electronic reprint
Acta Crystallographica Section F

Structural Biology
and Crystallization
Communications
ISSN 1744-3091

Editors: H. M. Einspahr and M. S. Weiss

Salvage and storage of infectious disease protein targets in the
SSGCID high-throughput crystallization pathway using
microfluidics
Jeff Christensen, Cory J. Gerdts, Mathew C. Clifton and Lance Stewart

Acta Cryst. (2011). F67, 1022–1026

This open-access article is distributed under the terms of the Creative Commons Attribution Licence
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ISSN 1744-3091

Volume 67
Part 1January 2011

Acta Crystallographica Section F

Structural Biology
and Crystallization
Communications
Editors: H. M. Einspahr and M. S. Weiss

Acta Crystallographica Section F: Structural Biology and Crystallization Communications
is a rapid all-electronic journal, which provides a home for short communications on
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Acta Cryst. (2011). F67, 1022–1026

Christensen et al. · Salvage and storage of protein targets

laboratory communications
Acta Crystallographica Section F

Structural Biology
and Crystallization
Communications

Salvage and storage of infectious disease protein
targets in the SSGCID high-throughput
crystallization pathway using microfluidics

ISSN 1744-3091

Jeff Christensen,a Cory J.
Gerdts,b Mathew C. Cliftonb andLance Stewartba

Emerald BioStructures, 7869 NE Day Road
West, Bainbridge Island, WA 98110, USA, andb
Emerald BioSystems, 7869 NE Day Road West,
Bainbridge Island, WA 98110, USA

The MPCS Plug Maker is a microcapillary-based protein-crystallization system
for generating diffraction-ready crystals from nanovolumes of protein. Crystallization screening using the Plug Maker was used as a salvage pathway forproteins that failed to crystallize during the initial observation period using the
traditional sitting-drop vapor-diffusion method. Furthermore, the CrystalCards
used to store the crystallization experiments set up by the Plug Maker are shown
be a viable container for long-term storage of protein crystals without adiscernable loss of diffraction quality with time. Use of the Plug Maker withSSGCID proteins is demonstrated to be an effective crystal-salvage and storage
method.

Received 12 February 2011
Accepted 14 June 2011

1. Salvaging protein targets
The Seattle Structural Genomics Center for Infectious Disease(SSGCID) seeks to determine the molecular structures of bacterial
proteins thought to be good targets for drug therapies, with a target of
75 structures per year over ﬁve years. These protein targets arrive at
the crystallization core facility at Emerald BioStructures in highly
puriﬁed and concentrated form as frozen aliquots. A schematic of the
workﬂow used in this study is shown in Fig. 1. 10–15 new targets
arrive at the facility each week and are immediately screened
for crystallization using traditional sitting-drop vapor diffusion.
Following the strategy proposed by Newman et al. (2005), all targets
are screened against two 96-condition screens, JCSG+ (Emerald
BioSystems) and PACT Premier (Molecular Dimensions), giving 192
unique conditions. The experiments are monitored for four weeks

Figure 1
Workﬂow schematic for the salvage study.

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Acta Cryst. (2011). F67, 1022–1026

laboratory communications
and any crystals that form enter an optimization pathway that
includes testing for protein crystal diffraction, subsequent crystal
optimization and structure determination. Inevitably, there areprotein targets that produce no crystals within four weeks or fail inthe optimization pathways. Typically, these targets are retired from
active observation to open space for the next set of incoming targets.
Within the Emerald BioStructures crystallization core facility, strategies have been utilized to attempt to salvage the structure determination of retired proteins. Such salvage pathways have included
limited proteolysis (McPherson et al., 2004) and exogenous nucleation (D’Arcy et al., 2003; Thakur et al., 2007), although both had very
limited success for the required effort involved.
With close to 900 retired protein targets currently available forsalvage, the MPCS (Microcapillary Protein Crystallization System)
Plug Maker (Emerald BioSystems) was used to thoroughly re-screen
a small set of retired proteins. The plug-based microﬂuidic technology
encompassed in the Plug Maker is able to thoroughly screen crystallization conditions by quickly and automatically generating concentration gradients for each condition that is screened. Details of
the Plug Maker and its underlying technology have been publishedpreviously (Gerdts et al., 2008, 2010; Li et al., 2006; Zheng et al., 2003,
2005). Using this technology, as many as 800 20 nl crystallization
experiments can be set up in one CrystalCard (Emerald BioSystems;
Fig. 2) using as little as 5 ml protein sample. Furthermore, although
they only use up 10 nl of protein each, the experiments are large
enough to produce diffraction-ready crystals (50–200 mm per side)

Figure 2
Image of a CrystalCard showing the two microcapillary channels.

