In Situ Proteolysis to Generate Crystals for Structure Determination: An Update
Citation: Wernimont A, Edwards A (
In Situ Proteolysis to Generate Crystals for Structure Determination: An Update
Amy Wernimont 0 1
Aled Edwards 0 1
Haiwei Song, Institute of Molecular and Cell Biology, Singapore
0 Funding: The Structural Genomics Consortium is a registered charity (Number 1097737) supported by the Canadian Institutes of Health Research, the Canadian Foundation for Innovation, and Genome Canada, through the Ontario Genomics Institute, GlaxoSmithKline, the Karolinska Institute, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research, and the Wellcome Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
1 Midwest Center for Structural Genomics and Structural Genomics Consortium, University of Toronto , Toronto, Ontario , Canada
For every 100 purified proteins that enter crystallization trials, an average of 30 form crystals, and among these only 13-15 crystallize in a form that enables structure determination. In 2007, Dong et al reported that the addition of trace amounts of protease to crystallization trials-in situ proteolysis-significantly increased the number of proteins in a given set that produce diffraction quality crystals. 69 proteins that had previously resisted structure determination were subjected to crystallization with in situ proteolysis and ten crystallized in a form that led to structure determination (14.5% success rate). Here we apply in situ proteolysis to over 270 new soluble proteins that had failed in the past to produce crystals suitable for structure determination. These proteins had produced no crystals, crystals that diffracted poorly, or produced twinned and/ or unmanageable diffraction data. The new set includes yeast and prokaryotic proteins, enzymes essential to protozoan parasites, and human proteins such as GTPases, chromatin remodeling proteins, and tyrosine kinases. 34 proteins yielded deposited crystal structures of 2.8 A resolution or better, for an overall 12.6% success rate, and at least ten more yielded well-diffracting crystals presently in refinement. The success rate among proteins that had previously crystallized was double that of those that had never before yielded crystals. The overall success rate is similar to that observed in the smaller study, and appears to be higher than any other method reported to rescue stalled protein crystallography projects.
The field of protein crystallography has seen great progress in
crystallization, data collection, phasing techniques, crystallization
screens, robotics, as well as in software for data reduction, phasing,
model building and refinement . However, the overall success
rate (as measured by the number of deposited structures per
number of selected targets) remains relatively low [2,3]. On
average, only about 1520% of protein targets that can be purified
will then crystallize in a form from which a structure can be
determined (http://www.targetdb.pdb.org; http://thesgc.com/
The field of protein crystallization, which previously focused
almost entirely on the optimization of crystallization strategies, is
now increasingly addressing the improvement of the crystallization
properties of the proteins themselves . This trend began in the
early 1990s, with the advent of molecular biology techniques and
mass spectrometry. The use of these techniques allowed scientists
to focus crystallization efforts on the most stable domains of target
proteins, as identified by their pattern of resistance to limited
proteolysis [5,6]. Stable domains crystallize more readily and often
result in better-diffracting crystals [7,8,5]. Success rates were
further increased by expressing many variations of the protein
domain, as differences of a few residues at the N- or C- termini
often have dramatic effects on soluble protein expression and
protein crystallization. Graslund et al. found that by screening ten
derivatives of a given protein domain instead of one, the
probability of generating a soluble protein increased two-fold
and the probability of generating a structure increased four-fold
Crystallization can also be promoted by changing the surface
properties of the protein to reduce the conformational entropy of
surface residues. The most straight-forward approach is to use
reductive methylation of surface lysine residues . In large,
systematic studies, lysine methylation rescued 6% of a set of
recalcitrant proteins [11,10]. Surface entropy can also be reduced
by site-directed mutagenesis of clusters of charged residues .
