Post on 26-Apr-2023
Protein crystallization by capillary counterdiffusion forapplied crystallographic structure determination
Joseph D. Ng,a,* Jos�ee A. Gavira,a and Juan M. Garc�ııa-Ru�ıızb
a Department of Biological Sciences and the Laboratory for Structural Biology, University of Alabama at Huntsville, Huntsville, AL 35899, USAb Laboratorio de Estudios Cristalograficos IACT, CSIC-UGRA Facultad de Ciencias, Granada 18002, Spain
Received 5 February 2003
Abstract
Counterdiffusion crystallization in capillary is a very simple, cost-effective, and practical procedure for obtaining protein crystals
suitable for X-ray data analysis. Its principles have been derived using well-known concepts coupling the ideas of precipitation and
diffusion mass transport in a restricted geometry. The counterdiffusion process has been used to simultaneously screen for optimal
conditions for protein crystal growth, incorporate strong anomalous scattering atoms, and mix in cryogenic solutions in a single
capillary tube. The crystals obtained in the capillary have been used in situ for X-ray analysis. The implementation of this technique
linked to the advancement of current crystallography software leads to a powerful structure determination method consolidating
crystal growth, X-ray data collection, and ab initio phase determination into one without crystal manipulation. We review the
historical progress of counterdiffusion crystallization, its application to X-ray crystallography, and ongoing tool development for
high-throughput protein structure determination.
� 2003 Elsevier Science (USA). All rights reserved.
Keywords: Protein crystallization; Counterdiffusion; Cryocrystallography; High throughput; Crystallization cassette
1. Introduction
In the present biotechnology revolution during which
genomic sequences of a variety of species have been de-termined (some examples include Pyrococcus furiosus
(Maeder et al., 1999), Saccharomyces cerevisiae (Mewes
et al., 1997), Caenorhabditis elegans (The C. elegans Se-
quencing Consortium, 1998), Drosophila melanogaster
(Adams et al., 2000), Arabidopsis thaliana (The Arabid-
opsisGenome Initiative, 2000), and human (Venter et al.,
2001), there is an enormous demand to decipher the
three-dimensional structure of their protein gene prod-ucts rapidly and accurately by means of X-ray crystal-
lography. The access to the molecular structure of these
gene products is crucial in building information that will
facilitate predictions of structure and potential function
for almost any protein from knowing its coding sequence.
Such information is essential in understanding the en-
semble of tens of thousands of proteins specified by any
organism�s genome.Advanced recombinant DNA methods, systematic
approaches for protein crystallization, and highly de-veloped X-ray diffraction instruments and procedures
contribute to the intricate link necessary to obtain in-
formation from gene to structure (Fig. 1). The crystal-
lographic structure determination of those proteins that
are able to be overexpressed in a recombinant system
and are soluble in aqueous solution is limited by the
ability to obtain protein crystals. Thus the bottleneck
step for protein structure determination is achievingcrystals that can diffract to atomic resolution (higher
than 3�AA) with reflections that can be readily indexedand reduced for structure factor calculations. In the case
of de novo protein structure determination without a
priori structure information, crystals must not only be
suitable for X-ray diffraction but also be deemed useful
for ab initio phasing (Ng, 2002).
In today�s era of structural genomics, the attitudetoward protein structure determination is unlike that of
the past. Historically, structural pursuits were directed
Journal of Structural Biology 142 (2003) 218–231
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StructuralBiology
* Corresponding author. Fax: 1-256-824-3204.
E-mail address: ngj@email.uah.edu (J.D. Ng).
1047-8477/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.doi:10.1016/S1047-8477(03)00052-2
at targeted proteins with known functions. Exhaustive
effort was expended in the details of each step, from
protein purification to crystallization and structure de-
termination. The entire focus was on deciphering the
structure of one molecule at a time, for which each
biochemical or crystallographic procedure was applied
with great care and precision taking whatever time wasrequired. The first protein structure solved, hemoglobin
from sperm whale, required over 100 years to attain
since it was first crystallized (H€uunefeld, 1840; Perutzet al., 1960).
Conversely, the current demand in structural aims is
to consolidate crystallographic procedures and assemble
methodological pipelines to grow as many protein
crystals as is feasible, determine their three-dimensionalstructures as rapidly as possible, and eventually decipher
the proteins� functions. In particular, proteins that canbe most easily isolated, purified, and crystallized for X-
ray diffraction are examined first while the troubling
ones are reserved for later studies. Thus the modern
approach is working on proteins that are the ‘‘low-
bearing fruits,’’ while the higher and unreachable ones
are not considered immediately for structural analysis.In considering the present demands for structural bi-
ology we have fabricated a technique to screen and op-
timize crystal growth conditions under a restricted
geometry configuration. Protein crystals suitable for X-
ray diffraction and ab initio structure determination canbe obtained by a counterdiffusion mechanism in which
the volumes of the precipitating agent and protein solu-
tion are arranged in juxtaposition to one another inside
an X-ray capillary (Gavira et al., 2002). The counterdif-
fusion technique is a novel approach to effectively screen
for optimal supersaturation conditions under which
protein crystals can be obtained for in situ X-ray analysis.
We describe here the history of this methodology and itsapplication to protein crystallography. In the frame of
high-throughput use, a unique prototype crystallization
cassette is presented here as a potential tool for structural
genomic endeavors.
