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Archives of Biochemistry and Biophysics 602 (2016) 3e11

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Current trends in protein crystallization

 A. Gavira
cos, IACT (CSIC-UGR), Avda. de las Palmeras, 4. 18100 Armilla, Granada, Spain
Laboratorio de Estudios Cristalogra

a r t i c l e i n f o a b s t r a c t

Article history: Proteins belong to the most complex colloidal system in terms of their physicochemical properties, size
Received 3 November 2015 and conformational-exibility. This complexity contributes to their great sensitivity to any external
Received in revised form change and dictate the uncertainty of crystallization. The need of 3D models to understand their func-
16 December 2015
tionality and interaction mechanisms with other neighbouring (macro)molecules has driven the
Accepted 22 December 2015
tremendous effort put into the eld of crystallography that has also permeated other elds trying to shed
Available online 31 December 2015
some light into reluctant-to-crystallize proteins. This review is aimed at revising protein crystallization
from a regular-laboratory point of view. It is also devoted to highlight the latest developments and
Protein crystallization
achievements to produce, identify and deliver high-quality protein crystals for XFEL, Micro-ED or
Batch neutron diffraction. The low likelihood of protein crystallization is rationalized by considering the
Counter-diffusion intrinsic polypeptide nature (folded state, surface charge, etc) followed by a description of the standard
Vapor-diffusion crystallization methods (batch, vapour diffusion and counter-diffusion), including high throughput ad-
Neutron vances. Other methodologies aimed at determining protein features in solution (NMR, SAS, DLS) or to
XFEL gather structural information from single particles such as Cryo-EM are also discussed. Finally, current
approaches showing the convergence of different structural biology techniques and the cross-
methodologies adaptation to tackle the most difcult problems, are presented.
Synopsis: Current advances in biomacromolecules crystallization, from nano crystals for XFEL and Micro-
ED to large crystals for neutron diffraction, are covered with special emphasis in methodologies appli-
cable at laboratory scale.
2015 Elsevier Inc. All rights reserved.

1. Introduction condition based on sequence similarities [3]. Furthermore, in the

last decades several initiatives have been developed to predict
Even today many crystallization or structural articles regard the protein crystallizability based on the protein sequence as reviewed
process to produce suitable crystals for X-ray diffraction more as an by Sanchez-Puig and co-workers [4] and will be covered further in
art than science. This belief is based on the unpredictability of the this specialized issue.
chemical cocktail and, generally, on the physicochemical conditions Besides the difculties to crystallize macromolecules, the
that may produce the coveted crystal. It is a fact that the crystalli- number of deposited structures at the PDB ( [5]
zation process is still the limiting step to obtain an accurate 3D has increased exponentially over the past few years mainly due
model of the biomacromolecule of interest by means of X-ray to the tremendous effort made by several large scale structural
(neutron) diffraction technique (XRD). Protein crystallization is a initiatives around the world. Indeed, there are currently over
multi-parameter process that is very difcult to predict [1]. The 100,000 PDBs entries. The large centres have adopted a high-
number of parameters affecting the nucleation and growth of a throughput (HT) approach encompassing the full pipeline propel-
well-formed crystal are too high and its prediction entails a degree ling many innovative methods and techniques from the cloning to
of complexity comparable to the simulation of the protein-folding the data collection of the target sequences. Even though the
process. In most cases, when facing a particular crystallization outcome of HT is still proportionally low and expensive [6] the
problem, the only information available is the aminoacid sequence. knowledge and technological gains generated are invaluable. The
Although not very helpful at the initial stage, the information that main issues of HT have already been described [7] while new
can be gathered from the primary sequences should not be over- strategies to increase the output of HT protein crystallization are
looked even if it is only used to interpret the results obtained from being investigated. Among them, the HT native protein fractioning
the crystallization screening [2] or to select an initial crystallization applied to the Escherichia Coli pool of proteins [8] or the direct
0003-9861/ 2015 Elsevier Inc. All rights reserved.
4 J.A. Gavira / Archives of Biochemistry and Biophysics 602 (2016) 3e11

