Vibrations and Sound 16. Volume of Crystallization Sample 17. Presence of Amorphous Material 18. Surfaces of Crystallization Vessels 19. Proteolysis 20. Contamination by Microbes 21. Pressure 22. Electric and Magnetic Fields 23. Handling by Investigator
Precipitating Agents
Salts Diminish electrostatic repulsion between proteins Promote hydrophobic interactions between proteins PEGs Compete for water molecules with proteins Organics Lower dielectric screening and increase electrostatic interactions Combinations of the above In different concentrations each At different pH values LEADING TO A NICE COMBINATORIAL EXPLOSION QUICKLY
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10. Reducing or Oxidizing Environment 11. Metal Ions
From: Alex McPherson
13 For each protein there are in principle millions of conditions to be explored How to walk efficiently through protein crystallization space??
Salts Used in Crystallization of Proteins
1. Ammonium or sodium sulfate 2. Lithium sulfate 3. Lithium chloride 4. Sodium or ammonium citrate 5. Sodium or potassium phosphate 6. Sodium or potassium or ammonium chloride 7. Sodium or ammonium acetate 8. Magnesium sulfate 9. Cetyltrimethyl ammonium salts 10. Calcium chloride 11. Ammonium Nitrate 12. Sodium formate Histogram showing the relative success of the most popular precipitation agents. PEG has now become the most popular reagent, with ammonium sulfate next
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McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press.
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From: Alex McPherson
Solubility of hemoglobin in concentrated phosphate buffers as a function of ionic strength and temperature.
Histogram of those ammonium sulfate concentrations producing macromolecular crystals
McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press.
Note: Ionic strength is defined as: = Ci zi2 Where Ci is the concentration of the i-th ion present in the solution and zi is its charge. 17 Summation is done for all charged particles present in the solution.
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McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press.
02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
PEG variations
Polyethylene glycols (PEGs) are among the most frequently used crystal growth agents. The molecular weights range from PEG200 to PEG20,000 Sometimes monomethyl PEG gives better results than PEG sometimes worse. Be aware of potential differences in purity and oxidizing agents in different PEG bottles Currently, it appears that medium Mw PEGs (3-4K) plus 100 to 500 mM salt are possibly the most succesful crystallization solutions for the average protein ( of course, not for your proteins). A wide variety of salts are available in so-called PEG-ion screens.
Solubility of various proteins in PEG-4000.
Measurements were made in 0.05 M potassium phosphate, pH 7.0, containing 0.1 M KCl
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McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press.
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Histogram of the PEG-6000 molecular weights producing macromolecular crystals.
Histogram of the PEG-6000 concentrations producing macromolecular crystals.
25 randomly selected proteins were set up by sitting drop vapor diffusion at pH 7.0 and otherwise identical conditions against PEG-200, -400, -1000,-2000,-4000,-8000,10000, and -20000. After 12 weeks of incubation, the trials were scored for crystals.
McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press.
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McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press.
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Organic Solvents Used Crystallization of Proteins
1. Ethanol 2. Isopropanol 3. 1,3-Propanediol 4. 2-Methyl-2, 4-pentanediol (MPD) 5. Dioxane 6. Acetone 7. Butanol 8. Acetonitrile 9. Dimethyl Sulfoxide 10. 2, 5-Hexanediol 11. Methanol 12. 1,3-Butyrolacetone, and many more such as: 13. 1,4-butane diol
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McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press.
Top twelve from: Alex McPherson
02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
Histogram of those MPD concentrations yielding macromolecular crystals.
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McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press. McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press.
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Vapor Diffusion
By far the most popular method
Step1. Mix (un)equal small volumes of protein solution and precipitant solution Step2. Let the mixture equilibrate through the vapor phase against a significantly (but not ridiculously) larger volume of the precipitant solution the reservoir
Typical volumes for the protein drop are 0.3+0.3 to 5+5 microliter Typical reservoir volume 0.1 to 2 milliliter Typical protein concentration 10 mgs/ml Several variant techniques:
Histogram showing the relative successful used of the various methods for macromolecular crystallization. Currently, vapor diffusion methods are far more popular than any other approach. 27
McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press.
Sitting drop Hanging drop
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Vapor diffusion - Hanging drop variant
Vapor diffusion - Sitting drop variant
Volumes in the hanging drop typically: 1 L protein solution plus 1 L reservoir solution but that can vary up and down by factor up to 10, and the ratio need not to be 1:1.