2. History of counterdiffusion crystallization
Counterdiffusion techniques have been well investi-
gated and explained for over 100 years since the first
description of periodic precipitation pattern formation
in gelled medium (Liesegang, 1897). The historical un-
dertakings highlighting the long-term effort in under-
standing the mechanism of counterdiffusion processes
and its application for protein crystallography are out-
lined in Table 1.Near the end of the 19th century, Liesegang had
observed a self-organizing pattern formed by the com-
plex interplay of diffusion, chemical reaction, and pre-
cipitation known as ‘‘Liesegang rings’’ or ‘‘bands’’ in
circular and linear geometries, respectively. These
structures can be formed by the nonhomogeneous spa-
tial distribution of crystals in a precipitation reaction in
a gel (Henisch, 1970). Ostwald proposed that Liesegangring formation was a coupled event between chemical
precipitation and diffusion in which nucleating particles
in a supersaturated medium would prevail at the ex-
pense of their surrounding products (Ostwald, 1897,
1899). As the result, a supersaturation wave is created,
forming a decreasing local supersaturation gradient with
a diminishing nucleation rate across the system. The
spacing between the regions of nucleation would pro-duce the patterns of rings or bands. Today it is possible
to predict, using mathematical models (Garcia-Ruiz,
1991; Henisch and Garcia-Ruiz, 1986), and simulate
(Ot�aalora and Garc�ııa-Ruiz, 1996, 1997) the spatial be-havior of a diffusion–precipitation system as a function
of time.
In the early 1970s, Zeppezauer and Zeppezauer
(1968) and Salemne (1972) showed that convection canbe minimized using X-ray capillaries and as a result the
quality of protein crystals can be improved. Similar re-
sults were obtained using gelled medium to prevent
convective mass transport (KalKura and Devanaraya-
nan, 1987; Robert and Lefaucheux, 1988). It was not
until about 20 years later that Garc�ııa-Ruiz proposedapplying the counterdiffusion techniques in gelled
Fig. 1. Procedure for direct crystallography from gene to protein
structure. The three-dimensional structure determination of a protein
by X-ray crystallography requires a pipeline of biochemical and crys-
tallographic events. A gene sequence coding for a protein must first be
cloned and produced in a recombinant overexpression system. After
the recombinant protein is purified, crystallization screens must be
performed to produce suitable crystals for X-ray diffraction and phase
calculation. These are the rate-determining steps for deciphering the
three-dimensional structure of the protein by X-ray crystallography.
To date protein crystallization can be performed by conventional
(vapor diffusion and batch) or counterdiffusion methods. Once phase
information is obtained, an initial electron density map can be calcu-
lated for the tracing of the initial protein model. The final structure is
obtained after molecular refinement and validation. The ultimate
model is reported and studied for biological applications.
J.D. Ng et al. / Journal of Structural Biology 142 (2003) 218–231 219
medium to the crystallization of biological macromole-
cules as an alternative method to carry out a wide
screening of experimental crystallization conditions in a
single experiment (Garcia-Ruiz, 1991). In 1993, Garc�ııa-Ruiz and collaborators developed the gel acupuncture
method (Garcia-Ruiz et al., 1993), in which the pre-
cipitant agent permeates through a cushion of gel into a
capillary filled with protein solution. As the counter-diffusion configuration became optimized the idea of
obtaining crystals directly in capillary for acquisition of
X-ray diffraction data was realized.
In the past year we have used the counterdiffusion
crystallization method in capillary to produce protein
crystals for X-ray crystallography. For the first time a
protein was crystallized in a capillary equilibrated si-
multaneously with precipitant, cryoprotectant, and aheavy atom derivative solution by counterdiffusion.
Consequently in situ X-ray data collection of the crystal
was possible and thereafter a de novo structure was
determined using phases calculated by single anomalous
scattering techniques (Gavira et al., 2002).
3. Counterdiffusion vs conventional crystallization ap-proaches
There are a number of devices and procedures to
bring a protein solution to a supersaturated state (for
review see McPherson, 1999). The most commonly used,
with the most success in obtaining protein crystals, are
the batch and vapor diffusion methods (Cudney et al.,
1994). Batch crystallization entails the direct mixing of
an undersaturated protein solution with a precipitant
solution. Supersaturation with respect to the protein is
immediately created as the result of changes in protein
solubility imposed by the precipitant solution (Fig. 2A).
Consequently a crystalline solid state may form if thechemical and physical parameters were well selected.
Vapor diffusion is conducted by mixing protein and
precipitant solutions together in a sitting or hanging
drop supported by some surface and set to equilibrate
against a reservoir of precipitant solution in an enclosed
chamber. As water or other volatile components in the
protein droplet are equilibrated through a vapor phase,
the protein and precipitant concentrations are increased,driving the system toward supersaturation. As the result,
crystals grow out of one single condition (i.e., selected
pH, salt concentration, temperature, etc.) (Fig. 2B).
