research papers
Acta Crystallographica Section D

Biological
Crystallography

The plug-based nanovolume Microcapillary Protein
Crystallization System (MPCS)

ISSN 0907-4449

Cory J. Gerdts,a,b,c Mark
Elliott,a,b Scott Lovell,b,d Mark B.
Mixon,b Alberto J. Napuli,eBart L. Staker,d Peter Nollertc
and Lance Stewarta,b,d*aAccelerated Technologies Center for Gene to
3D Structure, USA, bdeCODE biostructures Inc.,
7869 NE Day Road West, Bainbridge Island,
WA 98110, USA, cEmerald BioSystems Inc.,
7869 NE Day Road West, Bainbridge Island,
WA 98110, USA, dSeattle Structural Genomics
Center for Infectious Disease, USA, ande
Department of Biochemistry, University ofWashington, Seattle, WA 98195, USA

Correspondence e-mail: lstewart@decode.com

The Microcapillary Protein Crystallization System (MPCS)
embodies a new semi-automated plug-based crystallization
technology which enables nanolitre-volume screening of
crystallization conditions in a plasticware format that allows
crystals to be easily removed for traditional cryoprotection
and X-ray diffraction data collection. Protein crystals grown inthese plastic devices can be directly subjected to in situ X-ray
diffraction studies. The MPCS integrates the formulation of
crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the
microfluidic circuitry of a plastic CrystalCard, 10–20 nl
volume droplets are generated, each representing a
microbatch-style crystallization experiment with a different
chemical composition. The entire protein sample is utilized in
crystallization experiments. Sparse-matrix screening andchemical gradient screening can be combined in one comprehensive ‘hybrid’ crystallization trial. The technology lends
itself well to optimization by high-granularity gradientscreening using optimization reagents such as precipitation
agents, ligands or cryoprotectants.

Received 17 June 2008
Accepted 2 September 2008

PDB Reference: methionineR-sulfoxide reductase, 3cxk,
r3cxksf.

1. Introduction
The field of structural biology is generating technologies that
increase throughput and efficiency each year. Such advances
have initiated a goal to progress from gene to three-dimensional structure in 3 d. In an effort to improve efficiency, one
may want to minimize the volume of protein required suchthat sufficient material for crystallization screening and optimization can be obtained from cell-free synthesis. With the‘three-day’ structure goal in mind, the Accelerated Technologies Center for Gene to 3D Structure (ATCG3D) is engaged
in the development of several technologies to increase efficiency in the gene-to-structure pipeline, including synthetic
gene design (Gene Composer; http://www.genecomposer.net/),protein crystal imaging (DETECT-X; Emerald BioSystems)
and microfluidic nanovolume crystallization (The Microcapillary Protein Crystallization System; MPCS). Here, we
describe the first working prototype of the MPCS, which
consists of a microsyringe pumping system, the pumping
system’s associated software (MicroPlugger) and plastic
CrystalCards that house the ‘3+1 mixer’ (where reagents are
combined) and microfluidic channel (where experiments are
stored). The CrystalCards are manufactured with materials
that enable a balance between X-ray transmission, opticalclarity, moldability, chemical resistance and surface energy.
This system produces diffraction-ready crystals by allowing
crystal extraction from the CrystalCard or in situ X-ray1116

doi:10.1107/S0907444908028060

Acta Cryst. (2008). D64, 1116–1122

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diffraction and structure solution using diffraction dataobtained from samples within the CrystalCard.