crystallization of proteins from crude extracts [9] should be high- consolidated techniques that have or may have a relevant contri-
lighted since they did not follow the well established requirement bution to current methodologies. Besides the tremendous contri-
of sample purity statements for macromolecule crystallization. bution of HT to the eld of biocrystallogenesis this article is written
With the advent of serial femtosecond X-ray crystallography (SFX) from the low throughput perspective (i.e. what to do in a regular
the mentioned HT approaches are pushed to the limit by growing laboratory with little or no access to automatize pipelines).
crystals in vivo using different types of cells and providing new Therefore crystallization will be treated from a traditional point of
paths for proteins reluctant-to-crystallize in vitro [10]. view. From now on we will call proteins to all biological macro-
To full our needs of 3D coordinates of the molecular machinery molecules including isolated proteins, proteineprotein or protein-
of life, crystallography is the most direct technique to provide ac- nucleic acid complexes to avoid long descriptions. If needed for a
curate models if crystals of sufcient quality are obtained. Yet, as we particular subject, we will use the specic mention of the particular
progress in deciphering genomes encoding new proteins, the system to which it applies. There are also several relevant subjects
remaining structures to be determined become more elusive and that will be treated along this issue-volume, including the pro-
difcult to attack with current methodologies. Although it is clear duction of large crystals for neutron diffraction or small crystals for
that both X-ray and neutron crystallography are the only tech- XFEL, the crystallization of membrane proteins or the production of
niques that could provide high resolution models for reluctant-to- protein-drug complexes.
crystallize systems (membrane proteins, proteineprotein or
protein-nucleic acid complexes, big particles, etc.), other tech- 2. Protein nature
niques such as nuclear magnetic resonance (NMR) [11], cryo-
electron microscopy (Cryo-EM) [12] and small angle scattering When facing the task of growing a protein crystal, undoubtedly
(SAXS) may provide valuable information about the unshaped the rst difculty is the intrinsic complexity of the biological
primary sequence. Table 1 summarizes the main benets and type macromolecule. There are several important points that need to be
of structural information that can be derived from each technique. considered if our intention is to grow crystals suitable for diffrac-
In this issue, readers will also nd dedicated articles on both SAXS tion studies. Firstly we have to take into account that proteins are
and electron microscopy applications and uses. The effort put in folded polypeptide chains that, at best, will remain folded at room
structural biology is not restricted to crystallography but has also temperature and, in the worst case scenario, will belong to the so
permeated other elds. In fact the EU has funded a project to speed called intrinsically disorder proteins (IDPs) family. IDPs are a
up the automated structural determination using NMR, Critical paradigm lacking any structural feature at the secondary and ter-
Assessment of Automated Structure Determination by NMR (CASD- tiary structural level for which the main characteristic is the
NMR) [13], while a repository of SAS experimental data and derived prevalence of highly charged or polar residues, as well as glycine
models (SASBDB) [14] and a dedicated database repository of and proline, with fewer hydrophobic residues [18]. Between those
intrinsically disorder proteins/regions (IDPs) related data (pE-DB) two extremes, compact packed or highly disordered proteins, one
[15] have also been created. Cryo-electron microscopy (Cryo-EM) is can imagine that partially folded or weakly folded proteins may
also gaining momentum and popularity in structural biology exist. One simple reason that justies these unstable states is that
studies. Advances in electron detectors and software for the pro- we have puried just a portion of the machine (i.e. a component of
cessing of thousand of images or the correction of beam-induced a bigger complex or just that our protein needs a partner i.e.
motion are supporting this development facilitating the gaining another protein, co-factor, ligand, etc).
of structural information at low resolution, currently around 3.0 This leads us to the point of protein purity, a major issue in
[12,16]. Single particle EM has recently gone beyond this limit protein crystallization, covered as the rst essential requirement to
reaching 2.2 resolution in the case of the b-galactosidase from ensure the desirable crystal. We will not go into detail here and will
E. Coli [17]. This wide-ranging effort made in different disciplines just remind the reader that the worst impurity in a protein sample
will have to converge to a common road of interdisciplinary labo- is the protein itself. As pointed out in several articles and summa-
ratories and groups able to conjugate different techniques in order rized in recent works [8,9], the problem of having other contami-
to attack the most difcult, but also the most interesting, bio- nant proteins in the media does not necessarily hamper the
macromolecular structural problems. formation of crystalline material, although it will denitively affect
In this article we will briey summarize the current methods crystal quality. Baker and co-workers [9] were able to crystallize
and protocols employed to search, improve and detect bio- seven proteins, including one membrane protein, from heteroge-
macromolecule crystals highlighting the most recent de- neous bovine rod outer segment (ROS) crude extract. Although only
velopments in this eld while also paying attention to emerging or two of them diffracted X-ray to a resolution suitable to determine

Table 1
Summary of main techniques employed to obtain biological macromolecules structural information.