The counterdiffusion technique is arranged such that
the volumes of the precipitating agent and protein so-
lution are juxtaposition to one another inside an X-ray
capillary (Fig. 2C). The two solutions are set to diffuse
against each other, resulting in a spatial–temporal gra-dient of supersaturation along the length of the capil-
lary. In contrast to conventional techniques, varying
supersaturation conditions leading to protein precipita-
tion, nucleation, and crystal growth can be obtained
simultaneously in the capillary. Therefore crystals
Table 1
Historical events leading to macromolecular crystallization by counterdiffusion
Date Event References
Inorganic materials
1896 Periodic precipitation pattern formation was first described Liesegang, 1897
1897 Self-organization of periodic precipitation formation was
explained
Ostwald, 1897, 1899
1970 Low-solubility substance was crystallized in gelled media Henisch, 1970
1986 Simulation of precipitation and diffusion mass-transport coupling
was demonstrated
Henish and Garc�ııa-Ruiz, 1986
Macromolecules
1968–1972 Protein crystallization was performed in glass capillaries by batch
methods
Zeppezauer and Zeppezauer, 1968; Salemne, 1972
1987 Gelled medium was first used for protein crystallization Robert and Lefaucheux, 1988; KalKura and
Devanarayanan, 1987
1991 Use of gelled medium in a counterdiffusion configuration for
protein crystallization was proposed
Garcia-Ruiz, 1991
1994 GAME, a hybrid technique that uses capillary and gel, was
developed for protein crystallization
Garcia-Ruiz et al., 1993
1996–1997 Precipitation behavior of a protein solution was predicted and
simulated in a counterdiffusion system
Ot�aalora and Garc�ııa-Ruiz, 1996, 1997
1998–2002 Silica and agarose gels were used to crystallize protein by
counterdiffusion
Garc�ııa-Ruiz et al., 1998, 2002
2001 Successful cryo-treatment of protein crystals contained in
capillary was demonstrated
L�oopez-Jaramillo et al., 2001
2002 First protein–nucleic acid complex was crystallized in agarose
medium by counterdiffusion crystallization in capillary
Biert€uumpfel et al., 2002
2002 First protein structure was determined by SAS from crystals
grown and contained in capillary
Gavira et al., 2002
220 J.D. Ng et al. / Journal of Structural Biology 142 (2003) 218–231
obtained by this manner are a consequence of selective
growth from different supersaturation environments
(Garc�ııa-Ruiz et al., 2001).The general strategy for protein crystallization is to
reduce the solubility of the macromolecule from the
solvent such that an extreme supersaturation state is
achieved with respect to the protein. At this point there is
a high probability that critical nuclei will spontaneously
form in solution. In a phase diagram for crystallization
(Fig. 2) the dark red regions of the curve represent the
extreme supersaturation region, often referred to as the
labile region (Feigelson, 1988; Miers and Isaac, 1907).The lesser value of supersaturation is highlighted in light
red in the phase diagram, representing the region where
nucleation events are low and crystal growth is favorable.
This portion of the diagram is the metastable region. In
traditional protein crystallization screens, chemical re-
agents in buffered solution can be randomly, systemati-
cally, or selectively chosen to mix and equilibrate with
varying protein concentrations. In this process, a super-
saturated condition targeting a single labile region for
spontaneous nuclei and a metastable region for thegreatest crystal growth can be acquired. Each crystalli-
zation preparation under conventional methods would
examine only one condition at a time.
The counterdiffusion crystallization procedure, on the
other hand, is able to compose a continuous supersat-
uration gradient. During the diffusion process a super-
saturation state with respect to the protein can be
attained in the labile region, while crystal growth canoccur in a continuous range in the metastable area of the
phase diagram (Fig. 2C). Hence, crystal formation is
caused by the progression of a nucleation front resulting
from the nonlinear interplay among mass transport,
protein crystal nucleation, and growth (Carotenuto
et al., 2002; Garc�ııa-Ruiz et al., 2001).
Fig. 2. Comparison between conventional vapor diffusion and capillary counterdiffusion crystallization. Phase diagrams are illustrated showing the
supersaturated areas in a range of increasing protein concentration as a function of the ionic strength in the protein solution. The labile and
metastable regions are highlighted in dark red and light red, respectively (A–C). The subsaturated region is highlighted in yellow. During the
crystallization process, the protein solution is driven to the supersaturated region by reducing the solubility of the macromolecule from the solvent.
Nucleation proceeds in the labile region and thereafter the solubility of the protein solution falls into the metastable region where crystal growth is
most favorable (a! b! c). Vapor diffusion techniques entail a protein droplet (sitting or hanging) on a coverslip to equilibrate with a precipitatingsalt. The protein droplet initially contains a partial volume of the precipitating solution. During the equilibration process, the protein solution is
reduced rendering the protein insoluble (B). In the counterdiffusion configuration, the precipitant and protein solution are prepared in juxtaposition
to each other inside an X-ray capillary. When the two solutions diffuse against each other, a spatial–temporal gradient of supersaturation is created.
Crystals form by the progression of a nucleation front (C). The capillaries used can range from 0.2 to 1.0mm in diameter with volumes of 1.9 to
47.1ll.
J.D. Ng et al. / Journal of Structural Biology 142 (2003) 218–231 221
4. Crystallization strategies
The tactics to crystallize proteins can be separated
into two parts assuming that a significant quantity of
pure homogeneous protein is available. First the initial
condition must be found under which the protein mol-
ecule can be displaced into a state of supersaturation
followed by an equilibration process that favors minimal
nucleation and optimal crystal growth. The methods ofcrystallizing proteins must usually be applied over a
broad set of conditions, adjusting chemical variations
such as pH, ionic strength, metal ions, or detergents.
Physical factors must also be considered during the
crystallization process, including temperature, gravity,
surfaces, viscosity, dielectric properties, or vibrations.
Biochemical issues also come into play in that the purity,
modification, or aggregation determines the fate ofsuccessful crystal growth. The factors considered here
that influence crystallization are by no means exhaus-
tive. A comprehensive list describing the factors affect-
ing protein crystal growth has been reviewed by
McPherson (1999).
