Archives of Biochemistry and Biophysics 602 (2016) 3e11

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Archives of Biochemistry and Biophysics

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Current trends in protein crystallization

A. Gavira
Jose

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
Keywords:
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

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

http://dx.doi.org/10.1016/j.abb.2015.12.010
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-
lighted since they did not follow the well established requirement
of sample purity statements for macromolecule crystallization.
With the advent of serial femtosecond X-ray crystallography (SFX)
the mentioned HT approaches are pushed to the limit by growing
crystals in vivo using different types of cells and providing new
paths for proteins reluctant-to-crystallize in vitro [10].
To full our needs of 3D coordinates of the molecular machinery
of life, crystallography is the most direct technique to provide ac-
curate models if crystals of sufcient quality are obtained. Yet, as we
progress in deciphering genomes encoding new proteins, the
remaining structures to be determined become more elusive and
difcult to attack with current methodologies. Although it is clear
that both X-ray and neutron crystallography are the only tech-
niques that could provide high resolution models for reluctant-to-
crystallize systems (membrane proteins, proteineprotein or
protein-nucleic acid complexes, big particles, etc.), other tech-
niques such as nuclear magnetic resonance (NMR) [11], cryo-
electron microscopy (Cryo-EM) [12] and small angle scattering
(SAXS) may provide valuable information about the unshaped
primary sequence. Table 1 summarizes the main benets and type
of structural information that can be derived from each technique.
In this issue, readers will also nd dedicated articles on both SAXS
and electron microscopy applications and uses. The effort put in
structural biology is not restricted to crystallography but has also
permeated other elds. In fact the EU has funded a project to speed
up the automated structural determination using NMR, Critical
Assessment of Automated Structure Determination by NMR (CASD-
NMR) [13], while a repository of SAS experimental data and derived
models (SASBDB) [14] and a dedicated database repository of
intrinsically disorder proteins/regions (IDPs) related data (pE-DB)
[15] have also been created. Cryo-electron microscopy (Cryo-EM) is
also gaining momentum and popularity in structural biology
studies. Advances in electron detectors and software for the pro-
cessing of thousand of images or the correction of beam-induced
motion are supporting this development facilitating the gaining
of structural information at low resolution, currently around 3.0
[12,16]. Single particle EM has recently gone beyond this limit
reaching 2.2 resolution in the case of the b-galactosidase from
E. Coli [17]. This wide-ranging effort made in different disciplines
will have to converge to a common road of interdisciplinary labo-
ratories and groups able to conjugate different techniques in order
to attack the most difcult, but also the most interesting, bio-
macromolecular structural problems.
In this article we will briey summarize the current methods
and protocols employed to search, improve and detect bio-
macromolecule crystals highlighting the most recent de-
velopments in this eld while also paying attention to emerging or
consolidated techniques that have or may have a relevant contri-
bution to current methodologies. Besides the tremendous contri-
bution of HT to the eld of biocrystallogenesis this article is written
from the low throughput perspective (i.e. what to do in a regular
laboratory with little or no access to automatize pipelines).
Therefore crystallization will be treated from a traditional point of
view. From now on we will call proteins to all biological macro-
molecules including isolated proteins, proteineprotein or protein-
nucleic acid complexes to avoid long descriptions. If needed for a
particular subject, we will use the specic mention of the particular
system to which it applies. There are also several relevant subjects
that will be treated along this issue-volume, including the pro-
duction of large crystals for neutron diffraction or small crystals for
XFEL, the crystallization of membrane proteins or the production of
protein-drug complexes.
2. Protein nature
When facing the task of growing a protein crystal, undoubtedly
the rst difculty is the intrinsic complexity of the biological
macromolecule. There are several important points that need to be
considered if our intention is to grow crystals suitable for diffrac-
tion studies. Firstly we have to take into account that proteins are
folded polypeptide chains that, at best, will remain folded at room
temperature and, in the worst case scenario, will belong to the so
called intrinsically disorder proteins (IDPs) family. IDPs are a
paradigm lacking any structural feature at the secondary and ter-
tiary structural level for which the main characteristic is the
prevalence of highly charged or polar residues, as well as glycine
and proline, with fewer hydrophobic residues [18]. Between those
two extremes, compact packed or highly disordered proteins, one
can imagine that partially folded or weakly folded proteins may
exist. One simple reason that justies these unstable states is that
we have puried just a portion of the machine (i.e. a component of
a bigger complex or just that our protein needs a partner i.e.
another protein, co-factor, ligand, etc).