Technique Application media Derived structural information

Diffraction (X-ray, neutron) - Crystals form (nano- to millimetre size). - Single 3D model at atomic resolution.
- No restriction in MW. - Depends on crystal quality.
- Dynamic information (Laue-diffraction).
XFEL (Serial femtosecond X-ray crystallography) - Nanometre size crystals (millions of specimens). - Single 3D model at atomic resolution.
- No restriction in MW.
NMR spectroscopy - Protein in solution. - Multiple 3D models, dynamic and interactions.
- MWs <40e50 kDa. - Highlight exible regions.
SAS (SAXS/SANS) - Protein in solution. - Molecular envelope i.e. size and shape.
- MWs few kDa to GDa.
Micro-EDa (Electron diffraction) - Thin protein crystals (hundreds of specimens). - High resolution (~2.0 ).
- No restriction in MW.
Cryo-EM (Electron microscopy) - Flash cooled single particle sample. - High resolution (~3.0 )
- MWs >250 kDa. - Dynamic 3D models.
Other EM techniques include transmission and scanning electron microscopy that are regularly used to produce 2D and 3D structures at medium to low resolution
(~3.0e10.0 ).
J.A. Gavira / Archives of Biochemistry and Biophysics 602 (2016) 3e11 5

their 3D structure, the other ve could be further optimized. It is protein-model systems. Mobility, protonation state and accessible
therefore important to pay attention to the protein of interest as a surface areas are modied/tuned by changing the temperature and
self-impurity. The different aggregation state for proteins with additives in the crystallization media i.e. buffer pH, salts, etc., and
tendency to form multimers, or the co-existence of ligated and un- therefore for each crystallization condition, for the given protein, it
ligated forms (prosthetic-groups, co-factors, etc.) are just two ex- is almost as if we started with a different protein. In the case of
amples of sources of heterogeneity (self-impurities) that will not lysozyme it is possible to get crystals under several crystallization
show up in a SDS-gel test but that can hinder nucleation or proper conditions with very different packing. In fact, lysozyme crystal-
growth of protein crystals. Nonetheless, when the targeted protein lizes in ve different space groups, hexagonal, tetragonal, ortho-
appears as a single band on a SDS-gel, that's a promising target. rhombic, monoclinic and triclinic just by changing the precipitant
Although further characterization may be required (isoelectric composition [27]. Moreover, tetragonal and orthorhombic space
focusing, mass spectrometry, etc.), crystallization trials may be groups can be obtained under identical chemical conditions just by
started but it is worth to spend some time to gather the information changing the temperature from 18 to 37  C, a type of behaviour
that can be relevant to either design the crystallization optimiza- typically observed in inorganic crystallization.
tion round or to interpret the results [4]. On one hand, it is useful to Apart from the diverse crystal contacts involved in each crys-
run the macromolecule sequence in several web-based servers to talline form, the most relevant feature of those polymorphs is the
obtain information concerning the isoelectric point, identication difference in the amount of water content in each crystal form,
of exible regions, known cofactors, ligands, etc., that may stabilize ranging from approximately 28%e50% for the triclinic and hexag-
the protein in solution. On the other hand, it is possible to inves- onal space groups respectively (Table 2 and Fig. 2). It is worth
tigate protein crystallizability through several servers [19,20] or to mentioning that three out of the four structures solved at resolu-
get information from the crystallization of homologous proteins tion higher than 1.0 belong to the triclinic space group and only
directly from the PDB [5]. one to the tetragonal polymorph from a crystal grown under
It is worth mentioning here that other dramatically different microgravity conditions [28]. Among them the highest resolution
methodologies are being investigated in order to maintain or even (0.65 , PDB ID. 2VB1) was obtained in the triclinic space group. As
enhance high throughput output or as a need to produce sufcient pointed out by Wang in his work, with data at ultrahigh resolution,
number of nano to micro-size crystalline materials for SFX. For the lysozyme molecule can be considered as a rigid protein,
example, the native protein fractioning technique has been applied nonetheless 45 side-chain residues with alternating conformations
to the E. Coli pool of proteins [8]. With this approach the pure have been found as well as eight sections of main-chain accounting