The second part presumes that initial crystallization
conditions have been found to produce protein crystal-
line materials but are not sufficient for X-ray diffractionand in turn not adequate for any subsequent structure
determination. Therefore the optimization of the initial
screening conditions must be performed to improve the
quality of the crystal satisfactorily enough for X-ray
analysis. This entails fine changes in any of the variable
parameters mentioned above that would optimize the
supersaturation state to produce the highest quality
crystal.Initial crystallization conditions have been success-
fully identified with ‘‘sparse matrix’’ or ‘‘incomplete
factorial’’ screens (Carter and Carter, 1979; Carter et al.,
1988) that evaluate a range of pH and precipitant
combinations with minimal macromolecule material. In
view of the principles of conventional vapor comparedto counterdiffusion crystallization techniques, it is easy
to see that more supersaturation conditions are explored
concurrently in the latter procedure during crystalliza-
tion screens. We can consider undertaking an explor-
atory sparse matrix screen with common proteins and
examine the number of successful crystallization trials
obtained using vapor diffusion compared to counter-
diffusion preparations.Model proteins, lysozyme, thaumatin, and insulin,
were individually screened for initial crystallization
conditions using a commercially available sparse matrix
screening kit (Hampton Research, Laguna, CA, USA).
Vapor diffusion crystallization experiments were pre-
pared in both hanging- and sitting-drop configurations
in which the protein solution was set to equilibrate
against a reservoir solution containing the sparse matrixprecipitants. Counterdiffusion crystallization was pre-
pared as described above (Gavira et al., 2002), with the
same precipitant solutions used in the vapor diffusion
trials. During the course of equilibration, all samples
were visually observed for protein crystals that may be
suitable for X-ray diffraction. Droplets or capillaries
that produced microcrystals to small and large single
crystals were considered to be ‘‘hits.’’ Using these modelproteins, there were on the average twice as many hits
acquired in capillaries compared to those obtained in
vapor diffusion droplets containing a variety of both
favorable and unfavorable habits (Fig. 3). These ob-
servations are consistent with our notion that capillary
counterdiffusion can survey broader supersaturation
conditions thereby providing a higher likelihood of
finding crystallization conditions compared to conven-tional techniques.
Once a preliminary set of crystallization conditions
has been established, the next step in producing adequate
crystals for X-ray diffraction is optimization. Conven-
tionally, conditions can be improved by attentively ad-
Fig. 3. Comparative crystallization screens of lysozyme, thaumatin, and insulin against the sparse matrix precipitants of Hampton Research Crystal
Screen I. Both hanging (left) and sitting (right) droplet vapor diffusion crystallization produced fewer occurrences of protein crystals compared to
those trials performed by the counterdiffusion method.
222 J.D. Ng et al. / Journal of Structural Biology 142 (2003) 218–231
justing concentrations of protein and/or precipitant
concentrations in a systematic fashion. In the case of
crystallization screens by vapor diffusion, the contents of
a droplet and/or precipitant reservoir are slightly altered
such that a change in supersaturation can be achieved.
More extensive optimization can include incrementing
other chemical or physical parameters affecting protein
solubility as mentioned previously. The optimization
Fig. 4. Gallery of protein crystals grown in capillary by counterdiffusion. The crystals shown are by no means exhaustive but rather examples of the
best crystals obtained. The sizes of the shown crystals range from 0.05 to 5mm in the longest dimension. In some cases the crystals fill up the diameter
of the capillary. Protein crystals of P. furiosus were obtained with Zhi-Jie Liu and Florian Schubot in the laboratory of B.C. Wang.
Fig. 6. The anomalous difference electron-density maps of iodine-derived thaumatin (left) and lysozyme (right) contoured at 3 r (blue) and 6 r (red).The signals shown correspond to iodine and sulfur scattering atoms.
J.D. Ng et al. / Journal of Structural Biology 142 (2003) 218–231 223
procedure still entails a considerable number of discretetrials with a range of crystallization conditions at fine
intervals.
Crystallization screens that take place in a single
capillary by counterdiffusion techniques, on the other
hand, represent already an integrated range of a protein
and a precipitant concentration mixture. In practice, the
initial concentration of proteins used for capillary crys-
tallization is usually two to three times as high as that inconventional vapor diffusion screening. Immediate pre-
cipitation is usually observed at the protein–precipitant
interface during the counterdiffusion process. If an ap-
propriate precipitant is chosen, within time the proper
protein-to-precipitant concentration proportion is es-
tablished along the length of the capillary where protein
crystals may grow. During the course of equilibration the
optimal condition to produce a useful protein crystal isrevealed and selected immediately for X-ray analysis.
Counterdiffusion techniques have been very effective
in crystallizing a wide number of macromolecules in-
cluding proteins, viruses, and nucleic acid–protein
complexes (for example, see Biert€uumpfel et al., 2002)(Fig. 4) with different molecular weights and isoelectric
points under a wide range of chemical and physical
conditions.