Protein crystallization can also be facilitated by the addition of
specific ligands or inhibitors, which bind to the protein and lower
its intrinsic heterogeneity . Finally, the addition of trace
amounts of protease to the crystallization trials in situ proteolysis
rescued 10 out of 69 different proteins (,14%) that had
previously failed in crystallization and structure determination
In situ proteolysis appears to be the most efficacious
crystallization rescue strategy. However, while the study of Dong et al. was
systematic and rigorous, all but one of the successful cases derived
from a single experimenter, and the proteins were predominantly
of bacterial origin. This prompted further inquiry into whether the
method (1) would be applicable to human proteins, (2) would be as
successful in other hands, and (3) would be useful for those proteins
for which dozens of variants had already been tested. This paper
describes an expanded study: applying in situ proteolysis to 270
new proteins since the last paper, from prokaryotes and
eukaryotes, and by dozens of scientists.
All protein constructs contained an N-terminal hexahistidine
tag, with a recognition site for TEV protease
MGSSHHHHHHSSGRENLYFQGH or MAPEHHHHHHDYDIPTTENLYFQGA). Proteins
were purified as described earlier [14,15,9]. All structures reported
here have been deposited into the Protein Data Bank (PDB, www.
Proteases were ordered from Sigma-Aldrich and stock solutions
made up as follows: a-chymotrypsin (C3142) was dissolved in
1 mM HCl and 2 mM CaCl2 at a concentration of 1 mg/mL.
Trypsin (T8003) was dissolved into 1 mM HCl and 2 mM CaCl2
at a concentration of 1.5 mg/mL. Elastase (E0127) was dissolved
into 200 mM Tris-HCl buffer (pH 8.8) at a concentration of
1 mg/mL. Papain (P5306) was dissolved into water at a
concentration of 1.2 mg/mL. Subtilisin A (P5380) was dissolved
into 10 mM Na acetate and 5 mM Ca acetate at a concentration
of 1 mg/mL. Endoproteinase Glu-C V8 (V8 protease) was
dissolved into water at a concentration of 2 mg/mL. Stock
solutions of protease were serially diluted into a buffer comprising
10 mM HEPES (pH 7.5) and 500 mM NaCl as needed.
In situ proteolysis
In situ proteolysis was performed essentially as described ,
with the working set of proteases expanded, on a case-by-case
basis, to include trypsin (at a range of 1:10000 to 1:10 v/v), V8
protease (at a range of 1:100 to 1:40 v/v), papain (at a range of
1:1200 to 1:10 v/v), thermolysin (at 1:20 v/v), and subtilisin (at
Pre-screening to identify a promising protease
Investigative limited proteolysis with a panel of proteases was
used occasionally to identify a protease that generated promising
degradation patterns, as detected by denaturing gel electrophoresis
or mass spectrometry. 510 mL of protein, dissolved at 1020 mg/
mL, was incubated with a range of proteases for thirty minutes at
room temperature. The proteases were used at dilutions of 1:10,
1:100, and 1:1000. The reactions were stopped by the addition of
SDS-Coomassie sample loading buffer for analysis by gel
electrophoresis, or by formic acid for analysis by mass
spectrometry. The protease(s) and concentration that yielded the largest,
most stable domain were chosen for subsequent crystallization
Results and Discussion
More than 270 proteins that failed to produce crystals or that
produced crystal forms unsuitable for structure determination
were subjected to in situ proteolysis crystallization trials over an
eight-month period in 2008. This set comprised about 200 yeast or
bacterial proteins, 70 human proteins, and 5 parasitic proteins. Of
these, 34 proteins generated crystals of sufficient quality for
structure determination (Table 1), for a rescue rate of ,13%. 10
additional crystals in the set are being optimized, so the number of
deposited structures may increase. We did not identify the protein
cleavage sites using mass spectrometry, as was done in Dong et al,
because of the effort involved. To indicate the approximate extent
of cleavage, we have included in the Supplementary Information
the sequence of each protein construct that entered crystal trials
and the regions of the protein for which electron density was
absent (Text S1).