Table 1
Concentrated conditions composing the MPCS screen (MPCS Sweet 16).
MPCS
Screen Wizard
No.
Full No. JCSG+
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

1
2
6
9
14
17
31
39
47
49
51
52
59
82
88
91

A-7
E-3
A-2
E-8
E-1
D-4
C-1
A-6
E-4
C-7
D-6
E-2
E-7
E-12
E-6
B-8

At 100 mM

At 200 mM

30%(w/v) PEG 8000 CHES pH 9.5
None
20%(v/v) 2-propanol HEPES pH 7.5
NaCl
30%(w/v) PEG 3000 Citrate pH 5.5
None
1.5 M (NH4)2HPO4 Acetate pH 4.5
None
1.4 M sodium citrate Cacodylate pH 6.5
None
40%(w/v) PEG 8000 Acetate pH 4.5
Li2SO430%(w/v) PEG 8000 Phosphate–citrate pH 4.2 NaCl
30%(w/v) PEG 1000 Phosphate–citrate pH 4.2 Li2SO4
2.52 M (NH4)2SO4
Tris pH 8.5
Li2SO420%(w/v) PEG 3000 Acetate pH 4.5
Zn(OAc)2
30%(w/v) PEG 8000 Tris pH 8.5
MgCl2
3.0 M (NH4)2SO4
Cacodylate pH 6.5
NaCl
20%(v/v) 2-propanol Cacodylate pH 6.5
Zn(OAc)2
20%(w/v) PEG 8000 Imidazole pH 8.0
None
30%(w/v) PEG 3000 Imidazole pH 8.0
Zn(OAc)2
20%(w/v) PEG 8000 Tris pH 7.0
MgCl2

that can be examined in situ or extracted from the peel-apart
CrystalCard (Gerdts et al., 2008, 2010; Yadav et al., 2005).
The JCSG+ screen used for the initial trials set up for SSGCID
targets arose from data mining conducted by the Joint Center for
Structural Genomics, which identiﬁed 96 conditions in commercial
screens that had been most successful in crystallizing proteins(Newman et al., 2005; Page & Stevens, 2004). 16 of these conditions
were taken from Wizard Screens I and II (Emerald BioSystems) inorder to compose the MPCS Sweet 16 Screen (Table 1).
Protein targets to be salvaged were screened against all or a
portion of the MPCS Sweet 16 Screen based on the availability ofremaining protein. No special criteria were used to select the targets
other than that they were listed in our tracking spreadsheet as having
failed to crystallize in either of the two standard vapor-diffusion trials
during the initial four-week screening period. Subsequent to theirselection for MPCS screening, the retired targets were re-examined intheir original vapor-diffusion plates or selected for additional vapordiffusion trials for other reasons and ended up crystallizing sometime
between two and six months after initial screening. These samples
were still included in this analysis because the comparison to the

Figure 3

Pictures of crystals obtained in batch-under-oil-style microﬂuidic experiments in the CrystalCards. The microcapillary width in all pictures is 200 mm. (a) MyavA01582cA1;(b) MyavA01582aA1; (c) MyavA01566cA1; (d) MyavA00213aA1; (e) MysmA01566cA1; (f) MyavA01582cA1; (g) MythA00746aA1; (h) GilaA01434aA1

Acta Cryst. (2011). F67, 1022–1026

Christensen et al.  Salvage and storage of protein targets

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laboratory communications
initial screening for retired targets remains valid (Table 3). One of theprotein samples was not set up in vapor-diffusion trials because thevolume delivered was inadequate and thus went directly to MPCS.
Crystallization experiments were set up in CrystalCards using the
Plug Maker instrument. These experiments are analogous to batch
crystallization, in which the crystallization reagents are combined ina drop (plug) surrounded by an oil and this initial condition is notexpected to change signiﬁcantly over time (see crystal pictures inFig. 3). In this system, each plug is separated by a small volume
of inert perﬂuorinated oil (Fluorinert FC-40, Sigma–Aldrich). The
CrystalCard contains two separate microcapillaries and a thoroughgradient of as many as 12 different crystallization conditions can bescreened in each microcapillary, covering a wide range of crystallization phase space.