extract was fractioned in 408 samples and subjected to crystalli- for approximately 35% of the protein structure [29]. This demon-
zation assays, 295 of which produced crystals and 152 were opti- strates the dynamic nature of protein molecules even in its crys-
mized. Among other relevant conclusions it is interesting to talline phase.
highlight that the purity is not a requirement to nd initial crys- We should now have a clearer picture of the problem of protein
tallization hits but it is denitely highly important to obtain good crystallization. On one hand, the characteristics of the intrinsic
diffracting crystals. This methodology has been further investigated polypeptide chain, that determine the folded state and stability,
in an attempt to crystallize mammalian proteins from crude extract will regulate the crystallizability under a particular set of condi-
from bovine rod outer segment (ROS), from which seven proteins tions since the formation of crystalline contacts are controlled by
were crystallized, one of them being a membrane protein [9]. In the state and position of supercial charged residues. Therefore this
these two cases the initial aim was to obtain protein crystals from may explain that the likelihood of forming a crystal is low. On the
natural sources but this idea has been further exploited to induce other hand, the number of required contacts to form protein crys-
the in vivo crystallization of heterologous gene overexpression of tals is low and the solvent content is very high. Both characteristics
proteins in mammalian [21] or insect-cells using baculovirus may facilitate the accommodation of more exible regions
shuttle vectors [10]. Driven by XFEL and Micro-ED needs we may increasing the likelihood of nding the right conditions to form a
expect an increasing development of in vivo crystallization tech- crystal. Crystals of these characteristics, high water content and low
nologies and methodologies. number of crystal contacts, will denitively be weak and sensitive
Freshly puried pure protein solutions are typically obtained in but can be obtained. In the next section we will review the current
the chemical cocktail required during the last chromatography crystallization techniques with special emphasis on the crystal
purication step, or after dialysis in the same buffer at low ionic improvement methods.
strength, but those may not be the best conditions for crystalliza-
tion purposes. Since a sufcient amount of material in solution is
needed in order to form a crystal, it is therefore good practice to 3. Protein crystallization
carry out a solubility screen test to nd the best condition to
maintain the protein happy in solution [22,23]. Not only the The crystallization process is typically divided into three
solubility of a protein increases when the pH is 2e3 units different different steps, namely, nucleation, growth and growth cessation.
from the isoelectric point (pI) [24] but also the nucleation region is These consecutive steps will determine the number, size and
wider [25]. The gain in stability by choosing the right pH may be quality of the generated crystalline material and are controlled by
easily quantied using the thermouor method [26] or by light the driving force, supersaturation. In order to precipitate a protein
scattering methods. Both methods are also suitable to identify from solution we need to drive the system into a supersaturated
possible ligands or partners following the thermal shift upon state in which the amount of energy gained after entering this zone
binding. of the phase diagram can be released by the formation of a pre-
Being at the best situation, a pure and homogeneous protein cipitate. To ensure the precipitation of a crystalline material, both,
solution, which is also stable under the current conditions, should the excess of energy gained (thermodynamic contribution) and the
be able to produce crystals. But if we take a closer look to the object rate at which this excess of energy has been reached (kinetic
under study, the protein, we may understand that packing these contribution), should be neither to high nor too fast. Both re-
elements within an ordered system, the crystal, is somehow quirements, the supersaturation level and the rate of supersatura-
fortuitous. In Fig. 1 it is shown the charge (A) and hydrophobicity tion, are explored differently depending on the crystallization
(B) distribution of the enzyme lysozyme, one of the most studied technique employed.
6 J.A. Gavira / Archives of Biochemistry and Biophysics 602 (2016) 3e11