Yeast and bacterial proteins
Of the initial 200 yeast and bacterial proteins, two-thirds had
previously resisted crystallization, and the remaining third had
formed crystals unsuitable for structure determination. These
crystals were too small, very thin, formed stacks, or diffracted
poorly and could not be improved upon with standard
optimization strategies. Occasionally, crystals diffracted well but
a very large number of copies in the asymmetric unit (more than
1MD in case of TM1086) made it impractical for structure
determination by SAD/MAD techniques. In situ proteolysis
treatment led to 20 structures from these recalcitrant proteins.
Before the in situ proteolysis process, 11 had not previously
crystallized at all (from ,135 tested), and the other 9 had formed
crystals unsuitable for structure determination (from ,65 tested).
From these data, the technique yields higher success rates within
the subset of proteins that had previously formed poor crystals.
This trend had been observed in the original study.
Human Proteins. Of the 70 human proteins targeted for
crystallization by in situ proteolysis, 54 had not previously formed
crystals, and 16 had formed poor-diffracting crystals. The set that
had not formed crystals comprised both those that had not been
tested previously as well as a set on which in situ proteolysis was
performed in parallel with conventional crystallization trials. In situ
proteolysis treatment led to 12 structures from these recalcitrant
proteins. Of these, 5 were from the set of 16 that had previously
formed crystals unsuitable for structure determination. This
success rate was notably high.
Of the five proteins from human parasites targeted for
crystallization by in situ proteolysis, none had previously
crystallized. In situ proteolysis treatment led to 2 structures.
Variations on in situ proteolysis experimental approaches
The intent of Dong et al. was to carry out a systematic,
statistically significant, and well-controlled test of the efficacy of in
situ proteolysis. Each new crystal was analyzed using mass
spectrometry to ensure that the success of the method could be
attributed directly to the use of proteases. Their paper strongly
suggested that the method should be adopted as a primary
crystallization strategy due to its high success rate, but was
qualified by the fact that the method was being employed in a very
The intent of this study was to examine the efficacy of the method
in practice, as carried out on a larger number of proteins by a larger
number of experimenters. This strategy had the advantage of
exploring the use of the method under less controlled conditions, in
which individual investigators adopted slightly different
methodologies and strategies. The disadvantage of this strategy is that any
conclusions drawn have more caveats, due to the inability to control
all aspects of the experiments. We describe here several variations to
the original method that have proven successful.
In situ proteolysis using a wider array of proteases. In
the study of 69 target proteins by Dong et al., chymotrypsin was
used at a single concentration. In a few cases trypsin was also
tested at a single concentration. Chymotrypsin and trypsin are
selective proteases: chymotrypsin cleaves on the C-terminal side of
bulky hydrophobic side-chains such as phenylalanine, tyrosine, or
tryptophan; and trypsin cleaves on the C-terminal side of basic
Crystal before protease
All structures deposited into the protein databank with corresponding accession code. Resolution numbers are rounded to the nearest tenth of an Angstrom.
C = chymotrypsin, T = Trypsin, Therm = Thermolysin, Pa = Papain, V8 = V8 protease.
residues. It is likely that many target proteins were not optimal
substrates for these enzymes, but in the original study, it was
impractical and prohibitively expensive to systematically explore
other proteases for the 70 target proteins.
In this study, although chymotrypsin and trypsin accounted for
the most successes, other proteases were used, including V8
protease, thermolysin and papain. In total, 26 of the 34 structures
derived from the use of chymotrypsin or trypsin, 5 structures
derived from the use of the V8 protease, which cleaves on the
Cterminal side of acidic residues, two structures derived from the use
of thermolysin, and one from papain.
