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Protein crystallization

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An essay on several aspects of protein crystallization research

Lysozyme

Mirjam Leunissen Department of solid state chemistry October 2001















Abstract

This essay is about the crystallization of proteins. Since the first published observation of crystallizing proteins about 160 years ago, protein crystal growth has developed into an extensive research field with many applications, for instance in the pharmaceutical industry. In this report, an overview of the unique physical-chemical characteristics of protein molecules, their properties in solution and their properties in the crystalline state, together with a description of some important aspects of protein crystallization and research in this f ield, is given. Seven proteins that are the main models of crystallization studies are presented, just as the general (thermodynamic) principles applying to protein crystal growth and the main parameters influencing the process. The most commonly used protein crystallization techniques are described, together with their underlying principles and the approaches used to find suitable conditions for crystallization of a particular protein. Focusing on the fundamental studies of protein crystallization on a molecular level, the current knowledge of nucleation processes, crystal growth mechanisms and kinetics is summarized. Concluding this essay, an inventory is made of knowledge that is still missing, but that is crucial in order to develop a more directed crystallization approach. Suggestions for future research, aimed at obtaining this insight, are given.















Contents Abstract Chapter 1

Introduction

1.1 The importance of protein crystal growth

1

1.2 A history of protein crystallization

2

1.3 Aspects of protein crystallization research

4

Chapter 2

Features and properties of proteins

2.1 Protein structure

6

2.2 Properties of proteins in solution

7

2.3 Properties of protein crystals

8

2.4 The complexity of protein crystallization

9

2.5 Protein model systems

9

Chapter 3

Principles of protein crystallization

3.1 The thermodynamics of crystal growth

13

3.2 Parameters influencing protein crystal growth

15

Chapter 4

Methods and approaches in protein crystallization crystallization

4.1 Protein crystallization methods

18

4.2 Approaches to find crystallization conditions

25

Chapter 5

Nucleation and growth processes of protein crystals

5.1 Methods of investigation

28

5.2 Formation of critical nuclei

28















Chapter 1 Introduction

This essay deals with the crystallization of the members of one particular, but very broad, class of biological macromolecules: the proteins. Protein crystallization forms a very extensive field of research, with many different aspects and applications. As you will learn in this essay, it is in some respects rather different from ‘conventional’ crystallization of inorganic, small molecule compounds, for instance where the crystallization techniques are concerned, while in other respects they have many characteristics in common. An example of such similarity is formed by the crystal growth mechanisms. In order to get a bit acquainted with the subject, in this introductory chapter, I will first tell something about the importance and applications of protein crystallization and the development of the field of protein crystal growth from its starting point until now. Furthermore, focusing on the research component of the protein crystal growth field, I will indicate its many different aspects and which of them will be treated in this essay.

1.1

The importance of protein crystal growth

So, question is: why would anybody want to spent (sometimes huge) efforts on the crystallization of proteins? While, in the past, purification of mixtures was the main goal in crystallizing experiments, at present the main answer to the question stated above is provided by the fact that studies of the atomic structure of biological macromolecules (i.e. proteins, DNA, RNA, etc.) have proved of great value in revealing structure/function relationships that are of importance in our understanding of how enzymes, nucleic acids and other macromolecules operate in biological systems. Recently, nuclear magnetic resonance (NMR) studies of protein solutions have yielded atomic structure information for some small proteins. However, for proteins with molecular weights in excess of 20,000 Da (Da=dalton=1.66x10 -27 kg, this unit of mass is specifically applied to proteins) only X-ray and neutron diffraction techniques can provide structure information to atomic resolution. Such studies require single crystals crystals of high structural perfection and with minimum dimension of several hundred micron. But, one may ask, is the amount of information obtained really worth the effort, time and money