Fig. 1. Charge (left) and hydrophobic (right) surfaces representation of HEWL (PDB ID. 1IEE). Electrostatic charge is represented from blue (positive) to red (negative) with white
colour representing the neutral charge. Similarly, the hydrophobicity is represented from red (hydrophilic) to green (hydrophobic) being white the regions of amphiphilic character.

Table 2
Summary of typical unit cell dimensions of lysozyme crystals in the ve space groups showed in Fig. 2 and the corresponding Mathew's coefcient and solvent content.

Space group Unit cell dimesnions Matthew's coefcient Solvent content (%)
a, b, c ()/a, b, g ( )

P1 26.65 30.80 33.63 1.70 27.59

88.30 107.40 112.10
P 21 28.00 31.00 34.00 1.85 33.52
89.00 72.00 67.80
P 21 21 21 31.00 56.00 73.00 2.22 44.48
P 43 21 2 78.50 78.50 37.80 2.04 39.59
P 61 2 2 86.00 86.00 68.00 2.54 51.54

Fig. 2. Example of lysozyme polymorphism: triclinic (A), monoclinic (B), orthorhombic (C), tetragonal (D) and hexagonal (E).

3.1. Crystallization methods amount of information can be found in the bibliography regarding
the different set-ups and modications to exert a better control of
There are several techniques that can be used to drive a system the systems [30e33]. It is essential to understand that each tech-
into the supersaturated region, but essentially there are three main nique screens the phase diagram differently inuencing the
approaches: batch, vapour diffusion (VD) and liquideliquid diffu- nucleation, and consequently the growth, although the nal
sion [1], all of which have been extensively discussed and a vast chemical composition of the system in all the techniques may be
J.A. Gavira / Archives of Biochemistry and Biophysics 602 (2016) 3e11 7

the same. Therefore using different crystallization methods should is also a good technique to exert a ne control of the crystallization
be considered when trying to improve crystal quality. conditions and therefore recommended to improve crystal quality
We will highlight only those points that may be relevant for by simple screening-grid crystallization in which one or two vari-
inquisitive people. The widespread use of each technique is ables are modulated. Automation of batch experiments has also
laboratory-dependent and is also linked, at least in big laboratories, been developed in parallel to VD and is set-up under-oil facilitating
to the availability of automatic/robotic facilities which multiply by a the preservation of the drops and avoiding drop desiccation during
factor of 10 (1 mL vs. 0.1 mL) the number of experiments per unit of plate preparation [34,35]. The gentlest way to mix a protein and
volume of the protein sample. precipitant solution is achieved when using capillaries or mem-
Vapour diffusion (VD) is by far the most common method for branes, which reduces convective mass transport. This way the two
screening purposes in any of the possible congurations, sitting- solutions can be juxtaposed and allow to equilibrate prior to crys-
drop (VD-SD) and hanging-drop (VD-HD). For this methodology a tallization [1]. This is called the free interface diffusion method
drop containing the protein and precipitant is equilibrated against a (FID) and is mainly applied nowadays within microuidic devices
reservoir typically containing double concentration of the precipi- [38].
tant cocktails. The reservoir then draws water from the drop, The counter-diffusion (CD) technique can be considered as a
through the air-gap separation, driving the protein/precipitant derivative of the FID concept but in which instead of driving the
solution-mixture to a higher concentration state that may even- system towards the equilibration of the solution prior to crystalli-
tually enter the supersaturated region (Fig. 3). The choice of zation, the system is strongly destabilized by driving it towards
conguration has been driven mainly by the easiness to sh crys- high supersaturation values at the beginning of the experiment
tals from HD, whereas in SD crystals tend to be stuck at the plastic [39]. With this mechanism of action the technique self-explores an
surface putting into risk the integrity of the sample. In contrast, SD extended region of the phase diagram while the intrinsic dynamics
is easier to set-up manually or using robotic systems since it re- permits to optimize the crystallization condition producing crystals
duces the number of steps. Even so, both approaches have been of high quality mainly due to the slow supply of molecule to the
explored and the market provides with robotic systems specialized growing crystal and the impurity ltering effect gained under the
in one of the set-ups or to be used with both of them [34,35]. diffusion mass transport regime [40e42]. Among all possible con-
Batch crystallization is understood in biocrystallogenesis as gurations and media, capillaries and microuidic devices have
micro-batch under-oil method in which the protein and the pre- been selected to develop a series of crystallization devices, tools
cipitant cocktail are mixed together and covered by a layer of and adapted crystallization screening kits for screening purposes
parafn oil. A single supersaturation value is initially investigated using the CD technique. One of the most interesting characteristics
in each experiment [36]. The screening path could be extended if a is that both devices, capillaries and microchips, allow in situ X-ray
mixture of parafn and water permeable oil, i.e. silicon, is used [37]. data collection at room temperature [40,43,44] or even under
In this case the main problem is to avoid complete desiccation of cryogenic conditions [45]. The most remarkable improvement is
the drop. Due to the small amount of sample, drying may also occur being done on the microuidic chip material from, the original
with pure parafn oil due to the water permeability of plastic. Batch poly-dimethylsiloxane (PDMS) or poly-methyl-methacrylate

Fig. 3. Shows a typical phase diagram for a protein-precipitant couple with the solubility and metastability curves guides. It is worth to remember that while the solubility curve is
thermodynamically dened, the metastability curve has a kinetic nature and depends on how fast the system moves towards the supersaturate region. The screening path of vapour
diffusion (green), batch (yellow) and counter-diffusion (red), represented as a hanging drop, micro-batch under-oil and capillary counter-diffusion set-ups, are shown with colour
arrows. Starting and nal equilibrium conditions are marked with empty and lled circles, respectively.
8 J.A. Gavira / Archives of Biochemistry and Biophysics 602 (2016) 3e11