2. Storing protein crystals
Long-term storage of protein crystals at room temperature may be
useful for the production of protein crystals for future research(ligand soaking) and may be useful owing to a lack of immediate

Table 2
Data-collection statistics.
Values in parentheses are for the highest of 20 resolution shells

Space group
Unit-cell parameters (Å,  )Wavelength (Å)
Resolution range (Å)
No. of unique reﬂections
Multiplicity
CompletenessRmerge†Mean I/(I)† Rmerge =MysmA.00391.bA1/3C

MysmA.01566.cA1

P21
a = 57.6, b = 95.6,
c = 115.3,  = 99.6
1.541850–2.8 (2.90–2.80)30844 (3076)
3.5 (3.4)
99.6 (100)0.13 (0.48)
6.94 (2.66)

R3/R32a = b = 87.8,
c = 183.9
1.541850–2.6 (2.69–2.60)
16285 (1623)
2.7 (2.7)
99.6 (99.9)
0.13 (0.49)
6.87 (2.2)

P P

P P

h

h

i jIi ðhÞ  hIðhÞij=i Ii ðhÞ.

access to X-ray data collection. Using traditional sitting-drop vapor
diffusion or batch-under-oil techniques, protein-crystal storage can be
difﬁcult owing to evaporation of the crystallization drop. A long-term
study of crystal stability in CrystalCards has shown that crystallization experiments inside CrystalCards can be stored without
measurable evaporation and without meaningful loss of protein
crystal diffraction quality. Crystals of BrabA.00113a.A1, an infectious
disease target from SSCGID, were grown at the same time inside six
CrystalCards and were subsequently examined using in situ X-ray
diffraction by mounting the CrystalCard on the goniometer (Fig. 4;
Gerdts et al., 2008; Yadav et al., 2005) periodically from April to
December 2010. While not quantiﬁed, the crystals appeared to beunchanged and showed no obvious degradation of protein crystal
diffraction quality over the course of eight months (Fig. 5). Furthermore, a 2.6 Å resolution data set was collected from one of theprotein targets salvaged in this study (MysmA00391bA1/3C) after the
crystals had been stored in the CrystalCard for approximately ﬁvemonths (Table 2).

3. Materials and methods
3.1. Crystallization screening conditions

Figure 4
Picture of a CrystalCard mounted on a goniometer for in situ diffraction
experiments.

The similarity of microcapillary crystallization to batch crystallization makes it advantageous to start with crystallization solutions
at higher component concentrations than are found in commercial
sparse-matrix screens; thus, when running a concentration gradient of

Figure 5Comparison of in situ diffraction of BrabA.00113a.A1 cystals after long-term storage. Diffuse X-ray scatter is a result of the plastic material of the CrystalCard.

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Christensen et al.  Salvage and storage of protein targets

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Acta Cryst. (2011). F67, 1022–1026

laboratory communications
Table 3
Summary of MPCS crystallization results.
MPCS CrystalCard in situ diffraction screening and data collection
SSGCID target protein

MPCS sample No.

AnphA.00481.aA1AnphA.00462.aA1BaheA.00227.aA1
BaheA.01161.aA1/3C
EnhiA.01533.aA1
GilaA.01434.aA1
LedoA.00425.aAG11
MyavA.00213.aA1
MyavA.00754.aA1
MyavA.00814.aA1
MyavA.00937.aA1
MyavA.01097.hA1
MyavA.01097.iA1
MyavA.01188.aA1
MyavA.01212.aA1
MyavA.01326.lA1
MyavA.01379.aA1
MyavA.01530.cA1
MyavA.01549.bA1
MyavA.01582.aA1
MyavA.01582.cA1MyavA.01625.aA1
MyavA.01649.cA1
MyavA.10520.bA1
MyavA.17065.aA1
MyleA.00778.aA1
MyleA.01155.aA1
MypaA.00249.aA1
MysmA.00337.bA1
MysmA.00391.bA1/3C
MysmA.01263.bA1
MysmA.01566.bA1
MysmA.01566.cA1
MythA.00746.aA1
Totals

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
34

MPCS crystals†

*
*
*
*
*

Protein diffraction‡

Likely protein*

Vapor-diffusion crystals
in retired trays§

*
*

XX
XX
X

*
*
*

X

*
*

*

XX

*
*

*
X
X

*
*

XX
XX

PDB 3qlj
*
*

X
X
X

*

*
*
*
*
*
*
23

Salt

X
X

*

*
*
*
*
*

Not salt

X

*

*
XXX

*
*
X

XXX
XX
8

11

2

2

*
*
14

† An asterisk indicates the presence of crystals. ‡ X, evidence of protein crystal diffraction in situ in the CrystalCard; XX, clear evidence of protein crystal diffraction in situ in the
CrystalCard; XXX, complete X-ray diffraction data set collected from a crystal extracted from the peeled-apart CrystalCard. § An asterisk indicates the presence of crystals. A
structure was deposited for MyavA.01582.aA1 with PDB code 3qlj.