5. Applied crystallography
One may rejoice when crystals suitable for X-ray
analysis have been obtained after screening and optimi-
zation of selected crystallization parameters. However,
for ab initio protein structure determination by X-raycrystallography, more work will be required to prove the
usefulness of the celebrated crystal. Since most protein
crystals are very sensitive to X-ray radiation and will not
survive a long-duration X-ray exposure (especially with a
synchrotron beam source), data collection must be per-
formed under supercooled conditions. Therefore, a
cryoprotectant solution must be used to treat the protein
crystal such that it can tolerate supercooling whilesustaining its ability to diffract X-rays. The crystallog-
rapher is faced with the additional task of trying a series
of cryogenic solutions at different concentrations and
soak times. Furthermore, strong X-ray scattering atoms
intrinsic either to the protein (such as sulfur or metal
ions) or a derivatized atom (such as halides or heavy
metals) must be incorporated in the crystals for ab initio
phasing (Blundell and Johnson, 1976). This imposesfurther rigorous searches whereby different heavy atoms
and their soaking conditions are tried without disrupting
the native crystal. Having strong scattering intensity of
the derived atom and knowing precisely where in the
crystallographic unit cell these atoms are located are
imperative to obtaining phasing information using single
isomorphous replacement (Green et al., 1954), multiple
isomorphous replacement (Harker, 1956), multiwave-
length anomalous scattering (Fanchon andHendrickson,
1991), single anomalous dispersion (Hendrickson and
Teeter, 1981; Smith and Hendrickson, 1983), or single-
wavelength anomalous scattering (Chen et al., 1991;
Wang, 1985).
The model proteins insulin, lysozyme, and thaumatin
have been used to demonstrate the feasibility of growingcrystals in a counterdiffusion geometry against a pre-
cipitating salt while concurrently incorporating a cryo-
protectant and a strong X-ray scattering atom. In all
Fig. 5. Preliminary X-ray analysis on model proteins grown by
counterdiffusion in capillary. Insulin, thaumatin, and lysozyme were
crystallized against precipitants containing cryoprotectant and iodine
or bromide in capillary by counterdiffusion. After 2 weeks of equili-
bration, the protein crystals grew along the length of the capillaries.
One example of a crystal from each protein is shown at the top left.
Insulin, thaumatin, and lysozyme are shown from left to right, re-
spectively, measuring up to 0.3mm in the longest dimension. In the
case of thaumatin and lysozyme, the crystals filled the diameter of the
capillary. The crystallization conditions for insulin have been described
by Gavira et al. (2002). The initial concentration of thaumatin was
100mg/ml in 100mM sodium phosphate, pH 7.0, equilibrated against
30% sodium tartrate, 100mM potassium iodine (or sodium bromide),
and 25% glycerol. In the case of lysozyme, 100mg/ml of the protein
was prepared in 50mM sodium acetate, pH 4.5, and equilibrated
against 20% sodium chloride in the same buffer with 100mM potas-
sium iodine (or sodium bromide) and 25% glycerol. In all cases, the
capillary containing the selected crystals was mounted on a goniometer
and flash-cooled immediately in a cryogenic stream at 100K as shown
at the top left. The region containing the optimal crystal for X-ray
diffraction was excised very carefully with a sharp glass cutter without
shattering the capillary or damaging the crystal. Diffraction data were
collected using an MSC R-AXIS IV image plate detector. A typical 1�oscillation diffraction photograph of the insulin crystal diffracting to
2.3�AA is shown at the bottom left. A high-redundancy data set for in-
sulin was collected for data indexing and reduction with HKL 2000
(Otwinowski and Minor, 1997). The bottom right displays the resulting
anomalous difference Patterson map identifying the iodine anomalous
signals. The anomalous peaks on the Harker section y ¼ 0:0 contouredfrom 3 to 11 r in 1-r increments were calculated and visualized withXTALVIEW (McRee, 1999).
224 J.D. Ng et al. / Journal of Structural Biology 142 (2003) 218–231
cases, iodine or bromide salts were mixed with the pre-cipitating solution including glycerol as the cryoprotec-
tant (examples of effective halide soaking have been
reported by Dauter et al. (2001)). The precipitating salt
mixtures were carefully deposited in the capillary,
making contact with the protein chamber, forming a
liquid–liquid free-diffusion system (for detail of prepa-
ration see Gavira et al., 2002). During the equilibration
process, a supersaturation wave is activated along theprotein chamber where the salts initially diffuse into the
protein volume allowing nucleation, crystallization, and
glycerol treatment to occur in sequential events.
Protein crystals can be observed in as early as a few
days along the length of the capillary. Even though
crystals suitable for X-ray diffraction can be obtained in
a few days, at least 2 weeks of equilibration time is re-
quired for sufficient cryoprotection. The crystals grownin the capillary range from 0.05 to 0.5mm in the longest
dimensions, at which the largest crystals filled the di-
ameter of the capillary (Fig. 5). Single X-ray diffraction
images can be obtained at room temperature for each
crystal in the capillary to initially evaluate the best
crystal quality in terms of resolution limit and mosaicity.We have observed and demonstrated (Gavira, 2000;
L�oopez-Jaramillo et al., 2001) that crystals along the spanof the capillary were not of equivalent quality when
analyzed. This is not entirely too surprising since crys-
tals grown along the capillary length will experience
different supersaturation environments.
The most striking advantages of the simultaneous
process of protein crystallization, high X-ray scatteringatom incorporation, and cryoprotection are threefold.