The elaborate information that can be obtained from the three-dimensional structure of a protein is useful in a variety of ways. From the basic biological view point, this information underlies our understanding of the mechanisms by which enzymes, receptors, hormones, etc. function in biological systems. Within the pharmaceutical industry, protein structure information can be helpful in the development of novel drugs. Since many pharmaceutical agents act by interacting with proteins, knowledge of the three-dimensional structure of a target protein can be used to design compounds that selectively bind to sites of this protein and thereby inhibit its activities. Another highly promising application of protein crystallography is in protein engineering. Using molecular biology techniques, investigators can specifically alter protein molecules by site-directed mutagenesis (i.e. by altering proteins in specific, selected regions by altering the DNA stretch encoding that part of the protein). In general, the most promising approaches to protein engineering depend upon detailed structural information about the proteins of interest. An additional use of protein crystallography is in the design of synthetic vaccines. Several recent studies have indicated that effective vaccines might be made from synthetic peptides (very small proteins) that are representative of protein segments found on the surfaces of target proteins. Protein crystallography provides one of the most effective techniques for locating those peptides. However, the crystallization of proteins is not only an excellent tool to obtain information about the three-dimensional protein structure, but also a very interesting subject for crystal growth studies in its own right. Compared to ‘conventional’, small molecule (often salts) crystal growth systems, proteins display relatively slow growth kinetics and have growth units with a large size (because individual protein molecules are very large). These facts allow one to more readily study crystal growth mechanisms, par example by means of in situ situ atomic force microscopy observations. Therefore, protein crystallization now serves as the best model system for general crystallization from solution. Concluding, one can say that there are two interests: a general interest in delineating processes that play a role in crystallization from solutions and an interest in the crystallization of certain proteins in particular, in order to obtain structural information about these proteins. These two interests do not stand on their own, however. As many proteins tend to resist their easy crystallization, more insight in the processes at work during crystal formation might enable a more directed approach with a higher chance of success. I will come back to this in the concluding chapter of this essay.

1.2

A history of protein crystallization crystallization

This section is based on information in [1] and references therein. In table 1.1 (page 3) a chronology of protein crystal growth is given. The first protein crystals The history of (recorded) protein crystal growth started about 160 years ago. The first published observation of the crystallization of a protein appears to be by Hünefeld in 1840. The protein, hemoglobin from the earthworm, was obtained as flat plate-like crystals when the worm’s blood was pressed between two slides of glass and allowed to dry very slowly. This observation clearly stated that protein crystals can be produced by the controlled evaporation of a concentrated

















successful and reproducible methods for the growth of hemoglobin crystals was Fünke, who published a series of articles on the purposeful growth of hemoglobin crystals. Following hemoglobin, the next class of proteins to be investigated, in the period from 1850 to about 1900, were the plant seed reserve proteins, principally the so c alled ‘globulins’. The methods that were developed to crystallize these proteins included: extraction of proteins into salt solutions followed by slow cooling, dialysis of a salt solution extract of the seeds exhaustively against distilled water and treatment of protein solutions with alcohol, acetone or ether. In these procedures we find for the first time the exploitation of several approaches now in common use: temperature variation under constant solution conditions, dialysis against low ionic strength solutions (to take advantage of the low solubility of many proteins at low ambient salt concentrations) and the use of organic solvents as ‘precipitating’ or ‘crystallizing agents’. At almost the same time the work with plant seed proteins was carried out, similar efforts were done to crystallize the proteins hen egg albumin and horse serum albumin. The procedures for their crystallization used many of the suggestions of Hofmeister regarding the ‘salting out’ of proteins by high concentrations of salt ions and the precipitation of proteins by careful regulation of pH (for an explanation, see section 3.2). The first enzyme, urease, was crystallized in 1925 by Sumner and at almost the same time the first hormone, insulin, was crystallized too. Crucial to the insulin crystallization was the addition of divalent zinc ions. This was one of the first examples of crystal growth promoted by the addition of metal ions.

Table 1.1 A chronology of protein crystal growth. Source: growth. Source: [1].

















A change of attitude Until the 1930s (but also beyond that for many years), the rationale for crystallizing proteins, particularly enzymes, was to supply a technique for purifying a specific protein from a complex extract, or to demonstrate the purity of a preparation. In the late 1930s, however, certain X-ray diffractionists began to turn their attention to protein crystals as a source of structural information about biological macromolecules for reasons stated in the previous section. This interest carries on till present time. The interest of X-ray diffractionists was influential in promoting efforts to reproducibly grow high quality protein crystals, but also led to efforts to increase success rates and to automate the crystallization process. The background for the latter is formed by the fact that with the extraordinary advances in data collection and computing techniques and with the revolution in pharmacology and biotechnology, the ask for new macromolecular crystals very soon greatly surpassed their supply. At present the bottleneck in solving the problem of limited crystallization yields is mainly formed by a lack of insight. As you will see in this essay, most protein crystallization approaches are based on the ‘trial-and-error’ principle, while insight in the fundamental processes at work is very restricted. As a consequence, many proteins still resist crystallization for unknown reasons. Clearly, at present, the challenge is to obtain enough knowledge about the processes at work and, based on this knowledge, to develop new, more directed methods in order to be able to readily crystallize any protein at will.