a crystallization condition against a target protein sample theresulting plugs in the gradient will cover a wider crystallization space.
During development of the MPCS Plug Maker system, custom
versions of Wizard Screens I and II were designed in which the same
reagents were used but at proportionally higher concentrations.
Using the 16 conditions common to both the Wizard and JCSG+
screens would allow us to compare the microcapillary results withthose of the initial vapor-diffusion trials; these conditions were
dubbed the ‘MPCS Sweet 16 Screen’ (see Table 1).

the detector and the plug containing the crystal was aligned in thebeam. Rotation of the card in the ! axis is limited to approximately
15 from 0 because of interference with the beam stop as well as theincreasing thickness of the card that the X-ray beam must penetrate
when the card is not perpendicular to the beam. A few 30 s/0.5 test
images were collected within this range and examined for protein
diffraction. If no obvious diffraction was noted a salt test was
performed.
3.5. Salt test

3.2. MPCS parameters

The protein samples were set up using the Plug Maker hybrid
protocol. Gradients of conditions 1–8 and 9–16 of the Sweet 16
Screen were run in channels one and two, respectively. Approximately 5 ml protein sample was required to screen all 16 conditions.

4. Results and discussion

3.3. CrystalCard storage

CrystalCards were stored in standard plastic microscope slide
boxes (VWR) with a small amount of water added to keep themhumidiﬁed. The boxes were kept at 289 K.
3.4. Diffraction testing

For in situ diffraction screening at room temperature, CrystalCards
were mounted on the goniometer using a special clip (Fig. 4). The
CrystalCards were oriented with the thin backing of the card facing
Acta Cryst. (2011). F67, 1022–1026

With the card mounted and aligned as above, the detector was
moved in to 50 mm and the 2 axis moved out to 45 . A wedge of1 /10 s images at least 15 wide was collected and if no small-molecule
reﬂections were observed the crystal was characterized as ‘not salt’.

Microﬂuidic salvage efforts were attempted on 34 targets. 23
produced crystals or crystal-like objects. Through in situ X-ray
diffraction screening eight of these showed obvious protein diffraction patterns, and complete data sets to 2.6 Å resolution werecollected for two targets from crystals harvested directly from the
CrystalCards (Table 2). Because the crystals screened at room
temperature may have sustained radiation damage, the data sets werecollected at 100 K using different crystals from the same cards. Of theremaining crystals, 11 were characterized as ‘likely protein’ owing to a
Christensen et al.  Salvage and storage of protein targets

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laboratory communications
few very low-resolution reﬂections close to the beam stop, two werecharacterized as ‘not salt’ after failing the salt test (see x3) and two
were conﬁrmed as being salt crystals (Table 3).
The initial vapor-diffusion screening of SSGCID samples against
only two 96-condition screens is minimal and has allowed the crystallization group to achieve the annual goals outlined in the goals of
the SSGCID. Screening these same samples against a larger numberof sparse-matrix screens may have resulted in crystals for a larger
percentage of samples, but would come with the additional burdenof more trials to set up, monitor and store, and a greater cost of
consumables. Using the capabilities of the Plug Maker to screen awide variety of concentrations of just 16 crystallization screening
conditions has resulted in two high-resolution data sets and legitimate
crystallization leads for many previously retired targets. While all of
the vapor-diffusion crystals reported here appeared after four weeks,many of the MPCS crystals appeared in the CrystalCards within days.
The results of these preliminary MPCS experiments are very
encouraging. Although nominally using a screen of only 16 conditions, the actual number of unique conditions is the number of plugs
in the CrystalCard (up to 800) because each crystallization solution
is run as a gradient. The ability to obtain crystallization leads, protein
crystal diffraction and full diffraction data sets using less than 10 ml
protein sample, and with a success rate comparable to standard
crystallization methods, represents a large saving in effort and indicates that efﬁcient microﬂuidic scanning of protein and crystallization
condition concentrations in initial crystallization trials using the
MPCS Plug Maker can be a highly effective means of producing
protein crystals.
In addition to soluble proteins, the Plug Maker may prove to be
useful for crystallizing nucleic acids and membrane proteins, both ofwhich represent special challenges. While preliminary experiments
have shown that membrane proteins solubilized with some detergents
form plugs normally, neither nucleic acids nor membrane proteins
have been tested extensively by us. Experiments are planned to more

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Christensen et al.  Salvage and storage of protein targets

thoroughly investigate this and the results of these experiments will
be reported in due course.
The authors thank the entire SSGCID team. This research was
funded under Federal Contract No. HHSN272200700057C from the
National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Department of Health and Human Services. This
work was also supported by the PSI-2 Specialized Center Grant
U54 GM074961, co-sponsored by NIGMS–NCRR for the Accelerated Technologies Center for Gene to Three-Dimensional Structure.
All protein targets were provided by the NIAID-supported Seattle
Structural Genomics Center for Infectious Disease.

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