This leads us to the point of protein purity, a major issue in
protein crystallization, covered as the rst essential requirement to
ensure the desirable crystal. We will not go into detail here and will
just remind the reader that the worst impurity in a protein sample
is the protein itself. As pointed out in several articles and summa-
rized in recent works [8,9], the problem of having other contami-
nant proteins in the media does not necessarily hamper the
formation of crystalline material, although it will denitively affect
crystal quality. Baker and co-workers [9] were able to crystallize
seven proteins, including one membrane protein, from heteroge-
neous bovine rod outer segment (ROS) crude extract. Although only
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.
a
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
their 3D structure, the other ve could be further optimized. It is
therefore important to pay attention to the protein of interest as a
self-impurity. The different aggregation state for proteins with
tendency to form multimers, or the co-existence of ligated and un-
ligated forms (prosthetic-groups, co-factors, etc.) are just two ex-
amples of sources of heterogeneity (self-impurities) that will not
show up in a SDS-gel test but that can hinder nucleation or proper
growth of protein crystals. Nonetheless, when the targeted protein
appears as a single band on a SDS-gel, that's a promising target.
Although further characterization may be required (isoelectric
focusing, mass spectrometry, etc.), crystallization trials may be
started but it is worth to spend some time to gather the information
that can be relevant to either design the crystallization optimiza-
tion round or to interpret the results [4]. On one hand, it is useful to
run the macromolecule sequence in several web-based servers to
obtain information concerning the isoelectric point, identication
of exible regions, known cofactors, ligands, etc., that may stabilize
the protein in solution. On the other hand, it is possible to inves-
tigate protein crystallizability through several servers [19,20] or to
get information from the crystallization of homologous proteins
directly from the PDB [5].
It is worth mentioning here that other dramatically different
methodologies are being investigated in order to maintain or even
enhance high throughput output or as a need to produce sufcient
number of nano to micro-size crystalline materials for SFX. For
example, the native protein fractioning technique has been applied
to the E. Coli pool of proteins [8]. With this approach the pure
extract was fractioned in 408 samples and subjected to crystalli-
zation assays, 295 of which produced crystals and 152 were opti-
mized. Among other relevant conclusions it is interesting to
highlight that the purity is not a requirement to nd initial crys-
tallization hits but it is denitely highly important to obtain good
diffracting crystals. This methodology has been further investigated
in an attempt to crystallize mammalian proteins from crude extract
from bovine rod outer segment (ROS), from which seven proteins
were crystallized, one of them being a membrane protein [9]. In
these two cases the initial aim was to obtain protein crystals from
natural sources but this idea has been further exploited to induce
the in vivo crystallization of heterologous gene overexpression of
proteins in mammalian [21] or insect-cells using baculovirus
shuttle vectors [10]. Driven by XFEL and Micro-ED needs we may
expect an increasing development of in vivo crystallization tech-
nologies and methodologies.
Freshly puried pure protein solutions are typically obtained in
the chemical cocktail required during the last chromatography
purication step, or after dialysis in the same buffer at low ionic
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
carry out a solubility screen test to nd the best condition to
maintain the protein happy in solution [22,23]. Not only the
solubility of a protein increases when the pH is 2e3 units different
from the isoelectric point (pI) [24] but also the nucleation region is
wider [25]. The gain in stability by choosing the right pH may be
easily quantied using the thermouor method [26] or by light
scattering methods. Both methods are also suitable to identify
possible ligands or partners following the thermal shift upon
binding.