1.3

Aspects of protein crystallization research

There are many areas of research related to protein crystallization, each focusing on different aspects. In this essay I will treat only a limited number of them. Chapter 2 provides an introduction to the subject common to all of the studies: protein molecules. The general physical-chemical characteristics of protein molecules, their properties in solution and their properties in the crystalline state are dealt with. From this information, the answer to the question why the crystallization of proteins is very complex will be extracted. Furthermore, in this chapter, the seven proteins that are the main models of crystallization studies are presented. In chapter 3, I will treat the general (thermodynamic) principles applying to (protein) crystallization and explain which parameters play an important role in the crystallization processes and how they exert their influences. Chapter 4 is an overview of modern protein crystallization techniques, their respective advantages and/or disadvantages and the underlying thermodynamic principles.

















if you are new to the field of protein crystallization I would highly recommend it. Other interesting references are: [2] and [3], if you are interested in protein molecular interactions in solutions and [4] for interactions in the crystalline state. For a general theoretical introduction to the problem of protein nucleation, see [4], [5], [6], [7], [8]. A comprehensive list of references on impurity effects and defect formation is provided in [9], but lots of information on these topics can also be found in [10] and [11]. A nice review of theoretical models that are used to study protein crystal formation and the major ideas used in their development is presented in [ 5]. Undoubtedly, the information in this essay and in the references given above will not cover the whole field of protein crystallization, but it will provide a good starting point for anybody interested in this extensive field.

















Chapter 2 Features and properties of proteins 2.1

Protein structure

Proteins are so called macromolecules, because the particle diameter is ~30-100 Å as compared to ~3 Å for most inorganic particles. Proteins can be considered as polymers of amino acids, linked together in a chain-like arrangement. The number of amino acids constituting one protein molecule ranges approximately from 100 to 27,000 for the proteins known. The specific sequence of amino acids in a protein is called its ‘primary structure’. a

R1

R2

H3N C COO COO-

+

+

H3N C COO COO-

+

H

H

R2

R1 O H3N C

+

C N C COO COOH H

H

b

+ H2O

Ri

O

H (NH CH C)n OH Fig. 2.1 Primary structure of proteins. a) Formation of a peptide bond between two amino acids that differ in their side chains (R1, R2). The carbon atom in the middle of each amino acid structure is called ‘Cα’. b) Overall formula for a polypeptide chain (backbone) with side chains Ri. Adapted from: [12].

Natural proteins are built up by multiple numbers of twenty different amino acids, sometimes referred to as ‘residues’. The general structure of an amino acid can be seen in figure 2.1a. In each amino acid, the amino and carboxyl groups make one bond each with the so called α-carbon atom, Cα. One of the other two bonds of Cα is occupied with hydrogen, while the other is occupied with a relatively small group, called the ‘side chain’, which is different for each of the twenty amino acids. These side chains consist of simple hydrocarbon groups, that may contain aromatic rings, nitrogen, oxygen and sulfur (figure 2.2). The so called ‘peptide bonds’ between the amino and carboxyl groups of neighboring amino acids (figure 2.1a) form the backbone along the polypeptide chain. The side chains form its short branches (figure 2.1b).

















figure 2.2). In their natural aqueous environment, the polar groups prefer to associate with water. In contrast to these hydrophilic groups, the nonpolar hydrophobic groups prefer to associate with themselves. As a result of these preferences the molecule lowers its free energy by folding to a specific three-dimensional structure, the so called ‘tertiary structure’. The pictures in section 2.5 provide many examples of this overall three-dimensional structure of proteins. Molecules with comparable numbers of polar and nonpolar residues fold into globular shapes in which the hydrophilic residues tend to concentrate on the surface and the hydrophobic groups in the core. This three-dimensional three-dimensional structure is stabilized by various close-range interactions between atoms that are distant on the backbone. These include electrostatic interactions between charges and dipoles, van der Waals forces, hydrogen bonds, hydrophobic interactions and (covalent) disulfide bonds. The three-dimensional structure described above represents the final folding of a polypeptide chain, but some proteins display another level of organization, the so called ‘quaternary structure’. This refers to the fact that many functional proteins are complexes of smaller subunits. Each subunit is a polypeptide chain with its own specific tertiary, three-dimensional structure. To form the final functional protein a number of subunits associate to one big complex by means of noncovalent interactions. This arrangement of subunits is called the quaternary structure of a protein. A very nice example of the quaternary quaternary structure of a protein protein is displayed in figure figure 2.9 (page 11). 11).