First, crystals remain in a stable environment at all times
while never being exposed to drastic chemical changes or
subjected to physical manipulation. Second, protein
crystals can be quickly evaluated by in situ X-ray dif-
fraction from their original growth environment. Fi-
nally, since the entire crystallization and incorporation
process is done in a closed chamber, the investigator isnever exposed to any harmful chemicals after the
equilibration process has been initiated. Therefore, the
entire setup is safe and easy to transport. Compared to
conventional methods, the tedious manipulation of
transferring protein crystals to different solutions fol-
Table 2
Data processing and ISAS data statistics from synchrotron data
Bromide Iodine
Insulin Lysozyme Insulin Lysozyme Thaumatin
Wavelength (�AA) 0.91948 0.91935 1.7712 1.7700 1.7712
Space group I213 P43212 I213 P43212 P41212
Unit-cell parameter (�AA) a ¼ 77:6262 a ¼ 79:1074,c ¼ 36:9802
a ¼ 78:5817 a ¼ 79:1527,c ¼ 37:0296
a ¼ 58:1588,c ¼ 150:7039
Resolution range (�AA) 20.0–1.6 25.0–1.3 25.0–2.3 25.0–2.1 20.0–1.85
No observations 109 256 228 978 18 906 115 943 159 172
No unique reflections 10 467 29 433 3725 7495 21 371
Completeness (%)
Overall 99.5 99.9 99.4 95.2 99.7
Lowest shell 94.9 (20.0–3.4�AA) 98.9 (25.0–2.8�AA) 98.0 (25.0–5.0�AA) 95.5 (25.0–4.5�AA) 99.5 (20.0–4.0�AA)
Highest shell 100.0 (1.66–1.60�AA) 100.0 (1.35–1.3�AA) 99.7 (2.38–2.30�AA) 75.0 (2.18–2.10�AA) 34.8 (1.92–1.85�AA)
Rmergeð%ÞaOverall 8.3 5.9 7.2 8.2 9.7
Lowest shell 6.3 2.9 5.9 9.1 8.5
Highest shell 34.7 32.9 32.2 17.7 24.5
I=rI
Overall 17.13 31.3 13.2 17.5 13.4
Lowest shell 17.8 50.2 15.9 14.9 18.7
Highest shell 6.8 6.9 3.6 6.7 5.3
Redundancy 10.4 7.8 5.1 15.5 7.5
Crystal mosaicity (�) 0.735 0.182 1.012 0.665 0.318
ISAS data statistics
No. of scatters 3 2 1 2 5
f.o.m 0.71 0.75 0.66 0.7 0.77
R (inverse map) 0.323 0.217 0.363 0.286 0.206
Corr. Coeff. 0.941 0.977 0.903 0.96 0.98
X-ray data were recorded at line X4A at the National Synchrotron Light Source with a Fuji BAS2000 scanner. The data were processed (indexed,
integrated, and scaled) with the HKL2000 package (Otwinowski and Minor, 1997).aRmerge ¼
Phkl
Pi jhIi � Iij=
Phkl
PiðIiÞ.
J.D. Ng et al. / Journal of Structural Biology 142 (2003) 218–231 225
lowed by crystal mounting on cryoloops prior to X-rayanalysis is completely eliminated.
5.1. Data collection
Procedures in X-ray data collection of crystals ob-
tained by counterdiffusion capillary crystallization are
very similar to those performed with conventional cap-
illary-mounted crystals. There are, however, some un-ique technical considerations for cryogenic X-ray data
collection of protein crystals in capillary. The authors
suggest the optimal size for growing crystals in capillary
and subsequent X-ray data collection is 0.3mm. Capil-
laries with diameters smaller than 0.3mm have a high
propensity to vibrate in the presence of a strong cryo-
stream, rendering X-ray data collection impractical. It is
recommended in this case to excise a small region of thecapillary containing the targeted crystal or crystals. Only
a region of about 1 cm of the capillary can be efficiently
supercooled in the cryostream. The truncated capillary
piece may then be easily mounted and centered in the X-
ray beam with the cryostream without any further dif-
ficulties (Fig. 5). If the capillary diameter is bigger than
0.3mm we often observe more ice buildup around the
crystals due to a significant temperature gradient. Fi-nally, the outer surface of the capillary must be clean
and free of any dust, oil, or other contaminants. In the
cryostream, there is a tendency for ice to nucleate on
dirty surfaces that will interfere with the X-ray data
collection. Usually, a quick casual wipe of the capillary
surface with ethanol will prevent this problem.
Complete data sets were recorded from a single
crystal for each of the three different proteins studiedwith over 10-fold redundancy using the laboratory as
well as synchrotron X-ray sources. All data were col-
lected with a selected rotation range that produced a
highly redundant anomalous data set without employ-
ing an inverse-beam technique, giving rise to excellent
data collection statistics. Those data that were collected
using synchrotron radiation are shown in Table 2. Fig. 5
displays an example diffraction pattern of insulinshowing the fine and low mosaicity reflection spots in
the resolution range analyzed. In all cases, there is
slightly more background noise observed in the diffrac-
tion images from crystals grown in the capillary com-
pared to those analyzed in a cryoloop. The capillary
glass and the excess solution contribute slightly to de-
creasing the intensity over background error ðI=sÞ in allresolutions. Nonetheless, the reflections from each dataset were easily indexed and reduced.
5.2. Structure determination
Ab initio phase determination and initial electron-
density map calculations were performed with the com-
plete X-ray data sets obtained from insulin, thaumatin,
and lysozyme crystals grown in capillary. Immediatelyafter data collection, crystals were evaluated as to whe-
ther they would be useful for structure determination by
calculating Bijvoet difference Patterson maps to identify
any significant anomalous signals. Fig. 5 displays an ex-
ample of the Patterson map generated by the insulin data
set on the Harker section at y ¼ 0:0. In all cases of themodel protein crystals analyzed, the initial halide atom
positions were identified and refined. Consequently, theinitial phases and their respective anomalous difference
Fourier (ADF) maps were generated to find other strong
electron-density peaks corresponding to additional ha-
lide sites. All the identified halide sites found in the three
types of protein crystals were used for the final phase
calculation for each molecule. Fig. 6 shows examples of
ADF maps calculated for thaumatin and lysozyme,
showing clearly the strong scattering atoms used forphase determination. Protein crystals derived with iodine
produced ADF maps better than those of bromide-
derived crystals as shown in our preliminary study with
insulin crystals collected with a home X-ray source
(Gavira et al., 2002).