2. BackgroundThe underlying technology used to perform microfluidic
nanovolume crystallization discussed here was developed at
the University of Chicago (in the ATCG3D-collaborating
Ismagilov laboratory). Aqueous droplets, here called plugs,
form spontaneously by combining microfluidic aqueous
streams with an immiscible and biologically inert fluorocarbon
solution (Song et al., 2003; Tice et al., 2003, 2004). Three
separate aqueous streams of protein, buffer and precipitant
flow simultaneously into a single channel, where a controlled
flow of immiscible and biologically inert fluorocarbon breaks
the combined aqueous streams into 10–20 nl microbatch-style
crystallization experiments (Fig. 1a; Zheng et al., 2003; Zheng,
Tice, Roach et al., 2004). The region where the streams
converge is called the ‘3+1 mixer’. The 10–20 nl crystallizationplugs formed at the ‘3+1 mixer’ are incubated and monitored
for crystallization (Fig. 1b). By controlling the flow rates of theaqueous streams, very fine concentration gradients are
generated over a series of plugs (Fig. 1c), allowing the crystallographer to carefully probe crystallization phase space forcrystal optimization. Plug-based crystallization permits sparsematrix screening (Zheng & Ismagilov, 2005; Zheng, Tice &Ismagilov, 2004) using a pre-formed cartridge of plugs, theformation of fine concentration gradients (Zheng et al., 2003)
via on-chip formulation of plug composition and a combination of sparse-matrix and gradient screening termed the‘hybrid method’ (Li et al., 2006), the latter of which enables
comprehensive screening of crystallization phase space in one
single crystallization trial. Plug-based microfluidic crystallization has also been used to probe scientific questions
regarding protein-crystal nucleation and growth (Chen et al.,2005; Zheng et al., 2005; Gerdts et al., 2006) and to generatecrystals for novel protein structures (Gerdts et al., 2006).

3. The MPCS
As a technology development center, ATCG3D has developed
the Microcapillary Protein Crystallization System (MPCS) in
order to make plug-based protein-crystallization technology
available to the scientific community. The MPCS instrumentation is composed of three essential parts (Fig. 2): (i) apumping system, (ii) the MicroPlugger control software andFigure 1
Plug-based protein crystallization using the MPCS. (a) A microphotograph of crystallization trials in plugs being formed in the CrystalCard.
(b) Dyed protein crystals in the MPCS CrystalCard. (c) A schematic
drawing (left) of a channel containing a smooth pH gradient (right)
generated using a low-pH buffer, a high-pH buffer and a universal pH
indicator.
Acta Cryst. (2008). D64, 1116–1122

Figure 2
MPCS components. (a) A picture of the components of the MPCS system.
(b) Screenshots of the operational modes of the MicroPlugger software.
(c) A picture of the MPCS CrystalCard filled with dyed protein crystals.
(d) A picture of the PDMS/Teflon CrystalCard typically used for the
hybrid method.
Gerdts et al.  Microcapillary Protein Crystallization System1117

research papers(iii) the CrystalCard. The pumping system is a four-channel
syringe-pump system that delivers precise and continuous
flows to the MPCS CrystalCard. This system allows independent control over each channel and can generate extremely
fine gradients. Pumps are controlled by the MicroPlugger
pump-control software (Fig. 2b). The MicroPlugger software
allows the user to control the pump system in four different
operational modes: Prime Mode (for priming the lines),
Constant Mode (for constant-flow experiments), GradientMode (for generation of fine gradients) and Hybrid Mode (forsparse-matrix screening using the hybrid method). These
operational modes allow the user to perform all MPCS
experiment styles. The third essential component is theCrystalCard (Figs. 2c and 2d), which is the ‘microfluidic chip’where the aqueous and fluorous solutions come together tospontaneously form the 10–20 nl plugs at the 3+1 mixer. The
CrystalCard was designed with a hydrophobic surface that
supports the formation of aqueous plugs and contains a long
microfluidic capillary for storage and inspection. Access todiffraction-ready crystals is gained via physical crystal
extraction from the CrystalCard or by in situ X-ray diffraction.
There are two versions of the MPCS CrystalCard: a plastic
CrystalCard and a PDMS/Teflon CrystalCard. The plastic
CrystalCard (Fig. 2c) is used primarily for fine-gradientscreening and the PDMS/Teflon CrystalCard (Fig. 2d) is
typically used for hybrid screening and detergent-based
membrane-protein formulations. (The PDMS/Teflon CrystalCard has the necessary connection schemes and surface
properties to be compatible with hybrid screening anddetergent-based crystallizations.)
Using the pumping system, software and CrystalCards, theMPCS generates hundreds of distinct crystallization experiments within a few minutes, free from cross-contamination,using only 5 ml protein solution. The MPCS does not waste
any precious protein, because there isno dead volume in the system: theprotein sample is aspirated directly intoa Teflon needle and then displaced in its
entirety into the CrystalCard. Additional features of the MPCS include theability to perform microfluidic seeding(Gerdts et al., 2006) and the control ofevaporation by controlling the humidityof the CrystalCard’s storage container(CrystalCards stored in a humidified
container remain unchanged for at least
two months).