Being at the best situation, a pure and homogeneous protein
solution, which is also stable under the current conditions, should
be able to produce crystals. But if we take a closer look to the object
under study, the protein, we may understand that packing these
elements within an ordered system, the crystal, is somehow
fortuitous. In Fig. 1 it is shown the charge (A) and hydrophobicity
(B) distribution of the enzyme lysozyme, one of the most studied
5
protein-model systems. Mobility, protonation state and accessible
surface areas are modied/tuned by changing the temperature and
additives in the crystallization media i.e. buffer pH, salts, etc., and
therefore for each crystallization condition, for the given protein, it
is almost as if we started with a different protein. In the case of
lysozyme it is possible to get crystals under several crystallization
conditions with very different packing. In fact, lysozyme crystal-
lizes in ve different space groups, hexagonal, tetragonal, ortho-
rhombic, monoclinic and triclinic just by changing the precipitant
composition [27]. Moreover, tetragonal and orthorhombic space
groups can be obtained under identical chemical conditions just by
changing the temperature from 18 to 37
C, a type of behaviour
typically observed in inorganic crystallization.
Apart from the diverse crystal contacts involved in each crys-
talline form, the most relevant feature of those polymorphs is the
difference in the amount of water content in each crystal form,
ranging from approximately 28%e50% for the triclinic and hexag-
onal space groups respectively (Table 2 and Fig. 2). It is worth
mentioning that three out of the four structures solved at resolu-
tion higher than 1.0 belong to the triclinic space group and only
one to the tetragonal polymorph from a crystal grown under
microgravity conditions [28]. Among them the highest resolution
(0.65 , PDB ID. 2VB1) was obtained in the triclinic space group. As
pointed out by Wang in his work, with data at ultrahigh resolution,
the lysozyme molecule can be considered as a rigid protein,
nonetheless 45 side-chain residues with alternating conformations
have been found as well as eight sections of main-chain accounting
for approximately 35% of the protein structure [29]. This demon-
strates the dynamic nature of protein molecules even in its crys-
talline phase.
We should now have a clearer picture of the problem of protein
crystallization. On one hand, the characteristics of the intrinsic
polypeptide chain, that determine the folded state and stability,
will regulate the crystallizability under a particular set of condi-
tions since the formation of crystalline contacts are controlled by
the state and position of supercial charged residues. Therefore this
may explain that the likelihood of forming a crystal is low. On the
other hand, the number of required contacts to form protein crys-
tals is low and the solvent content is very high. Both characteristics
may facilitate the accommodation of more exible regions
increasing the likelihood of nding the right conditions to form a
crystal. Crystals of these characteristics, high water content and low
number of crystal contacts, will denitively be weak and sensitive
but can be obtained. In the next section we will review the current
crystallization techniques with special emphasis on the crystal
improvement methods.
3. Protein crystallization
The crystallization process is typically divided into three
different steps, namely, nucleation, growth and growth cessation.
These consecutive steps will determine the number, size and
quality of the generated crystalline material and are controlled by
the driving force, supersaturation. In order to precipitate a protein
from solution we need to drive the system into a supersaturated
state in which the amount of energy gained after entering this zone
of the phase diagram can be released by the formation of a pre-
cipitate. To ensure the precipitation of a crystalline material, both,
the excess of energy gained (thermodynamic contribution) and the
rate at which this excess of energy has been reached (kinetic
contribution), should be neither to high nor too fast. Both re-
quirements, the supersaturation level and the rate of supersatura-
tion, are explored differently depending on the crystallization
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
There are several techniques that can be used to drive a system
into the supersaturated region, but essentially there are three main
approaches: batch, vapour diffusion (VD) and liquideliquid diffu-
sion [1], all of which have been extensively discussed and a vast
amount of information can be found in the bibliography regarding
the different set-ups and modications to exert a better control of
the systems [30e33]. It is essential to understand that each tech-
nique screens the phase diagram differently inuencing the
nucleation, and consequently the growth, although the nal
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
be considered when trying to improve crystal quality.
We will highlight only those points that may be relevant for
inquisitive people. The widespread use of each technique is
laboratory-dependent and is also linked, at least in big laboratories,
to the availability of automatic/robotic facilities which multiply by a
factor of 10 (1 mL vs. 0.1 mL) the number of experiments per unit of
volume of the protein sample.