Identifying promising proteases in advance of
crystallization trials. In situ proteolysis was implemented in
three different experimental strategies. In the first, exemplified by
Dong et al, chymotrypsin and/or trypsin were added to the target
protein at a fixed concentration. In the second, promising
proteases were identified in advance of in situ proteolysis, by
prescreening proteases in a range of concentrations and analyzing the
results by mass spectrometry or gel electrophoresis. Of the 34
structures, 4 derived from the use of a protease identified in this
manner. For example, the structure of N-carbamoylsarcosine
amidase from Thermoplasma acidophilum was determined after in situ
trypsinolysis. For this protein, previous attempts at removing the
N-terminal 6-His tag with TEV protease were unsuccessful. The
seleno-methionine protein was then subjected to limited
proteolysis with chymotrypsin, trypsin, V8, and papain prior to
analysis by mass spectrometry. Trypsin yielded the largest stable
domain and was used for crystallization assays. One condition
generated crystals of excellent quality, and the structure was
determined at 2.35 A using SAD from a single data set (Luo et al.,
The third approach, used largely when the protein was very
abundant, is to screen a range of proteases using crystallization,
rather than gel electrophoresis, as the metric. This strategy has the
advantage that crystallization screens sample a wide range of
solution conditions in which the proteases may have more
favourable activities. This approach has proven particularly
successful for human small GTPases. Commonly, these protein
targets were crystallized with four different proteases in parallel
typically chymotrypsin, trypsin, subtilisin, and V8 at a 1:100 v/v
concentration. The target/protease mixtures were allowed to
incubate on ice for thirty minutes, at which point they were
subjected to crystallization trials. This technique was applied to 30
different target proteins; 8 structures resulted (PDB codes 3BFN,
3BOR, 3BPJ, 3C5C, 3CBQ, 3CON, 3DZX, and 3EAP), and
several more are in process.
Sites of cleavage after in situ proteolysis. In most cases
described here, as in the study of Dong et al., the cleavage sites appear
to reside at the N- and/or C-termini. For instance, the
bromodomain of the PF10_0328 protein from Plasmodium
falciparum would not crystallize using typical crystallization
techniques. Eight different constructs were designed and many
different protein/crystal screen permutations were tested, with and
without tag, and in the presence of six different proteases. Crystals
appeared in conditions with chymotrypsin and the structure was
solved from selenomethionine-containing protein. From the original
construct of 166 residues, including a N-terminal His tag, only 120
residues could be seen in the electron density map. Mass
spectrometry analysis of the crystals revealed a large protein peak
at 17703 Daltons, which corresponds to chymotrypsin cleavage just
C-terminal to Trp463, and no cleavage at the N-terminus (Figure 1).
There are a few instances in which the proteins were cleaved at an
internal loop. For example, CGD6_3220 is a GTPase from
Cryptosporidium parvum that failed to crystallize despite repeated
attempts with many different constructs. After trypsin treatment, the
protein readily crystallized and the structure was solved to 2.2 A
(PDB code 2RHD). All of the protein could be modeled into the
electron density map except for one loop corresponding to residues
6876. Mass spectrometric analysis of the crystals revealed two
internal cleavage events at residues 71 and 73, respectively.
Interestingly, this loop is located at the interface between two
molecules in the crystal lattice (Figure 2). When a highly homologous
structure was superimposed on the model and the packing analyzed,
the intact loop would have clashed with the symmetry-mate
molecule and have inhibited this crystal formation.
In situ proteolysis removes large amount of protein. As
stated earlier, the most common cleavage events occurred near the
N and C termini, and usually 2040 residues were removed.
However, in one previously reported case  and in one instance
reported in this work, a larger polypeptide fragment was removed.
For the eukaryotic translation initiation factor 3 (EIF3J; PDB code
3BPJ), 12 different protein constructs were designed in attempts to
refine the optimal domain boundaries for successful crystallization.
None of the proteins purified from these constructs yielded a
diffraction-quality crystal. Chymotrypsin treatment generated a
crystal that provided a 1.8 A dataset from which the structure was
solved. The structure revealed that almost half of the protein had
been removed; the initial construct contained residues 76220 but
only the C-terminal fragment (141220) was ordered in the crystal
structure. Secondary structure prediction using the JPred server
 suggested this region was buried, and thus this position would
likely not have been selected for construct design.