styles of crystallization screening: fine-gradient screening andhybrid screening.
3.1.1. Fine-gradient/optimization screening. The MPCS
gradient mode allows the crystallographer to very finely scan aregion of crystallization phase space. Since the aqueous
streams used in an MPCS experiment are independently
controlled using the MPCS pumping system and the MicroPlugger software, concentration gradients over a series ofplugs are easily formed by changing the flow rates of theindividual streams (Fig. 1c; Zheng et al., 2003). As the precipitant stream decreases in flow rate, the buffer streamincreases in flow rate such that that the sum of the flow rates
remains constant (although the MicroPlugger software allows
the user to design a customized experiment to suit their needs,
including changing the plug size or the sum of the aqueous
flow rates). Using a fine gradient, the crystallization phasespace of a particular protein–precipitant combination can be
carefully interrogated for crystal optimization or mapped out
to show the transition from precipitation to microcrystals tosingle crystals (Fig. 3). In the same manner, so-called reverse
screening can be carried out utilizing two crystallization
cocktails that have been found to promote the formation of
crystals (Stura et al., 1994). On-chip formulation of concentration gradients can be used for the optimization of precipitants, ligands, protein partners or cryoprotectants. By reducing
the volume of protein required for crystal optimization, finegradient formation can decrease protein-production requirements when crystals of different protein mutants or DNA
complexes are required.
3.1.2. Hybrid screening. Hybrid screening combines onchip formulation of gradients with sparse-matrix screening (Liet al., 2006). Sparse-matrix screening in plugs is achieved bygenerating a ‘pre-formed cartridge’ of plugs composed ofdifferent crystallizing agents (Zheng & Ismagilov, 2005). In the

3.1. MPCS experiment styles

Plugs formed using the MPCS aremicrobatch-style crystallization experiments in which protein and crystallizing
agent are combined and incubatedwhile surrounded by an oil (fluorocarbon, FC40; Li et al., 2006; Yadav etal., 2005). The MPCS facilitates two

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Figure 3
Mapping of crystallization phase space using a fine gradient for ribose-phosphate pyrophosphokinase. A precipitant gradient demonstrates a smooth transition from precipitation (lower left)
to microcrystalline precipitation (lower right) to microcrystals (middle left) to a few crystals (upper
right) to single crystals (upper left) of ribose-phosphate pyrophosphokinase.

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Acta Cryst. (2008). D64, 1116–1122

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Table 1
Crystallographic data.
Values in parentheses are for the 2.00–1.90 Å resolution shell for lysozyme andthe 1.76–1.70 Å shell for methionine-R-sulfoxide reductase.
Lysozyme
Data collection
Unit-cell parameters (Å)

Methionine-Rsulfoxide reductase

a = 42.00, b = 45.17,
a = b = 79.18,
c = 45.40,  = 88.4,
c = 38.38,
 = 83.7,  = 69.1
 =  =  = 90
P43212 (No. 96) P1 (No. 1)
50–1.90
50–1.700.979500.9793254338
11818110151
32539
11.4 (2.9)
23.1 (2.2)
13.7 (58.4)
6.8 (42.3)
98.8 (98.5)
95.3 (87.4)
5.4 (5.0)
3.6 (3.3)
24.1
22.1

Space group
Resolution (Å)
Wavelength (Å)
Total reflections
Unique reflectionshI/(I)i
Rmerge† (%)
Completeness (%)
Redundancy
Wilson B factor (Å2)
Refinement
Resolution (Å)
50–1.90
Reflections (working/test)
9412/480
Rworking/Rfree‡ (%)
19.6/23.0
No. of atoms (protein/solvent)1001/45
R.m.s. deviations
Bond lengths (Å)
0.016
Bond angles ( )1.607
Average B factor (Å2)
All atoms
28.8
Protein
28.5
Water
35.9
Coordinate error based
0.149
on Rfree (Å)
Ramachandran analysis (%)
Most favored (chain A/B)
89.4
Additionally allowed (chain A/B) 10.6