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This is a nice slide but in actual practice it is hardly every done this way any more (see a more up to date slide a little later). But for mass production of the same crystals it might be (re)considered perhaps??
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02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
Vapor diffusion sitting drop variant
A simplified view of the principle of vapor diffusion. Water molecules travel thru space until equilibrium is reached. But in precise terms, thermodynamic equilibrium is reached when the thermodynamic potential of water, H2O, is the same throughout the system.
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McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press. McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press.
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Micro-batch
Is a very simple procedure:
Step 1. Add protein solution to the reservoir Step 2. Add the precipitant solution to the reservoir Step 3. Seal reservoir.
Microbatch-under-oil
Procedure in outline:
Step 1: pipette oil into a well Step 2: pipette protein solution under oil Step 3 : pipette ppt solution under oil Step 4 : centrifuge plate for good mixing (not always)
Typical volumes for the protein drop are 0.1+0.1 to maybe even 5+5 microliter Typical protein concentration 10 mgs/ml A variant gaining popularity:
Micro-batch-under-oil
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Its main advantage is that it lends itself nicely to robotization with the small volumes under oil not subject to rapid evaporation When using water-permeable oils one has an extra effect: the concentration of protein and ppt is increasing gradually over time
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Microbatch Under Oil Crystallization Screening in a 1536 Well Microassay Plate
Free-interface Diffusion
More recently the free-interface diffusion procedure is getting new attention
Step 1: deliver protein to a reservoir usually cylindrically in shape such as e.g. a capillary Step 2: deliver precipitate solution to reservoir
Its main advantage is that it allows different positions in the reservoir to have different changes in protein and ppt concentration over time i.e. different rates of nucleation are explored When using water-permeable container material one has an extra effect: the concentration of protein and ppt is increasing gradually over time
35 From : George DeTitta and co-workers HWMRI, Buffalo. 36
02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
Free-interface Diffusion in Capillaries
Free-interface and Vapor Diffusion Combined
Note: Volumes can be smaller than below
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Photographs of a Nalgene 870 PFA capillary containing crystals. (a) A tube showing a combination of free-interface liquid-liquid and vapor diffusion crystallization techniques. On the left is 60 l of a precipitant solution containing polyethylene glycol in phosphate buffer, while separately on the right is a droplet of precipitant and protein solutions (5l each) in contact with each other.
If placed vertically, it makes sense to have the layer with greatest density being the lowest layer. Three, or more, layers are also possible
1= after adding the volumes to capillary 2= after making layers touch often by (mild) centrifugation 3= after crystals grow - often by magic. 37 (Often NOT at interface)
(b) A close-up view of the crystallization droplet that contains yellow crystals of dihydrolipoamide dehydrogenase. One of the characteristics of crystallization using this technique in these tubes is the initial formation of crystals specifically on the meniscus rather than attached to the tube wall.
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Use of plastic capillaries for macromolecular crystallization, Potter, R., Hong, Y.S. and Ciszak, E.M., J. Appl. Cryst. 37: 500-501 (2004)
Free-interface Diffusion and Microfluidics
Free-interface Diffusion and Microfluidics
(E) Prototype protein crystallization chip 144 parallel reaction chambers
A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion Hansen, 39 PNAS (2002) 99: 1653116536.
(A) Section of a device showing three pairs of compound reaction chambers. Control channels are filled with 20 mM Orange G (Aldrich). Buried control channels of the elastomer chip are separated from openbottom flow recesses by a 15-m elastomer membrane. Hydraulic actuation of the control channel deflects the membrane and pinches off the flow line, creating a fluidic seal. Containment valves (Upper and Lower) allow isolation of compound wells during incubation. (Scale bars, 1 mm.)
A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion Hansen, 40 PNAS (2002) 99: 1653116536.
Free-interface Diffusion and Microfluidics
A COMMENT REGARDING ALL METHODS
Finally, with regard the purity of a protein sample, never trust anyone*. No matter who the source, analyze and the protein sample yourself to insure that it is not so obviously heterogeneous to begin with that your efforts will be hopeless. Be skeptical of claims of purity unless you have seen the evidence yourself.