Vapour diffusion (VD) is by far the most common method for
screening purposes in any of the possible congurations, sitting-
drop (VD-SD) and hanging-drop (VD-HD). For this methodology a
drop containing the protein and precipitant is equilibrated against a
reservoir typically containing double concentration of the precipi-
tant cocktails. The reservoir then draws water from the drop,
through the air-gap separation, driving the protein/precipitant
solution-mixture to a higher concentration state that may even-
tually enter the supersaturated region (Fig. 3). The choice of
conguration has been driven mainly by the easiness to sh crys-
tals from HD, whereas in SD crystals tend to be stuck at the plastic
surface putting into risk the integrity of the sample. In contrast, SD
is easier to set-up manually or using robotic systems since it re-
duces the number of steps. Even so, both approaches have been
explored and the market provides with robotic systems specialized
in one of the set-ups or to be used with both of them [34,35].
Batch crystallization is understood in biocrystallogenesis as
micro-batch under-oil method in which the protein and the pre-
cipitant cocktail are mixed together and covered by a layer of
parafn oil. A single supersaturation value is initially investigated
in each experiment [36]. The screening path could be extended if a
mixture of parafn and water permeable oil, i.e. silicon, is used [37].
In this case the main problem is to avoid complete desiccation of
the drop. Due to the small amount of sample, drying may also occur
with pure parafn oil due to the water permeability of plastic. Batch
is also a good technique to exert a ne control of the crystallization
conditions and therefore recommended to improve crystal quality
by simple screening-grid crystallization in which one or two vari-
ables are modulated. Automation of batch experiments has also
been developed in parallel to VD and is set-up under-oil facilitating
the preservation of the drops and avoiding drop desiccation during
plate preparation [34,35]. The gentlest way to mix a protein and
precipitant solution is achieved when using capillaries or mem-
branes, which reduces convective mass transport. This way the two
solutions can be juxtaposed and allow to equilibrate prior to crys-
tallization [1]. This is called the free interface diffusion method
(FID) and is mainly applied nowadays within microuidic devices
[38].
The counter-diffusion (CD) technique can be considered as a
derivative of the FID concept but in which instead of driving the
system towards the equilibration of the solution prior to crystalli-
zation, the system is strongly destabilized by driving it towards
high supersaturation values at the beginning of the experiment
[39]. With this mechanism of action the technique self-explores an
extended region of the phase diagram while the intrinsic dynamics
permits to optimize the crystallization condition producing crystals
of high quality mainly due to the slow supply of molecule to the
growing crystal and the impurity ltering effect gained under the
diffusion mass transport regime [40e42]. Among all possible con-
gurations and media, capillaries and microuidic devices have
been selected to develop a series of crystallization devices, tools
and adapted crystallization screening kits for screening purposes
using the CD technique. One of the most interesting characteristics
is that both devices, capillaries and microchips, allow in situ X-ray
data collection at room temperature [40,43,44] or even under
cryogenic conditions [45]. The most remarkable improvement is
being done on the microuidic chip material from, the original
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-
copolymer (COC), which has proved to be very effective in avoid-
ing evaporation while keeping a low contribution to the scattering
of X-rays allowing structural determination from Single Anomalous
Dispersion (SAD) data [46,47].
High throughput crystallization (HTC) has brought bio-
macromolecular crystallization to a new level in the search for
initial conditions, crystal handling and detection. HTC implies the
used of robotic systems to set-up the crystallization experiments
(may include the preparation/distribution of precipitant cocktail)
[34,35] and could be the rst choice for initial screening when
available at the home laboratory or when it is commercially
accessible at reasonable prices. It is certainly the best way to screen
a sufcient number of different crystallization cocktails with low
consumption of sample [48]. Nonetheless the selection of the initial
screen will be the rst blind selection to be done on the crystalli-
zation loop. There are several hundreds of screening kits account-
ing for approximately 16,000 different cocktails, many of which
contain repeated conditions. Fortunately Fazio and co-workers
have done an extensive study comparing the redundancy of con-
ditions across different screening kits (256 kits 15,906 condi-
tions) and identifying the screening kits with the most successful
commercial conditions [49].