The ab initio phase determination and initial electron-
density map calculations were best performed with an
iterative algorithm (ISAS) that extracts phases from in-tensity information of single-wavelength anomalous
scattering data (Wang, 1985). The ISAS procedure was
conducted in seven steps as described by Wang (1985)
and Liu et al. (2000) to estimate the image of the protein
using the anomalous scattering signal of incorporated
iodine or bromide by solvent flattening (Gavira et al.,
2002). The initial experimental ISAS electron-density
maps for insulin and thaumatin are shown in Fig. 7, re-vealing the clear definition of the solvent region and high
degree of connectivity for unambiguous model tracing.
6. Considerations for high-throughput crystallography
The goal of today�s structural genomics projects is tocrystallize as many recombinant proteins as possible forX-ray analysis and subsequently decipher their three-
dimensional structures rapidly and efficiently. Once
protein crystals suitable for X-ray diffraction are
achieved, the crystallographic rate-determining step is
obtaining phase information. Currently, there are many
softwares for macromolecular crystal structure deter-
mination available that can be pipelined for high-
throughput goals. Crystallographic modules in structuredetermination can be tied together with user-friendly
interfaces among common crystallographic packages
with common formats.
We have concentrated our efforts on producing pro-
tein crystals appropriate for providing phase informa-
tion from anomalous signals. Once an adequate data set
has been indexed and reduced from a selected crystal
226 J.D. Ng et al. / Journal of Structural Biology 142 (2003) 218–231
grown in capillary, we have commonly used the com-
bination of XTALVIEW (McRee, 1999), PHASES(Furey and Swaminathan, 1997), and the CCP4 (Col-
laborative Computational Project 4, 1994) package to
identify and refine anomalous scattering-atom positions.
In our case, known positions of incorporated halides
(sulfur in some cases) are used in the ISAS package to
produce an electron-density map for initial model
building visualized by O (Jones et al., 1991) or XTAL-
VIEW. The sequential modules in phase determinationcan be tied together with a user-friendly interface such
that information acquired from any one step can be fed
back to the preceding steps for optimization. Fig. 8
outlines a strategy commonly used in calculating phase
information leading to an initial model. Using this
manual-feedback procedure an initial electron-density
map adequate for model building can be obtained in as
fast as 30min starting with an adequately scaled dataset. The processes described here can easily be auto-
mated and streamlined.
7. Counterdiffusion crystallization cassette
There is a great demand to develop tools to expedite
the process of growing protein crystals and determine
which of those crystals can provide information on both
the amplitudes and the phases of the diffracted X-rays
for structure determination. We have constructed a
crystallization cassette that uses counterdiffusion crys-tallization to obtain protein crystals that can be imme-
diately used for X-ray analysis.
The crystallization cassette is a device with which
multiple capillaries can be used to take up many sam-
ples of proteins at a time and mix them with several
crystallization reagents simultaneously. The cassette has
a cylindrical design and is made up of two sections that
are joined together. The upper section contains multiplecapillary tubes that extend downward. The lower
section is a disc having multiple slots, with each slot
corresponding to a capillary. The slots are about 50 lland are designed to hold the precipitant solution
cushioned with a wax that is placed in the bottom of
the slots. The precipitant solution includes the precipi-
Fig. 7. Experimental ISAS electron-density maps contoured at 1 r for thaumatin (left) and insulin (right) at 2.5 and 3.0�AA resolution, respectively,from synchrotron data. The experimental density shows a high degree of connectivity and definition of the solvent region fulfilling two main re-
quirements for ab initio structure determination. To illustrate the accuracy of the density map, the refined coordinates of the model proteins obtained
from the Protein Data Bank (code 1THW for thaumatin and 5LYZ for lysozyme) were laid over the experimental density.
Fig. 8. Process commonly used in calculating phase information
leading to an initial model. Sequential modules of crystallographic
software (capital letters) are tied together such that information
(lowercase letters) obtained from one step can be fed back to preceding
steps for optimization (ahttp://shelx.uni-ac.gwdg.de/SHELX/; bFurey
and Swaminathan, 1997; cMcRee, 1999; dWang, 1985; eJones et al.,
1991; fCollaborative Computational Project 4, 1994; gTerwilliger and
Berendzen, 1999; and hLamzin and Wilson, 1993.
J.D. Ng et al. / Journal of Structural Biology 142 (2003) 218–231 227
tating salt and can contain the cryogenic reagent and/orthe heavy atom salt. After the desired precipitant so-
lution is placed within each slot a latex tape is placed
across the surface of the disc covering each slot. A drop
of protein solution is placed on the latex tape above
each slot and the upper section is attached to the lower
section so that the tips of the capillaries are in contactwith the protein solution. The protein solution is then
taken up into the tube by capillary action. After the
protein solution enters the capillary, the upper section
pierces the latex tape so that the capillary tips contact
the precipitant solution and equilibration between the
Fig. 9. Crystallization cassette using counterdiffusion in capillary. The entire hardware showing the attached upper and lower chambers is shown in
two different views (top left). The process of loading, activating, and deactivating during the crystallization events is illustrated in sequential order
from left to right, top to bottom. The entire apparatus is built from plastic and X-ray glass capillary tubes.