From: Alex McPherson
(B) Loading of reagents using pressurized outgas priming method. The interface valve (Center) is actuated, and reagents are loaded into adjacent sides of compound wells. (Lower) Wells are being deadend- loaded with water. (Upper) Wells have been loaded with 13mMbromophenol blue sodium salt (Aldrich). (C) A gradient of dye concentration. The containment valves (Upper and Lower) isolate compound wells, and the interface valve is released to allow diffusive mixing. The image shows complete mixing after 2 h. (Scale bars, 1 mm.)
A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion Hansen, 41 PNAS (2002) 99: 1653116536.
42 *including yourself
02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
Vapor Diffusion
Walking through Phase Diagrams
Precipitant Concentration
Different Crystallization Methods
Precipitate
Nucleation Zone
Clear Drop
Protein Concentration
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Each Individual Well a Unique Vapor-Pressure End Point
Classical Micro-Batch Under Oil (Non-permeable oil and trays assumed)
Modified Micro-batch Under Oil
(Water-permeable oil)
Precipitant Concentration
Precipitant Concentration
Precipitate
Precipitate
Nucleation Zone Clear Drop
Nucleation Zone Clear Drop
Protein Concentration
Protein Concentration
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Each well a fatal and dry End Point
Microbatch Under Oil
(with Defined End Point)
Free Interface Diffusion
(impermeable reservoir material)
Precipitate
Precipitant Concentration
Precipitant Concentration
Precipitate
Nucleation Zone Clear Drop
Nucleation Zone Clear Drop
Protein Concentration
Protein Concentration
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In Vapor-Batch trays each well same vapor pressure end point 47
Equal Start volumes assumed
02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
Free Interface Diffusion
(permeable reservoir material : no defined end point) ( or air gap between two solutions in capillary: defined end point)
Special methods
Dialysis
Has the great that advantage the same protein solution can in principle be subjected to many different precipitant solutions in particular when the crystals grown can be readily dissolved again Is quite labor-intensive to set up
Precipitant Concentration
Precipitate
Nucleation Zone Clear Drop
Protein Concentration
Changing pH Epitaxial Growth
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Equal Starting volumes assumed
Dialysis
Advantages:
Tremendous control over the composition of the precipitant solution; Precipitant solution easy to change any time, if desired.
Dialysis
Thinwalled capillary Protein solution
Protein solution
Protein solution spun down
Precipitant solution
Disadvantages:
Quite cumbersome to set up and to inspect Real small volumes not easy.
Membrane
Plastic Ring
Precipitant solution
Membrane
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Small volume Dialysis in thin-walled glass capillaries If crystals grow can go straight into X-ray Beam (at Room temp)
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Dialysis
Altering the pH by diffusing in a volatile buffer though vapor phase
Popular with electron microscopy experts to grow 2D crystals in the presence of lipids.
Vapor diffusion of a presumed volatile base from the dessicator reservoir into the capillaries with protein Reservoir with aminoethanol in ethanol-buffer mixture
Ethanol Protein solution
Microdialysis buttons (Zeppezauer)
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McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press.
Used for the crystallization of papain, a plant sulfhydryl protease, by Jan Drenth et al. in 60s.
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02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
EPITAXIAL GROWTH
EPITAXIAL GROWTH
Often thought of as a possible way to initiate nucleation The surface, however, has to match the repeat distances of at least one surface of the (unknown) protein crystal The surface also has to have the proper physical chemical and properties to induce nucleation of the protein
Protein crystals
Mineral for nucleation
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From a paper by Alex McPherson in either Nature or The Scientific American, or both
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Crystallization: a multi-step process
Step 1 : Initial Screening Determine lead conditions for crystallization Using a coarse screen also called random screens Step 2 : Optimization of hits Optimize lead conditions to produce diffraction quality crystals by optimization matrices around initial hits
Temperature Variation
A subtle way to obtain (much better) crystals (Even 14 C can give different crystals than 20 C)
Actually : Often many optimization-generations needed
Room Temperature
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Cold Room Temperature
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Lori Anderson & SGPP
SEEDING
A splendid way to obtain (much better) crystals form poor initial crystals
MICROSEEDING
As seeds can serve:
(Way) too small crystals Crushed larger crystals Mutant protein crystals SulMet crystals for SelMet protein, and vice versa
Various seeding methods
Micro-seeding - such as Streak seeding usually with a particular hair from a particular domesticated animal - often with a range of dilutions from the solution with seeds Macro-seeding - Clean and grow again - partial dissolving of crystals to clean the surface, then increase protein concentration
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McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press.