HTC implies a fast growth in the number of conditions that have
to be periodically followed and examined. This new challenge has
been addressed in different ways by adapting methodologies from
other elds or developing new techniques to help identifying
protein crystals of small size. The oldest methodology of crystal
crushing or staining with a dye, are impossible or difcult to use
within the small volumes of HTC assays. Crystal sample identi-
cation and location are nowadays a main issue not only at labora-
tory scale [50] but also when facing the problem of automatic X-ray
data collection at synchrotron beamlines [51]. Therefore a number
of techniques have been implemented to identify and locate protein
crystals. Intrinsic UV uorescence, mainly from tryptophan, or from
uorescent dyes is being exploited [52]. Besides the intrinsic
characteristic of the instruments developed, there are general
concerns regarding the attenuation of different materials supplied
to set-up crystallization trials, the lack of tryptophan in many ge-
nomes or the radiation damage (time dependent) provoked to the
sample. At the same time a longest excitation wavelength of
300e390 nm, from a mercury arc lamp, instead of the typically
used 280 nm, has been proposed as a possible solution to minimize
protein radiation damage and hardware attenuation [53]. The au-
thors demonstrate that the proposed conguration is easy to adapt
to any existing microscope and compatible within pre-existing DLS
instruments [54]. Other approaches have focused on the use of
external uorescent dyes (Ru(bpy)2dcbpy) covalently bound to the
protein in order to identify the best hits, which are further opti-
mized in the absence of the dye [55,56]. This concept is also
exploited as a source of identication of different crystal compo-
nents in protein complex crystals by using different dyes. More
recently, new instrumentation based on nonlinear optics (NLO)
methods have also been developed [57]. Haupert has reviewed and
summarized the instrumentation, technical approaches and cur-
rent methods, from the application of second order nonlinear op-
tical imaging of chiral crystals (SONICC) based on second harmonic
generation (SHG) microscopy to the identication of protein crys-
tals [58]. Among other interesting properties of SHG-based tech-
niques, is the low inuence of the selected working wavelength
near the infrared (0.8e1 mm), which signicantly reduces the en-
ergy absorbed by protein molecules and is relatively low sensitive
to optical scattering (SHG signals is forbidden in solutions and
amorphous aggregates lacking long-range order), making it well-
suited for measurements of crystallizations in lipidic mesophases
[57]. Similarly, Two-Photon Excited Ultraviolet Fluorescence (TPE-
UVF) microscopy capable of being integrated within SONICC in-
struments has also been developed [59]. The use of visible light for
the excitation and the near-UV for detection avoids any interfer-
ence with the crystallization hardware and envisage a clear future
for this technique. As for today a new prototype combining both
NLO techniques has been assayed at the Advanced Photon Source
(APS) synchrotron source and its future application for the identi-
cation, location and automatic centring of crystals has been dis-
cussed [51]. Crystal identication becomes even harder when
reaching sizes in the nanometer range. A recent review covers the
current approaches to overcome this limitation [60] and will be
discussed in this issue.
3.2. Improving and handling protein crystals
Initial hits are in most cases a good starting point for crystal
improvement but not suitable to obtain good quality X-ray
diffraction data. A simple way to explain this common result is that
to overcome the nucleation energy barrier the supersaturation of
the system needs to be high enough, typically two or three times
higher than for regular growth. Since it is not possible to fully
control crystallization experiments, the system is usually overshot
producing a shower of crystals and/or a fast crystal growth i.e. small
size crystals and/or low quality crystals. Therefore two main ap-
proaches are being explored to improve crystal quality [61e63]:
separating crystal nucleation from growth and inducing nucleation
under low supersaturation conditions.
If screening for protein crystals is somehow unpredictable, once
an initial crystalline precipitate is obtained, trying to improve
crystal quality can be tackled in a more rational way. Once initial
crystalline material has been obtained, it can be used to seed it into
a much lower supersaturated system to allow the growth at slower
rate. Seeding crystals into metastable systems has been widely used
to improve protein crystal quality [62]. The seeding methodology
has also been employed in combination with matrix screening
experiments to increase the likelihood of nding new crystalliza-
tion conditions that may provide new polymorphs or crystals of
better quality [64]. The microseed matrix screen (MMS) method
has become a standard methodology to search for new hits since it
is easily automatized and can be applied to cross-seed between
related and un-related proteins [64].