2.2

Properties of proteins in solution

The properties of a protein are to a great extent determined by the amino acid side chains present on its surface. Within the group of polar residues there are two classes of charged amino acids: acidic and basic. At neutral pH, which centers the normal physiological range, the acidic amino acids are negatively charged and the basic amino acids carry a positive charge. Therefore, at pH ~6-7 and temperatures 0-40°C, typical of biological conditions, the molecular surface usually is charged. Like all polar side chains, the charged groups interact extensively with water and tend to solvate the protein. Moreover, exposed hydrophilic groups on macromolecules can bind, both transiently and in a stable manner, not only water molecules but also a variety of ions, both cations and anions. Thus, as a consequence of their polyionic character, the surfaces of macromolecules display a variegated pattern of positive and negative charge. As an example, the charge distribution on the surface of an arbitrary protein is visualized in figure 2.3.



















At the iso-electric point, pI, (at pH=11.3 for lysozyme [14], between 5 and 7 for most other proteins) there are equal numbers of positively and negatively charged residues and as a consequence at this pH value the net surface charge vanishes. However, one must realize that this does not mean that individual surface patches do not carry a charge. The net surface charge of a protein molecule changes with the pH of the solution and consequently, as one can imagine, the solubility of the protein changes with the pH too. For a particular protein, with its own specific sequence of amino acid side chains, the solubility dependence on the solution pH is given by the following equation [15]: [ P ]

[ P ]

[ P i ] i

[ P j ] K [1 j

( K i /[ H ]γ i )

γ i

[P] = the total concentration of dissolved molecules [P±] = the zwitterionic state of the protein molecule [Pi] = negatively charged states of the protein molecule [P j] = positively charged states of the protein molecule

([ H ] / K jγ j )] exp(

γ

α 0 s 0 / kT )

j

= the surface free energy energy of a protein molecule in solution s0 = the surface area of a protein molecule in solution γ = activity coefficient Kx = (de)protonation (de)protonation reaction constant side chain

α0

According to this formula, solubility reaches a minimum at proton concentrations corresponding to the isoelectric point and steeply increases at both lower and higher pH. The temperature dependence of solubility is determined by the temperature dependence of the protonation and deprotonation reaction constants of the side chains. For proteins, which exist predominantly in an aqueous environment, one free energy minimum is represented when they are fully solvated, but in extremely concentrated solutions where there is insufficient water to maintain hydration, the molecules may aggregate as an amorphous precipitate or they may crystallize. Let us therefore consider some properties of protein crystals.



















Protein-protein contacts in crystals are complex, involving a delicate balance of specific and nonspecific, intrinsically flexible interactions [5], [20]. Hydrogen bonding and electrostatic interactions, involving the participation of flexible amino acid side-chains on the protein’s surface, together with numerous solvent molecules or ions that are immobilized between molecules during crystal lattice formation, are examples of specific interactions. Nonspecific interactions are van der Waals interactions and hydrophobic interactions. It is not clear from the literature whether any particular type of interaction makes a dominant contribution to the energy of protein-protein contacts in crystals. It appears that interactions between proteins represent a delicate balance of many contributions whose relative importance varies from case to case.

2.4

The complexity of protein crystallization

In general, the crystallization of proteins is a very complex process. Experiences of many investigators point out that most proteins are difficult to crystallize and even if a protein tends to crystallize relatively easily there are many parameters that must be t aken into account. From the preceding sections one can identify multiple reasons for the difficulty of protein crystal growth. Apparently, protein molecules are very complex (large, flexible molecules often composed of several subunits), relatively chemically and physically instable (unfolding, hydration requirements, temperature sensitivity) and they have dynamic properties. If the solution changes, the molecule properties (e.g. conformation, charge and size) change too. Furthermore, every macromolecule is unique in its physical and chemical properties because every amino acid sequence produces a unique three-dimensional structure having distinctive surface characteristics. Thus, lessons learned by investigation of one protein only marginally apply to others. For a crystal to form, interactions between protein molecules must be suitable in their geometric arrangement, degree of specificity and strength. The size and complexity of proteins is reflected in



















dimensions and angles. Information on other proteins and their crystal structures can be found in the ‘Protein Data Bank’ [21]. This is also the source of all pictures in this section.