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02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
STREAK SEEDING
MACROSEEDING
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McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press. McPherson, A. (1999). Crystallization of Biological Macromolecules, Cold Spring Harbor Laboratory Press.
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MACROSEEDING
MACROSEEDING
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Lipoamide dehydrogenase Bram Schierbeek
Lipoamide dehydrogenase Bram Schierbeek
MACROSEEDING
ADDITIVES
Another way to obtain better crystals form poor initial crystals
The idea is that small-molecule ligands either:
stabilize the protein which usually means a significantly increased probability of obtaining crystals (the so-called freezers) assist in forming crystal contacts (the so-called glues)
(Sometimes the same small molecule can serve as freezer and as glue) (Sometimes additives are also called co-crystallants)
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Lipoamide dehydrogenase Bram Schierbeek
02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
ADDITIVES
Frequently used additives:
Substrates and products Substrate fragments
e.g. ADP for an NAD-binding enzyme
Co-crystallants and Crystallization
Pfal008421WES Native Pfal008421WES Native plus Acyclovir
Metals Any Ligand or Inhibitor Detergents like -octylglucoside Heavy-atom compounds
HAC-native gels can be useful
Any compound!
(There are special additive screens on the market)
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0.1 M Ammonium Sulfate 0.1 MOPS pH 6.5 25% PEG 3350 Drop size 1ul + 1ul
0.2 M Ammonium Sulfate 0.1 M HEPES pH 7.5 25% PEG 3350 2.1mM Acyclovir Drop Size 0.4ul + 0.4ul
UNMOUNTABLE
MOUNTABLE
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The effect of a known inhibitor
Specific Synthetic Ligands Change Crystals
(but in this remarkable case left cell dimensions the same!)
LT B-pentamers & MDT crystal contacts mediated by seven co-crystallant molecules.
Native ASU
Cyclosporin Co-crystal - SAME(!!)ASU
Hovey, B., Verlinde, C. L. M. J., Merritt, E. A. & Hol, W. G. J. (1999). Structure-based discovery of a pore-binding ligand: Towards assembly inhibitors for cholera and related AB5 toxins. J. Mol. Biol. 285, 1169-1178.
Tcru 13382 A cyclophylin homolog : without & with Cyclosporin 69
Jonathan Caruthers & SGPP
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Do Not Underestimate E.coli !
6-pyruvoyl-tetrahydropterin synthase (PTPS)
PFAL004546 from SGPP
Surprisingly frequently heterologous expression of proteins into E coli results in ligands bound to the protein of interest! Metal ions, like zinc, are frequent donations from E. coli. In some cases the donated ligand can even be labeled with selenium!
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Human homolog Subunit size Resolution Structure solution Structure
: 14% & 28 % identity : 173 residues :2 : MAD : 3 subs/ASU, biological hexamer
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SGPP & Juergen Bosch
02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
6-pyruvoyl-tetrahydropterin synthase (PTPS)
PFAL004546 from SGPP
Putative Methyl Transferase
LMAJ004091AAA from SGPP
Monomer
6-pyruvoyl-tetrahydropterin in 1.5 density (magenta = Zn2+)
Human homolog Subunit size Structure solution Resolution Structure Cofactor: : 36 % (81/224) : 250 : MAD : 1.9 : 1 subunit / ASU : S-Adenosyl-homocysteine
73 Zn and 6-pyruvoyl-tetrahydropterin at active site
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SGPP & Mark Robien
Putative Methyl Transferase
LMAJ004091AAA from SGPP
Anomalous Difference Fourier contour levels: showing without ambiguity that this is a Selenium atom
ROBOTICS
Advantages:
Less human errors Easier record keeping (Very) easy repeat use of protocols Frees scientists for the challenging tasks Smaller volumes possible
Some types of crystallization methods lend themselves well for robotics:
The sitting drop method The microbatch-under oil procedure
A bit more cumbersome is:
The hanging drop method
Not only the initial set-up needs to be roboticized, but also the imaging of the many drops to quickly see if crystals have appeared
S(e)-adenosyl-Selenohomocysteine..
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Also Preparation of Solutions can benefit greatly from Robotics
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The HTP Search Lab in Buffalo
a b
SGPP Crystal Optimization Robots
a. The Robbins Scientific Hydra 384 pipetting robot b. A 1536 well Greiner plate
c
c. The HTP robotic photo stand d. Macroscope thumbnail photos
Refined Optimization Matrices Made by Hand
Hydra II Plus 1
Acapella
e d
e. An enlargement of an interesting thumbnail, showing information about the crystallization cocktail.