Other approaches have been tested to overcome the nucleation
energy barrier, as for example inducing heterogeneous nucleation
with different types of the so-called nucleants [65]. Typically
these materials expose a surface that may stabilize the pre-critical
nucleation cluster or even the dense liquid droplet, observed
sometimes under supersaturated conditions, lowering the nucle-
ation energy barrier [66]. There is a wide variety of materials that
have been tested in the search for the universal nucleant [2], from
natural to synthetic. A review of the materials tested as nucleants
can be found in the supplementary material of Fermani and co-
workers [66].
A second approach to screening for initial crystallization con-
ditions and improving crystal quality in a single experiment is
provided by the use of counter-diffusion methods. Because of how
the technique works, starting at high supersaturation values far
from the equilibrium, the coupling of the precipitation and mass
transport under a diffusion controlled regime allows for the low
nucleation density and the slow crystal growth at positions far from
the salt incorporation point. Crystals at this position are typically of
higher quality and bigger size than at positions near to the salt
incorporation point (x0, x1 in Fig. 3) [40,41,67]. The capability of this
approach has been exploited to produce crystals of large size for
neutron diffraction purposes while allowing a gentle interchange of
 J.A. Gavira / Archives of Biochemistry and Biophysics 602 (2016) 3e11
perdeuterated solution [68]. Any condition that produces a micro-
crystalline material in batch or vapour diffusion experiments could
be easily explored to search for single crystals using the CD method
in single capillaries or in commercially available microchips. In
addition to the more extensive exploration of the phase diagram,
the diffusion mass transport regime provides a media that reduces
the incorporation of impurities [42,69,70] and can be exploited also
in combination with seeding techniques to increase the number of
hits or as crystal improvement methodology [71].
Crystallization in gel media will serve in this section as a link
between crystal improvement and crystal handling. Although gel
crystallization is not a frequent practice, the benets of using gel
media (agarose, silica, PEGs base hydrogel, sephadex, etc.) for the
crystallization of macromolecules are multiple - i.e. prevents con-
vection and crystal sedimentation, acts as impurity lter, etc. - and
have been proven to produce crystals of higher quality [72,73].
Since this subject has been recently covered in connection with the
use of peptide-based hydrogels for the production of high quality
protein crystal [74] and the formation of new polymorphs [75] it
will not be discussed here in detail. There are many types of
hydrogels, depending on their nature, that can be used and most of
them are compatible with any crystallization method. Among them
agarose is probably the most widely used due to its simple prep-
aration protocol and availability in any biochemistry laboratory.
Agarose can be found with a wide range of melting and gelling
points and can be used at concentrations below its critical gelling
point, thus facilitating its mixing with the protein solution [76].
Moreover, gels also provide an excellent media for the soaking of
ligands, co-factors, cryo-protectant, heavy atoms, etc. [45,77] while
preserving the integrity of the macromolecule crystals even when
exposed to organic solvents (DMSO) [78]. The use of hydrogels
avoids the manipulation of the crystals and therefore the osmotic
and mechanical stress exerted on the crystals during handling.
Crystal handling is indeed a main issue in protein crystallization.
Since it has an effect in crystal quality, avoiding the loss of crystal
integrity is a way of improving the quality of the diffraction data.
Protein crystals are exposed to mechanical and osmotic stress
during crystal preparation risking its integrity and quality. Room
temperature (RT) data collection is being pursued to avoid the use
of crystal treatment methods such as soaking with a cryo-
protectant or the use of heavy atoms, etc. Owing to the latest ad-
vances on data processing (integration and scaling), especially for
serial femtosecond crystallography, the completion of full datasets
from many different crystals is not an issue [79,80]. Another recent
improvement involves the use of high-transparency micro-pat-
ternable chips, made of silicon nitride, photoresist and polyimide
lm, to load the sample in their mother liquid, facilitating X-ray
data collection while keeping a low background scattering [81].
Other approach involves the use of the graphene-wrapped protein
crystals for the measurement of diffraction data in vacuo, which
minimizes the background and zero absorption of incident and
scattered beams [82].