Immediate successes after exhaustive trials. Our default
strategy for human proteins is to design 1015 different constructs
for each protein and to attempt to crystallize each one that can be
purified, in both the presence and absence of the histidine tag .
If this first round fails, more constructs with slight variations at the
N- and C-terminal positions are often created, though this strategy
is met with significantly diminishing returns. In situ proteolysis is
now being used to resuscitate some of the failed projects. In one
case, 36 constructs were purified for the histone methyltransferase
SETDB1 protein, and none crystallized, either with or without the
histidine tag. In the first experiment with in situ proteolysis,
excellent crystals were obtained directly from a matrix screen, and
after minor optimization led to crystals diffracting to 1.8 A (PDB
The heme-binding transcription factor rev-erbb and its
Drosophila orthologue E75 were the subject of extensive efforts
over a 23 year period. Dozens of constructs, including a number
of cysteine mutants and internal deletions, were tested to no avail.
Two of the constructs that were expressed to high levels
precipitated during concentration for crystallization. A small
amount of trypsin was added to facilitate concentration. After
successful concentration, the samples were analyzed by denaturing
gel electrophoresis, which showed that both proteins had been
digested to a single, large, stable domain. Accordingly, in situ
proteolysis trials with trypsin were pursued with the protein
constructs in the presence of the ligand, heme. Initial crystals from
a minimal screen were optimized and the protein to protease ratio
adjusted, and excellent crystals were obtained from which the
structure was solved to 1.9 A resolution (PDB code 3CQV; Pardee
et al, submitted).
Crystallization efforts for the protein TA0507 were initiated in
2001. Several rounds of protein purification and crystallization
trials yielded tiny needle clusters that could not be improved in size
or quality. The project was abandoned and the protein stored at
280 C for seven years until the in situ proteolysis method became
more widely used in the laboratory. At that point, the original
protein was thawed and chymotrypsin was added. Single crystals
were generated from the first crystal screen, and the conditions
refined to produce large, single crystals that diffracted to 2.1 A
(PDB code 3DTZ).
Reproducibility. Despite initial concerns, crystallization
using in situ proteolysis appears to be reproducible. In all the
experiments reported here, no trouble was observed in
reproducing additional crystals for improvement or data
collection. On some occasions, the ability to regenerate crystals
was remarkable. For example, in the case of SETDB1, the tudor
domain of human histone-lysine N-methyltransferase (PDB code
3DLM), excellent native crystals were obtained with
chymotrypsin. The experimenter was able to repeatedly obtain
crystals with this treatment for extensive testing. Subsequent in situ
proteolysis trials with the selenomethionine-incorporated protein
formed crystals of the same morphology; there was no need to
retest the protease concentration or to re-screen the protein.
Alternate protein conformations after in situ
proteolysis. Crystallization can trap proteins into rare,
perhaps non-physiological, conformations; this can also be the
case for proteins crystallized after in situ proteolysis. The
crystallization of rev-erbb provides such an example. Rev-erbb
crystallized in a number of crystal forms, one of which diffracted to
1.9 A. In this structure, the heme ligand is coordinated by a
histidine residue in the middle of the protein and a cysteine residue
in the N-terminal region of the fragment; both residues had been
suggested to be ligands for the heme in biochemical and genetic
studies [17,18]. One of the earlier crystal forms of rev-erbb
diffracted to about 3.5 A. In this crystal form, (Figure S1), the
Nterminal portion of the protein had partially unfolded and the
protein dimerized around the heme group which was coordinated
by the histidine residue in each monomer. The in depth analysis of
this crystal form was not pursued, and it is not known if its
formation was dependent on proteolysis, or if the conformation
represents a physiologically-relevant state.
Greater efficacy of method in cases where protein has
crystallized previously. The method of in situ proteolysis, in its
various iterations, has now been applied to over 300 soluble
proteins that had previously failed to yield a structure. Of these
proteins, ,200 had never before generated crystals and ,100 had
formed crystals that were unsuitable for structure determination.