Table 2.1 Crystal structures of frequently used model p roteins. Only angles ≠ 90° are denoted. Space a b c α β γ

Protein

Lysozyme

Canavalin

Concanavalin A

Thaumatin

(Apo-)ferritin

group

[Å]

[Å]

[Å]

[deg]

[deg]

[deg]

P1 P21 P212121 P6122 P43212 P63 P213 R3 C2221 I222 P21212 P212121 C2221 P1 P212121 C2 P41212 F432 P4212

27.28 28.00 59.40 87.01 79.10 126.35 105.99 136.80 136.50 88.70 61.36 71.29 118.70 78.80 44.30 117.70 58.60 182.90 147.23

31.98 62.29 68.70 87.01 79.10 126.35 105.99 136.80 150.30 86.50 85.46 71.65 101.38 79.30 63.70 44.90 58.60 182.90 147.23

34.29 60.50 30.80 70.4 37.90 51.64 105.99 75.70 133.40 62.50 91.46 190.96 111.97 133.30 72.70 38.00 151.80 182.90 152.58

88.5

108.6 90.8

111.8

120 120 120

97.10

90.20 94.00

97.50



















a

Canavalin

b

Canavalin is the major reserve protein of the seeds of the jack bean. It is composed of three identical subunits, each with M R ~47,000 Da, arranged about about a perfect threefold axis. axis. Each of the subunits consists of two chains, called A and B (figure 2.6). References: the crystallization of canavalin was first published by Sumner and Howell [32]. More information about its crystallization can be found in [33] and [34]. Studies of the solubility of canavalin are [23] and [35].

Fig. 2.6 Canavalin. a) Ribbon model displaying displaying the rather complex quaterquaternary structure of canavalin. The protein is a complex of three subunits, densely packed together. b) Each of the subunits is composed of two chains, called A and B.

Concanavalin A

Concanavalin A is a derivative of the enzyme chitinase and is, just as canavalin, obtained from the jack bean. The protein consists of two chains (A and B) of 237 amino acids each ( and MR ~25550 Da). Figure 2.7 is a ribbon model of this protein. References: the crystallization of concanavalin A was first published by Sumner [36]. A later investigation of the protein’s structure can be found in [37]. Solubility information was published by Mikol and Giegé [38]. Fig. 2.7 Concanavalin A. Ribbon model displaying the folded structure of the complex of two chains that form concanavalin A. Pronounced secondary

Fig. 2.8 Thaumatin. Ribbon model. This relatively simple



















Insulin

Insulin is a hormone from the pancreas. It consists of two chains (A and B) of respectively 21 and 30 amino acids (figure 2.10a). These chains are linked together by two (covalent) disulfide bonds. The protein’s molecular weight is ~ 7,300 Da. Insulin molecules have a tendency to form dimers in solution due to hydrogen-bonding between the B chains. One such dimer is depicted in figure 2.10b. In the presence of zinc ions insulin may even form hexamers, as displayed in figure 2.10c. References: the crystallization of insulin was first published by Abel et al. in [43]. Further investigations on crystalline insulin are presented in [44] and [45].

a

a)

b)

c)

Fig. 2.10 Insulin. Ribbon model. The insulin monomer monomer consists of two chains (A and B) covalently linked together via so called disulfide bonds. In aqueous environment environment insulin tends to form dimers due to hydrogen bonding between the B chains of the monomers. In the presence of divalent divalent zinc ions hexamers are formed. The zinc ions are visible in the structure as grayish balls.

b

c

Catalase

Catalase is a detoxifying, heme-containing enzyme from the liver. It is responsible for the elimination of hydrogen peroxide without formation of free radicals. Catalase consists of 4 identical subunits of 484 residues and 55472 Da each (figure 2.11).





















Chapter 3 Principles of protein crystallization In this chapter, I will introduce the general (thermodynamic) crystal growth principles and parameters influencing crystallization processes. The crystallization methods described in t he next chapter are based on these principles and parameters. For a description of the specific nucleation processes, crystal growth mechanisms and kinetics of proteins I would like to refer to chapter 5.