228 J.D. Ng et al. / Journal of Structural Biology 142 (2003) 218–231
protein solution and the precipitation solution occurs.After equilibration the capillary tips are placed in
contact with the soft clay contained in the bottom of
the slots, thereby sealing the capillaries, and the cassette
containing the protein crystals is now ready for X-ray
diffraction. Fig. 9 illustrates the sequences in the load-
ing, activation, equilibration, and termination events
executed by the capillary crystallization cassette.
The crystallization cassette holds several outstandingfeatures. First the lower disc of the apparatus is designed
to be preloaded with common precipitants for one-time
use only and completely disposable. Each disc can ac-
commodate any 12 different precipitant solutions ready
to diffuse against a protein solution prepared in a cap-
illary. It is feasible to use four discs to examine 48 dif-
ferent conditions as is commonly done with sparse
matrix crystallization screens. Second, loading the pro-tein solution is fast and easy, relying solely on capillary
action to draw in the protein. Finally, the loading of the
protein solution, union of the precipitant solution, and
final closure of the system are done in continuous steps
without step-wise manipulations.
Each capillary can be withdrawn from the cassette
and crystals grown can be immediately subjected to X-
ray diffraction analysis. The cylindrical geometry of thecassette has been designed to be attached to a rotating
adapter by which each capillary can be positioned in
front of the X-ray beam for diffraction analysis without
its removal from the cassette (Ng et al., 2003). The
search for initial crystallization conditions or crystal
growth optimization (including cryogenic soaking and
incorporation of atoms useful for phasing) of a large
number of proteins can be performed in the capillarycrystallization cassettes for direct X-ray analysis and
structure determination.
8. Conclusions
Structural genomics will depend by and large on
high-throughput determination of structures by X-ray
crystallography. As the result, X-ray crystallographers
are carrying out large-scale programs to determine the
three-dimensional structures of all proteins (Burley and
Bonanno, 2002). This entails searching for new toolsand methods for effective high-throughput processes.
The need to get through the bottleneck steps more effi-
ciently in macromolecular structure determination has
prompted us to investigate old concepts involving pre-
cipitation and diffusion mass transport in a restricted
geometry for X-ray crystallography applications.
The method of using counterdiffusion in capillary to
crystallize macromolecules has been well known and isnot a novel concept for today. However, it has been only
in recent years that this technique was shown to be
useful for determining three-dimensional structures of
biological molecules by direct crystallographic analysis.The underlying goal of present-day structural biology is
to automate and formulate pipeline processes from
crystal growth to structure determination. Currently a
pipeline that bridges automated crystallization, crystal
harvesting, and crystallographic data analysis does not
exist.
High-throughput protein crystallization has been re-
alized by a new generation of robots that are capable ofperforming tens of thousands of trials a day in minia-
turized nanoliter-volume experiments. Once crystals are
obtained, further screens must be performed on larger
scales and the resulting crystals must be harvested and
prepared for data collection (including heavy atom
soaking or cryogenic freezing). The task of harvesting
and transferring crystals from multiwell plates has al-
ways been a severe automation problem. Transport ofcrystals then becomes an issue since, in most cases,
structural genomics efforts will rely on synchrotron ra-
diation for efficient collection of diffraction data. Fi-
nally, prior to data collection at a radiation source,
crystals must be carefully mounted and centered.
Automated approaches to data collection have be-
come possible with the availability of dependable syn-
chrotron sources with stable X-ray optics, high-precisiondiffractometers, and fast scanning detectors. Once data
sets of acceptable quality have been collected and pro-
cessed, automated techniques for phase determination,
electron-density map calculations, model building, and
refinement at high resolution are now emerging such that
manual manipulation is no longer required.
The utilization and tool development for protein
crystallization by counterdiffusion in a restricted geom-etry can be the link that bridges automated crystallization
to crystallographic analysis. In the process of creating a
continuous pipeline frommacromolecular crystallization
to crystallographic structure solution, the counterdiffu-
sion crystallization procedure is an effective means to
produce protein crystals suitable for X-ray analysis
without harvesting and transporting challenges. Thus
developing tools to crystallize macromolecules bycounterdiffusion along with the advances in protein ex-
pression and purification, X-ray radiation, and crystal-
lographic software packages, the full streamlining of
tedious and time-consuming events in determining three-
dimensional structures will be possible.
Acknowledgments
This work was supported by NASA, Alabama
Structural Biology Consortium NSF-EPSCoR, NIH
NIGMS Structural Genomic Project, Alabama SpaceGrant Consortium, and the Secretaria de Educaci�oon yCiencia of Spain. We thank Zhi-Jie Liu and Bi-Cheng
Wang for the usage and guidance of the ISAS crystal-
J.D. Ng et al. / Journal of Structural Biology 142 (2003) 218–231 229
lography package. We also express our gratitude toCraig Ogata for his invaluable assistance at beam line
X4A at the National Synchrotron Light Source,
Brookhaven National Laboratory, and Zbigniew Da-
uter and Edward Meehan for many insightful discus-
sions. We greatly appreciate the technical support of
Joyce Looger and Diana Toh. The fabrication of the
crystallization cassette would not have been possible
without the engineering expertise of Mark Wells andGreg Jenkins. Finally we thank the laboratories that
have shared their results and experiences with counter-
diffusion capillary crystallization.
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