50–1.7030828/1650
16.6/19.9
2082/180
0.015
1.408
28.3
27.5
37.0
0.095

91.7/90.8
7.3/8.3

P P
P P† Rmerge =hkl
i jIi ðhklÞ  hIðhklÞij=hkl
i Ii ðhklÞ, where Ii(hkl) is the intensity
measured for the ith reflection P
and hI(hkl)iis the average intensity P of all reflections withindices hkl. ‡ Rworking =hkl jFobs ðhklÞj  jFcalc ðhklÞj =hkl jFobs ðhklÞj; Rfree iscalculated in an identical manner using 5% of randomly selected reflections that were
not included in the refinement.

along their longest axis (the channel dimensions are 200 
200 mm). Because the crystals are diffraction-ready, it is criticalto have method(s) for obtaining diffraction data without
scaling up the experiment or translating the crystal hit toanother experimental technique. The MPCS offers two
methods for obtaining X-ray diffraction data from crystals
grown in plugs: traditional cryocooling by crystal extraction
and in situ diffraction.
4.1. Protein-crystal extraction from the MPCS CrystalCard

Protein crystals can be extracted directly from individual
plugs in the MPCS CrystalCards. A thin plastic layer is bonded
to the plastic part that contains the microfluidic circuitry. This
thin layer has a bond that is designed to be strong enough toprevent fluid from leaking out of the CrystalCard during the
crystallization trial but weak enough to be manually peeled off(Figs. 4a and 4b). Additionally, the plugs are retained in theplastic part containing the microfluidic circuitry. After the thinplastic layer has been peeled back, the crystals are exposed
and the desired crystal(s) are accessible to microtools such as
cryoloops (Figs. 4c and 4d). Two techniques can be used toremove the crystals for cryoprotection: (i) the crystal(s) can be
scooped out with a cryoloop and placed in the desired cryoprotectant or (ii) 1 ml of the desired cryoprotectant can be
pipetted on top of the plugs containing crystals so that when
the crystal is scooped out it is already immersed in cryoprotectant. Using the first technique, evaporation of the plugs
is delayed by 5 min by the layer of fluorocarbon oil. Using
the second technique, evaporation of the plugs is delayed forat least 20 min, providing ample time to extract many crystals.

hybrid method of the MPCS, the pre-formed cartridge
containing a series of 100 nl plugs flows into one of the three
aqueous inlets of the 3+1 mixer. Using the Hybrid Mode of theMicroplugger software, a concentration gradient of eachcrystallizing agent in the pre-formed cartridge is formed bycoordinating the flow-rate change between the pre-formed
plugs and the buffer stream. Performing sparse-matrix andgradient screening in the same trial allows the MPCS to scan asubstantial portion of crystallization phase space, generating
20–40 individual crystallization experiments at different
concentrations from each pre-formed crystallant plug.

4. Producing diffraction-ready crystals
The MPCS is more than a low-volume protein-crystallization
screening tool: it produces diffraction-ready crystals. The
experimental volumes (10–20 nl; the exact volumes are
dependent on the CrystalCard and the flow rates) are verysmall compared with traditional crystallization techniques.
However, 10–20 nl plugs contain enough protein to generatecrystals that are large enough to obtain useful X-ray diffraction data. Crystals grown in plugs are typically 40–200 mm
Acta Cryst. (2008). D64, 1116–1122

Figure 4
Extracting diffraction-ready crystals from the MPCS CrystalCard. (a) A
picture of the thin plastic cover being removed from the MPCS
CrystalCard. (b) A picture of the opened MPCS CrystalCard with allof the crystallization experiments remaining in the plastic part containing
the microfluidic circuitry. (c) A microphotograph of a crystal in a plug justbefore being scooped out of the CrystalCard with a cryoloop. (d) A
microphotograph of the crystal in the cryoloop.
Gerdts et al.  Microcapillary Protein Crystallization System1119

research papers
Crystal extraction was used in combination with an MPCS
fine-gradient screen to generate crystals of methionineR-sulfoxide reductase (Fig. 5a). Crystals formed in a concentration optimization gradient screen of 30%(v/v) MPD,
25%(w/v) PEG 1500, 0.1 M acetate pH 4.5. The protein
concentration was 22 mg ml1 in 20 mM HEPES pH 7.0,
500 mM NaCl, 5% glycerol, 2 mM DTT. Crystals were
removed from the CrystalCard using a 0.1–0.2 mm cryoloop,
cryocooled (using the crystallant as the cryoprotectant) andsubjected to X-ray diffraction experiments. A 1.7 Å data set
was collected on SBC-CAT beamline 19BM located at theAdvanced Photon Source at Argonne National Laboratories
and the structure was subsequently solved and refined (Figs. 5band 5c; Table 1). The final coordinates and structure factors
were deposited in the Protein Data Bank (PDB code 3cxk).