3.1

The thermodynamics thermodynamics of crystal growth

Crystallization is a complex process, involving multiple equilibria between different states of the crystallizing species [6], [47], [48]. The three stages of crystallization common to all molecules are nucleation, crystal growth and cessation of growth. During nucleation enough molecules associate in three dimensions to form a thermodynamically stable aggregate, the so called critical nucleus. These nuclei provide surfaces suitable for crystal growth, which can occur by a couple of different mechanisms. Crystal growth ceases when the solution is sufficiently depleted of protein molecules,



















Fig. 3.2 Reversible molecular association reactions involved in the assembly of crystals. Monomers initially combine into small aggregates (here, called chains). The association of monomers into chains leads to the formation of prenuclear aggregates that continue to grow by further addition of monomers or chains. When sufficient molecules associate in three dimensions, a thermodynamically stable critical nucleus is formed. The addition of monomers and/or chains to critical nuclei eventually leads to the formation of macroscopic c rystals. Source: [6].

Monomers



















3.2

Parameters Parameters influencing protein crystal growth

A wide variety of physical, chemical and biochemical parameters affect protein crystallization processes, as can be seen in table 3.1. Of course the physical-chemical characteristics of the crystallizing protein, described in chapter 2, are important, just as the purity of the sample, the properties of the solution used for crystallization (e.g. composition, temperature and pH) and the method of sample handling. In the following, I will shed some light on the role of many (mainly physical and chemical) factors, given a specific protein sample (with a particular purity, sequence, modifications etc.). Some factors will also be shortly commented on in section 4.1. An extensive review of the influence of all factors, including the biochemical ones, can be found in references [48] and [50]. Table 3.1 Crystallization parameters. parameters. Physical, chemical and biochemical factors affecting the outcome of a protein crystallization experiment. Adapted from: [48] and [49].

Physical factors

Chemical factors

Biochemical factors

Temperature Methodology

Precipitant type Precipitant concentration

Sample purity Macromolecular Macromolecular impurities



















In section 2.2, it was explained that the temperature dependence of protein solubility is determined by the temperature dependence of the protonation and deprotonation reaction constants of the amino acid side chains in the protein structure [15]. In spite of the fact that most proteins display a clear solubility dependence on temperature, this parameter is not very often used to control supersaturation, but is in the majority of the cases kept constant during the entire experiment. Crystallization has been reported to occur over the entire range from 0 to 40°C and in some cases even 60°C, although it is usually conducted at either 4°C or at room temperature, 25°C. Just as is the case with the pH, extremes in temperature tend to cause denaturation of proteins [1]. Precipitants Protein solubility can also be decreased by changing the composition of the solution, for instance by inclusion of additives such as alcohols, hydrophilic polymers and detergents. These solubilityinfluencing agents are commonly known as ‘precipitants’ [1], [54]. Protein precipitants fall into four broad categories: salts, organic solvents, (long-chain) polymers and non-volatile organic compounds. In table 3.2 the most common precipitants in each of these categories (the last two have been taken together) are enlisted.



















Fig. 3.3 Lysozyme solubility. a) Solubility of lysozyme (mg/ml) as a function of salt concentration, expressed in moles/liter (M) at 18°C and pH 4.5. b) Solubility of lysozyme (mg/ml) as a function of salt concentration, expressed in moles/liter (M) at 18°C and 40°C and at pH 4.5. Lysozyme/NaCl data are given as an internal reference. Source: [1].





















Chapter 4 Methods and approaches in protein crystallization 4.1

Protein crystallization methods

In the previous chapter, many parameters that affect a protein’s solubility and its crystallization



















Fig. 4.1 Protein crystallization methods. Histogram showing the relative successful use of the various methods for macromolecular crystallization. Currently, vapor diffusion methods are far more



















Batch crystallization Very simple too, are ‘batch crystallization methods’ (figure 4.2). All components are directly combined into a single, supersaturated protein solution, which is then left undisturbed. The technique can be miniaturized by immersing protein droplets as small as 1 µl into an inert oil. The oil prevents evaporation evaporation of the sample. This is the so called ‘microbatch’ ‘microbatch’ method [65]. Besides Besides the very limited amounts of sample needed, the latter method has as further advantage that the



















Vapor diffusion

Supersaturation



















Seeding Given the fact that ideal conditions for nucleation and growth differ (i.e. higher supersaturation for nucleation than for growth), a logical crystallization strategy involves the separate optimization of these processes. This can be accomplished by seeding, a technique where crystals are transferred from nucleation conditions to those that will support only growth.

































































































































































































































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