Crystal Track Designs Matrices
RoboDesign
Crystallographer Review Manual Scoring Database and Image Archive
MicroScope II
Harvestable Crystals
Initial Screening for Leads
(George DeTitta, February 2001)
77 Structure Determination Units
78 Larry DeSoto & SGPP
02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
How to make the most of your crystals??
A protein crystal can be VERY precious in particular when only a few could be obtained! Procedures to obtain the most diffraction and phasing data out of your crystals:
Cryo-protect Anneal Dehydrate Expose different pieces of the same crystal - in particular on socalled micro focus synchrotron beams Soak in Heavy-Metal Compounds, even repeatedly Save exposed crystals for SEEDING
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CRYO PROTECTION
Protein crystals suffer at room temperature from serious X-ray radiation damage Cryo-cooling the crystals to 100 K is a way to dramatically reduce the radiation damage The cooling process needs to avoid the formation of ice crystals in the protein crystal since these destroy the order of the protein molecules in the protein crystal Some mother liquors are amenable to cryo-cooling without any additions many others need additives to save the crystals during the cooling process
Hope, H. Acta Crystallogr B. (1988) 44:22-26. Hope, H. et al, Acta Crystallogr B. (1989) 45: 190-199. Rodgers, D., Structure (1994) 2: 1135-1140.
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CRYO PROTECTION
CRYO PROTECTION
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CRYSTAL ANNEALING
Variations:
Flash Annealing
Yeh & Hol, Acta Cryst. (1998) D54, 479-480.
FLASH ANNEALING
In its simplest form : simply block the liquid nitrogen stream for around 30 seconds This allows the crystal to warm up Maybe the crystalline mosaic can reshuffle and reorder in this process The crystal attracts/looses water during this process so that changes in water content of the crystal might also be a factor
Procedure independently invented in many labs first publication seems to be: Yeh, J. I. & Hol, W. G. J. (1998). A flash annealing technique to improve diffraction limits and lower mosaicity in crystals of glycerol kinase. Acta Cryst. D54, 479-480.
New Solution Annealing
Harp et al. Acta Cryst. (1998). D54, 622-62
Dehydration (& Rehydration) Controlled Humidity Changes at Room Temperature
Huber group
General Ref:
Harp, J. et al: Acta Cryst. (1999). D55, 1329-334: Macromolecular crystal annealing: evaluation of techniques and variables.
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02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
FLASH ANNEALING
FLASH ANNEALING
Glycerol Kinase Diffraction pattern - Prior to Flash Annealing
85 Joanne Yeh
Joanne Yeh
..and after annealing
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FLASH ANNEALING
Literature examples: post-crystallization treatments can improve diffraction quality and resolution
Dehydration, cryocooling, annealing, rehydration Sometimes only crucial improvement in mosaicity Sometimes only minimal resolution improvement by 0.3 Sometimes real significant resolution improvement by 1.5 Up to 8 resolution gain reported..
Joanne Yeh
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The Power of Flash-Annealing
DsbG: 10>2
Initial diffraction: 10 , streaky spots
Tried stepwise equilibration into cryo, annealing. ~90% solvent
Before
EF-Tu-Ts
Complex of 2 elongation factors from E. coli (EF-Tu is a guanine-nt-binding Initial diffraction: 5 , 61% solvent Dehydration technique:
protein, EF-Ts is a guanine-nt-exchange protein)
Dehydration method
Transfer crystal from crystallization drop (20% PEG 4K) to 5l hanging drop of dehydrating solution (30% PEG 4K), equilibrated against 1 ml dehydrating solution overnight at 4C
Treated crystals were more robust than untreated crystals
After
Equilibrated in 2 steps (10 min each) against dehydrating solution + 15% and 25% glycerol
Over 24 steps, exchange ML (20% PEG4000 + 90 mM (NH4)2SO4) for cryo solutions with more concentrated PEGs and no (NH4)2SO4 Crystals first developed cracks parallel to one crystal axis; cracks disappeared after 21st transfer step
New diffraction: 2 , no more streaks! 53% solvent.
Heras, et al Structure 11: 139-145 (2003?)
New diffraction: 2.7, 55% solvent
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02 BSTR521 2007 - Crystal Growth, Freezing, Annealing - Wim Hol
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Eukaryotic transcription factor mediating immune response in mammals; important in ex...