4. Current trends in protein crystallization
There are many open fronts in the eld of biocrystallization
mainly derived from the incredible amount of instrumentation that
is being developed including the manipulation of micro/nano vol-
umes and particle size, improvement efciency of detection sys-
tems (UVevisible, x-ray, uorescence, SONIC, DDLS, etc.) or the
advent and fast development of various characterization tech-
niques (XFEL, Micro-ED, etc.). The emergent technologies on XFEL,
neutron diffraction, Micro-ED, etc. are expanding and diversifying
the number of approaches that can be used to get the required 3D
model while, their requirements of crystalline material covers from
9
the nano to the millimetre sizes range. Whereas XFEL needs to use
nano-crystals of homogeneous size to be continuously delivered at
room temperature, neutron diffraction requires perdeuterated
crystals of big size and Micro-ED micrometre size crystals under
cryogenic conditions [83,84]. This diversication is pushing on the
demand for a better comprehension theory of biomacromolecules
crystallization. Meanwhile new technical and methodological so-
lutions are being assayed to meet the needs of this diversity of
crystals size.
On this note, serial synchrotron-radiation diffraction at micro-
focus beamlines based on helical scan of single crystals [85] or more
recently from many microcrystal (grown in vivo) mounted on a
standard loop [80], have been used to generate wedges of data that
can be scaled together to obtain complete datasets. In addition to
the great advance in micro-crystallography [83], which implies
cryo-protection, room temperature data collection is also being
routinely used for testing and ultimately for data collection of
multiple crystals thanks to the high efciency of pixel array de-
tectors used in dedicated MX beamlines.
In the last decades, microuidic devices (microchips) have
emerged as a powerful technology in protein crystallization [86].
This technology combines low reagent consumption and high
density of experiments while providing a media in which mass
transport occurs mainly under a diffusion regime. The use of mi-
crochips has enabled the miniaturization of crystallization experi-
ments using sample volumes much smaller than needed with
robotic systems [87] with a high operational speed while maxi-
mizing the number of screening conditions. As already mentioned
standard crystallisation techniques can be implemented using
these devices but its potential is even higher. For example, micro-
chips have been used to carry out fundamental studies such as the
determination of phase diagrams or to study nucleation [88], to
decouple nucleation and growth [86,89,90] or to gather thermo-
dynamic and kinetic data [91]. A step further has been given by
adapting the counter-diffusion (CD) method [46,92] that, as
mentioned above, extensively explores the phase diagram for a
given combination of protein and precipitant cocktail. The avail-
ability of new materials such as a co-polymer of cyclic olen (COC)
with higher transparency to X-rays than poly(dimethylsiloxane)
(PDMS) have aided the construction of a new generation of mi-
crochips for in situ data collection from crystals obtained by
microbatch [44] and counter-diffusion [43,47] methods. The com-
bination of materials (COC-PDMS-Duralar) has been used to facili-
tate the de novo structure determination of a protein from single-
wavelength anomalous diffraction (SAD) data without crystal
manipulation [93]. We may therefore anticipate that microuidic
devices will play a central role in protein crystallization studies
ranging from phase diagram determination, screening and crystal
improvement to in situ data collection including time-resolved
serial crystallography from Laue diffraction [94,95]. Furthermore,
other advances are expected to extend the lifetime of protein
crystals under the beam from a deeper understanding of the
chemistry inside irradiated crystals [96].
Along this line, crystallization in hydrogel gel media should also
be taken into account not only for improving crystal quality but also
to exert control over the nucleation and growth processes. The role
of agarose to induce nucleation and silica gel to inhibit it have
already been observed and documented [40,97,98]. Even more, gel-
grown protein crystals have been recently shown to be excellent
candidates to produce crystals of bigger size for neutron diffraction
studies [99]. In this study protein crystals grown in agarose gel
were seeded into a metastable solution to grow big size crystals. In
a different investigation agarose gels have been proposed as media
for the continuous delivery of nano/micro-metre size crystals
needed for serial femtosecond crystallography for XFEL
10 J.A. Gavira / Archives of Biochemistry and Biophysics 602 (2016) 3e11

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