The success rates of in situ proteolysis differed dramatically
between these subsets. Of the proteins that had failed to
crystallize, 24 structures were obtained, for a success rate of
,12% (24 structures from 200 proteins). For the proteins that had
previously generated crystals, the success rate was almost double at
,21% (21 structures from 100 proteins).
This large discrepancy between successes of this technique with
the two populations could possibly be explained by inherent
stability of the purified protein. A protein that had crystallized
previously must have been stable enough over the time period
required to form crystals. However, the population of proteins that
never crystallized might contain a population of unstable proteins,
prone to denature or aggregate over the crystallization period. We
analyzed possible relationships among the group of proteins that
could not crystallize, such as pI or predominant predicted
secondary structure, but could not find any common trend among
Conclusions. In this greater sampling of human and
bacterial proteins, in situ proteolysis has proven effective when
even dozens of constructs failed to produce a protein amenable to
crystallization. The method has rescued proteins stored for years
and has proven remarkably reproducible. Although we have
shown that other proteases can also be used effectively, it is as yet
unknown if their use on a large scale is economical, or whether the
use of chymotrypsin and trypsin would capture most of the cases
that would be successful. The method appears doubly effective
when applied to proteins that have already formed crystals as
compared to proteins that have never crystallized.
Original sequences of proteins subjected to in situ
Figure S1 Crystal structure figure of a possible
non-physiological dimer. Possible non-physiological dimer obtained from a
3.5 A dataset collected on a crystal of rev-erbb.
Found at: doi:10.1371/journal.pone.0005094.s002 (0.48 MB TIF)
Complete list of authors
Midwest Center for Structural Genomics: Changsoo Chang,
Maksymilian Chruszcz, Marianne Cuff, H. Cui, Marcin Cymborowski,
Rosa DiLeo, Olga Egorova, Elena Evdokimova, Ekaterina Filippova,
JunGu, Jennifer Guthrie, Alexandr Ignatchenko, Andrzej Joachimiak,
Natalie Klostermann, Youngchang Kim, Yuri Korniyenko, H. Krause, M.
Kudritska, Hai-Bin Luo, Wladek Minor, J. Osipiuk, Keith Pardee, Qiuni
Que, J. Reinking, Alexei Savchenko, A. Schuetz, Tatiana Skarina, Kemin
Tan, Alexander Yakunin, Xiaohui Xu Adelinda Yee, Veronica Yim,
Rongguang Zhang, Heping Zheng, Matthew D. Zimmerman
Structural Genomics Consortium: Maria Amaya, Cheryl
Arrowsmith, George V Avvakumov, Helena Berglund, Alexey Bochkarev, C.
Butler-Cole, Lars-Goran Dahlgren, Sirano Dhe-Paganon, Slav Dimov,
Ludmila Dombrovski, Aiping Dong, Patrick Finerty Jr., Susanne Flodin,
Alex Flores, Susanne Graslund, Martin Hammerstrom, H. Hao, Maria
Dolores Herman, Bum-Soo Hong, Raymond Hui, Ida Johansson, T.
Karlberg, Yongson Liu, R. Loppnau, F. MacKenzie, Martina Nilsson,
Lyudmila Nedyalkova, Par Nordlund, Tomas Nyman, Jinrong Min, Hui
Ouyang, Hee-won Park, Chao Qi, A. Schuetz, A. Seitova, Limin Shen,
Yang Shen, Deepthi Sukumard, Wolfram Tempel, Yufeng Tong, Lionel
Tresagues, Masoud Vedadi, John R Walker, Johan Weigelt, Martin Welin,
Hong Wu, Ting Xiao, L. Xu, Hong Zeng, Haizhong Zhu.
Conceived and designed the experiments: AE. Analyzed the data: AKW
AE. Wrote the paper: AKW AE.
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