mounted directly onto a goniometer for X-ray diffraction
experiments at room temperature. Additionally, the CrystalCard can be translated along its x and y axes to collect datafrom multiple crystals for combination to yield complete data
sets. The CrystalCard can be rotated through 75 without
hitting the beamstop. In order to demonstrate this technique,
we mounted a CrystalCard containing lysozyme crystals on agoiniometer head at NE-CAT beamline 24ID-C located at theAdvanced Photon Source at Argonne National Laboratories(Fig. 6a) and collected X-ray diffraction data at room
temperature from three crystals in the CrystalCard. A portionof the electron-density map generated by molecular replacement is shown in Fig. 6(b). Crystallographic data are provided
in Table 1.

4.2. In situ X-ray diffraction

5. Materials and methods

The CrystalCard has been designed to allow in situ X-ray
diffraction. This allows the crystallographer to assess thequality of the crystals grown before alteration by the cryoprotection process (Luft et al., 1999; McPherson, 2000; Ng etal., 2003; Yadav et al., 2005; Lunde et al., 2008) and to collectdata for structure determination from robust crystals. The
CrystalCard is sufficiently transparent to X-rays to be

5.1. Preparation of the plastic CrystalCard

Plastic CrystalCards were manufactured by injection
molding (Siloam Biosciences Inc.). Plug formation in the
CrystalCard requires a low surface-energy (hydrophobic)
surface. This ensures that the fluorocarbon oil preferentiallywets the walls of the microcapillary. The polycarbonate
CrystalCard arrives from the manufacturer with a highFigure 5
Methionine-R-sulfoxide reductase. (a) A microphotograph of a methionine-R-sulfoxide reductase crystal. (b) Fo  Fc OMIT map contoured at 3 for
Cys residues and a Zn ion. (c) Ribbon diagram of the NCS dimer of methionine-R-sulfoxide reductase from Burkholderia pseudomallei. Zinc ions are
represented as gold spheres and acetate ions in ball-and-stick representation. The structure was deposited as PDB entry 3cxk.

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surface-energy (hydrophilic) microcapillary surface. To prepare the microcapillary surface for plug formation, Cytonix
PFC 502AFA solution is used to coat the inside of the
microcapillary. Cytonix PFC 502AFA adheres to the polycarbonate surface and generates a coating with a surface
energy of 6–10 dyn cm1 (1 dyn cm1 = 1  103 N m1). To
apply the coating, the CrystalCard is filled from the outlet with
Cytonix 502AFA solution and incubated on ice for 2 h (theCrystalCard inlets are prone to crack if incubated at higher
temperatures). The 502AFA solution is removed from the
CrystalCard via a vacuum and clean dry air is forced throughthe CrystalCard at 34.5 kPa for 1 h. After drying, the CrystalCard is cured at 333 K for 1 h and is then ready for use.
5.2. Macro–micro interface

A macro–micro interface connection between the syringes
and the CrystalCard is required for the MPCS to function
properly. A 360 mm inner diameter, 760 mm outer diameter(ID/OD 360/760) section of Teflon tubing is used to connect
the syringe to the CrystalCard. One end of the Teflon tubing
slides onto the syringe needle and the other end slides into a
760 mm inner diameter piece of silicon tubing connected to theplastic CrystalCard. For the PDMS/Teflon CrystalCard, holes
are manufactured in the PDMS to allow a tight-fitting seal
with the ID/OD 360/760 Teflon tubing.