(PMMA) to the most recent development with cyclo-olen- [57]. Similarly, Two-Photon Excited Ultraviolet Fluorescence (TPE-
copolymer (COC), which has proved to be very effective in avoid- UVF) microscopy capable of being integrated within SONICC in-
ing evaporation while keeping a low contribution to the scattering struments has also been developed [59]. The use of visible light for
of X-rays allowing structural determination from Single Anomalous the excitation and the near-UV for detection avoids any interfer-
Dispersion (SAD) data [46,47]. ence with the crystallization hardware and envisage a clear future
High throughput crystallization (HTC) has brought bio- for this technique. As for today a new prototype combining both
macromolecular crystallization to a new level in the search for NLO techniques has been assayed at the Advanced Photon Source
initial conditions, crystal handling and detection. HTC implies the (APS) synchrotron source and its future application for the identi-
used of robotic systems to set-up the crystallization experiments cation, location and automatic centring of crystals has been dis-
(may include the preparation/distribution of precipitant cocktail) cussed [51]. Crystal identication becomes even harder when
[34,35] and could be the rst choice for initial screening when reaching sizes in the nanometer range. A recent review covers the
available at the home laboratory or when it is commercially current approaches to overcome this limitation [60] and will be
accessible at reasonable prices. It is certainly the best way to screen discussed in this issue.
a sufcient number of different crystallization cocktails with low
consumption of sample [48]. Nonetheless the selection of the initial 3.2. Improving and handling protein crystals
screen will be the rst blind selection to be done on the crystalli-
zation loop. There are several hundreds of screening kits account- Initial hits are in most cases a good starting point for crystal
ing for approximately 16,000 different cocktails, many of which improvement but not suitable to obtain good quality X-ray
contain repeated conditions. Fortunately Fazio and co-workers diffraction data. A simple way to explain this common result is that
have done an extensive study comparing the redundancy of con- to overcome the nucleation energy barrier the supersaturation of
ditions across different screening kits (256 kits 15,906 condi- the system needs to be high enough, typically two or three times
tions) and identifying the screening kits with the most successful higher than for regular growth. Since it is not possible to fully
commercial conditions [49]. control crystallization experiments, the system is usually overshot
HTC implies a fast growth in the number of conditions that have producing a shower of crystals and/or a fast crystal growth i.e. small
to be periodically followed and examined. This new challenge has size crystals and/or low quality crystals. Therefore two main ap-
been addressed in different ways by adapting methodologies from proaches are being explored to improve crystal quality [61e63]:
other elds or developing new techniques to help identifying separating crystal nucleation from growth and inducing nucleation
protein crystals of small size. The oldest methodology of crystal under low supersaturation conditions.
crushing or staining with a dye, are impossible or difcult to use If screening for protein crystals is somehow unpredictable, once
within the small volumes of HTC assays. Crystal sample identi- an initial crystalline precipitate is obtained, trying to improve
cation and location are nowadays a main issue not only at labora- crystal quality can be tackled in a more rational way. Once initial
tory scale [50] but also when facing the problem of automatic X-ray crystalline material has been obtained, it can be used to seed it into
data collection at synchrotron beamlines [51]. Therefore a number a much lower supersaturated system to allow the growth at slower
of techniques have been implemented to identify and locate protein rate. Seeding crystals into metastable systems has been widely used
crystals. Intrinsic UV uorescence, mainly from tryptophan, or from to improve protein crystal quality [62]. The seeding methodology
uorescent dyes is being exploited [52]. Besides the intrinsic has also been employed in combination with matrix screening
characteristic of the instruments developed, there are general experiments to increase the likelihood of nding new crystalliza-
concerns regarding the attenuation of different materials supplied tion conditions that may provide new polymorphs or crystals of
to set-up crystallization trials, the lack of tryptophan in many ge- better quality [64]. The microseed matrix screen (MMS) method
nomes or the radiation damage (time dependent) provoked to the has become a standard methodology to search for new hits since it
sample. At the same time a longest excitation wavelength of is easily automatized and can be applied to cross-seed between
300e390 nm, from a mercury arc lamp, instead of the typically related and un-related proteins [64].
used 280 nm, has been proposed as a possible solution to minimize Other approaches have been tested to overcome the nucleation
protein radiation damage and hardware attenuation [53]. The au- energy barrier, as for example inducing heterogeneous nucleation
thors demonstrate that the proposed conguration is easy to adapt with different types of the so-called nucleants [65]. Typically
to any existing microscope and compatible within pre-existing DLS these materials expose a surface that may stabilize the pre-critical
instruments [54]. Other approaches have focused on the use of nucleation cluster or even the dense liquid droplet, observed
external uorescent dyes (Ru(bpy)2dcbpy) covalently bound to the sometimes under supersaturated conditions, lowering the nucle-
protein in order to identify the best hits, which are further opti- ation energy barrier [66]. There is a wide variety of materials that
mized in the absence of the dye [55,56]. This concept is also have been tested in the search for the universal nucleant [2], from
exploited as a source of identication of different crystal compo- natural to synthetic. A review of the materials tested as nucleants
nents in protein complex crystals by using different dyes. More can be found in the supplementary material of Fermani and co-
recently, new instrumentation based on nonlinear optics (NLO) workers [66].
methods have also been developed [57]. Haupert has reviewed and A second approach to screening for initial crystallization con-
summarized the instrumentation, technical approaches and cur- ditions and improving crystal quality in a single experiment is
rent methods, from the application of second order nonlinear op- provided by the use of counter-diffusion methods. Because of how
tical imaging of chiral crystals (SONICC) based on second harmonic the technique works, starting at high supersaturation values far
generation (SHG) microscopy to the identication of protein crys- from the equilibrium, the coupling of the precipitation and mass
tals [58]. Among other interesting properties of SHG-based tech- transport under a diffusion controlled regime allows for the low
niques, is the low inuence of the selected working wavelength nucleation density and the slow crystal growth at positions far from
near the infrared (0.8e1 mm), which signicantly reduces the en- the salt incorporation point. Crystals at this position are typically of
ergy absorbed by protein molecules and is relatively low sensitive higher quality and bigger size than at positions near to the salt
to optical scattering (SHG signals is forbidden in solutions and incorporation point (x0, x1 in Fig. 3) [40,41,67]. The capability of this
amorphous aggregates lacking long-range order), making it well- approach has been exploited to produce crystals of large size for
suited for measurements of crystallizations in lipidic mesophases neutron diffraction purposes while allowing a gentle interchange of
J.A. Gavira / Archives of Biochemistry and Biophysics 602 (2016) 3e11 9

