Protein crystallization by capillary counterdiffusion for applied crystallographic structure...

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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Journal of

StructuralBiology

* Corresponding author. Fax: 1-256-824-3204.

E-mail address: [email protected] (J.D. Ng).

1047-8477/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.doi:10.1016/S1047-8477(03)00052-2

mail to: [email protected]

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.


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.


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.


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).


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.


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


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.


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.


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-


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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