& Minor, 1997). For the lysozyme structure, intensities were
integrated separately for each of the three data sets using the
MOSFLM package (Leslie, 1992). The structures of lysozyme
and methionine-R-sulfoxide reductase were solved by molecular replacement using MOLREP (Vagin & Teplyakov, 1997)
with PDB entries 1iee and 3cez as the search models,
respectively. Structures were refined with REFMAC5(Murshudov et al., 1997) and model building was performed
with Coot (Emsley & Cowtan, 2004).

5.5. X-ray absorption of the CrystalCard

A simple test was conducted to analyze the absorption of
X-rays by the CrystalCard. To do this, the beam current in theion chamber normalized to the APS ring current (I/I0) was
measured with and without the CrystalCard inserted at awavelength of 0.979261 Å (12.66099 keV). I/I0 without the
CrystalCard was 1.91671  106 and I/I0 with the CrystalCard

5.3. Preparation of the hybrid method pre-formed cartridge(Li et al., 2006)

A 5–10 cm piece of ID/OD 360/760 Teflon tubing wasconnected to a 50 ml syringe and the other end to a 10 cm pieceof 200 mm inner diameter/230 mm outer diameter (ID/OD
200/230) Teflon storage capillary. The connection was sealedwith capillary wax (Hampton Research). The syringe andtubings were filled with FC40 (3M) and the syringe wasmounted on a manual aspirator (Stoelting No. 262M=, Wood
Dale, Illinois, USA). The end of the ID/OD 200/230 Teflon
storage capillary was dipped into the first precipitant and150 nl of fluid (15 units on the aspirator) was aspirated. A
50 nl air bubble (5 units on the aspirator) was aspirated. Thissequence was repeated until all of the desired precipitants (upto 24) had been aspirated. 2 ml FC40 (200 units on theaspirator) were aspirated into the tubing and the syringe wasplaced on the pump for the experiment. The end of the ID/OD
200/230 Teflon storage capillary containing the pre-formed
cartridge was connected to the precipitant inlet of the PDMS/
Teflon CrystalCard by cutting the channel with a razor blade
and inserting the capillary horizontally. The end of the ID/OD
200/230 Teflon storage capillary was inserted into the PDMS
chip (up to the point in the inlet where it becomes tapered)and sealed using capillary wax.
5.4. Structure determination

Data sets were collected at the Advanced Photon Source:
beamline 19BM at 100 K for methionine-R-sulfoxide reductase and beamline 24-IDC at room temperature for lysozyme.
Data were integrated and scaled with HKL-2000 (Otwinowski
Acta Cryst. (2008). D64, 1116–1122

Figure 6
In situ diffraction using the MPCS. (a) A picture of the MPCS
CrystalCard mounted on the goniometer head of an X-ray source. (b)A portion of the electron-density map obtained by combining in situ
diffraction data from three crystals inside the MPCS CrystalCard.
Gerdts et al.  Microcapillary Protein Crystallization System1121

research papers(perpendicular to the beam) was 1.5511  106. This constitutes a 19% X-ray absorbance by the CrystalCard.
This work was supported by the NIGMS–NCRR cosponsored PSI-2 Specialized Center Grant U54 GM074961 for
the Accelerated Technologies Center for Gene to 3D Structure. Methionine-R-sulfoxide reductase was provided by the
NIAID-supported Seattle Structural Genomics Center for
Infectious Disease (SSGCID), Contract HHSN266200700057C. We thank Frank Murphy, Kanagalaghatta Rajashankar and Stephen Ginell at the Advanced Photon Source(APS) for assistance with in situ diffraction studies and for
helpful discussions. We thank Aniruddha Puntambekar andcoworkers at Siloam Biosciences for their contributions. We
also thank Rustem Ismagilov and members of his team at the
University of Chicago for their support and advice, as well as
Yiping Xia for his work on the MicroPlugger software. Part ofthis work is based upon research conducted at the Northeastern Collaborative Access Team beamlines of theAdvanced Photon Source, supported by award RR-15301
from the National Center for Research Resources at the
National Institutes of Health. Use of the Advanced Photon
Source is supported by the US Department of Energy, Officeof Basic Energy Sciences under contract No. W-31-109-ENG38. Some results shown in this report are derived from work
performed at Argonne National Laboratory, Structural
Biology Center at the Advanced Photon Source. Argonne isoperated by the University of Chicago Argonne LLC for the
US Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357.

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