perdeuterated solution [68]. Any condition that produces a micro- the nano to the millimetre sizes range. Whereas XFEL needs to use
crystalline material in batch or vapour diffusion experiments could nano-crystals of homogeneous size to be continuously delivered at
be easily explored to search for single crystals using the CD method room temperature, neutron diffraction requires perdeuterated
in single capillaries or in commercially available microchips. In crystals of big size and Micro-ED micrometre size crystals under
addition to the more extensive exploration of the phase diagram, cryogenic conditions [83,84]. This diversication is pushing on the
the diffusion mass transport regime provides a media that reduces demand for a better comprehension theory of biomacromolecules
the incorporation of impurities [42,69,70] and can be exploited also crystallization. Meanwhile new technical and methodological so-
in combination with seeding techniques to increase the number of lutions are being assayed to meet the needs of this diversity of
hits or as crystal improvement methodology [71]. crystals size.
Crystallization in gel media will serve in this section as a link On this note, serial synchrotron-radiation diffraction at micro-
between crystal improvement and crystal handling. Although gel focus beamlines based on helical scan of single crystals [85] or more
crystallization is not a frequent practice, the benets of using gel recently from many microcrystal (grown in vivo) mounted on a
media (agarose, silica, PEGs base hydrogel, sephadex, etc.) for the standard loop [80], have been used to generate wedges of data that
crystallization of macromolecules are multiple - i.e. prevents con- can be scaled together to obtain complete datasets. In addition to
vection and crystal sedimentation, acts as impurity lter, etc. - and the great advance in micro-crystallography [83], which implies
have been proven to produce crystals of higher quality [72,73]. cryo-protection, room temperature data collection is also being
Since this subject has been recently covered in connection with the routinely used for testing and ultimately for data collection of
use of peptide-based hydrogels for the production of high quality multiple crystals thanks to the high efciency of pixel array de-
protein crystal [74] and the formation of new polymorphs [75] it tectors used in dedicated MX beamlines.
will not be discussed here in detail. There are many types of In the last decades, microuidic devices (microchips) have
hydrogels, depending on their nature, that can be used and most of emerged as a powerful technology in protein crystallization [86].
them are compatible with any crystallization method. Among them This technology combines low reagent consumption and high
agarose is probably the most widely used due to its simple prep- density of experiments while providing a media in which mass
aration protocol and availability in any biochemistry laboratory. transport occurs mainly under a diffusion regime. The use of mi-
Agarose can be found with a wide range of melting and gelling crochips has enabled the miniaturization of crystallization experi-
points and can be used at concentrations below its critical gelling ments using sample volumes much smaller than needed with
point, thus facilitating its mixing with the protein solution [76]. robotic systems [87] with a high operational speed while maxi-
Moreover, gels also provide an excellent media for the soaking of mizing the number of screening conditions. As already mentioned
ligands, co-factors, cryo-protectant, heavy atoms, etc. [45,77] while standard crystallisation techniques can be implemented using
preserving the integrity of the macromolecule crystals even when these devices but its potential is even higher. For example, micro-
exposed to organic solvents (DMSO) [78]. The use of hydrogels chips have been used to carry out fundamental studies such as the
avoids the manipulation of the crystals and therefore the osmotic determination of phase diagrams or to study nucleation [88], to
and mechanical stress exerted on the crystals during handling. decouple nucleation and growth [86,89,90] or to gather thermo-
Crystal handling is indeed a main issue in protein crystallization. dynamic and kinetic data [91]. A step further has been given by
Since it has an effect in crystal quality, avoiding the loss of crystal adapting the counter-diffusion (CD) method [46,92] that, as
integrity is a way of improving the quality of the diffraction data. mentioned above, extensively explores the phase diagram for a
Protein crystals are exposed to mechanical and osmotic stress given combination of protein and precipitant cocktail. The avail-
during crystal preparation risking its integrity and quality. Room ability of new materials such as a co-polymer of cyclic olen (COC)
temperature (RT) data collection is being pursued to avoid the use with higher transparency to X-rays than poly(dimethylsiloxane)
of crystal treatment methods such as soaking with a cryo- (PDMS) have aided the construction of a new generation of mi-
protectant or the use of heavy atoms, etc. Owing to the latest ad- crochips for in situ data collection from crystals obtained by
vances on data processing (integration and scaling), especially for microbatch [44] and counter-diffusion [43,47] methods. The com-
serial femtosecond crystallography, the completion of full datasets bination of materials (COC-PDMS-Duralar) has been used to facili-
from many different crystals is not an issue [79,80]. Another recent tate the de novo structure determination of a protein from single-
improvement involves the use of high-transparency micro-pat- wavelength anomalous diffraction (SAD) data without crystal
ternable chips, made of silicon nitride, photoresist and polyimide manipulation [93]. We may therefore anticipate that microuidic
lm, to load the sample in their mother liquid, facilitating X-ray devices will play a central role in protein crystallization studies
data collection while keeping a low background scattering [81]. ranging from phase diagram determination, screening and crystal
Other approach involves the use of the graphene-wrapped protein improvement to in situ data collection including time-resolved
crystals for the measurement of diffraction data in vacuo, which serial crystallography from Laue diffraction [94,95]. Furthermore,
minimizes the background and zero absorption of incident and other advances are expected to extend the lifetime of protein
scattered beams [82]. crystals under the beam from a deeper understanding of the
chemistry inside irradiated crystals [96].
4. Current trends in protein crystallization Along this line, crystallization in hydrogel gel media should also
be taken into account not only for improving crystal quality but also
There are many open fronts in the eld of biocrystallization to exert control over the nucleation and growth processes. The role
mainly derived from the incredible amount of instrumentation that of agarose to induce nucleation and silica gel to inhibit it have
is being developed including the manipulation of micro/nano vol- already been observed and documented [40,97,98]. Even more, gel-
umes and particle size, improvement efciency of detection sys- grown protein crystals have been recently shown to be excellent
tems (UVevisible, x-ray, uorescence, SONIC, DDLS, etc.) or the candidates to produce crystals of bigger size for neutron diffraction
advent and fast development of various characterization tech- studies [99]. In this study protein crystals grown in agarose gel
niques (XFEL, Micro-ED, etc.). The emergent technologies on XFEL, were seeded into a metastable solution to grow big size crystals. In
neutron diffraction, Micro-ED, etc. are expanding and diversifying a different investigation agarose gels have been proposed as media
the number of approaches that can be used to get the required 3D for the continuous delivery of nano/micro-metre size crystals
model while, their requirements of crystalline material covers from needed for serial femtosecond crystallography for XFEL
10 J.A. Gavira / Archives of Biochemistry and Biophysics 602 (2016) 3e11

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