Photon Factory Activity Report 2016 #34 (2017)

BL-5A, BL-1A /2016G554

Influence of precipitants on molecular arrangement and space group of protein crystals
Tyuji Hoshino1,* Satoshi Fudo1 Yuji Komukai1 and Michiyoshi Nukaga2
1
Graduate School of Pharmaceutical Sciences, Chiba University,
1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
2
Faculty of Pharmaceutical Sciences, Josai International University,
Gumyo 1, Togane-shi Chiba 283-8555, Japan
1 Introduction
In our previous work, we carried out the cluster
analyses of crystal structures for a single same kind ofprotein, exemplifying human immune-deficiency virus
type 1 (HIV-1) protease, hemoglobin, myoglobin, humanserum albumin, and hen egg-white lysozyme. In eachkind of proteins, crystal structures were separated into
several groups based on their structural similarities. From
this analysis, it was found that the crystal structures
belonging to the same group were crystallized by using
the same kind of precipitants. It was also shown that thecrystal structures belonging to the same group almost
always had a common space group. These results suggestthat precipitants strongly affect how each protein
molecule makes contacts with other protein molecules incrystal and the molecular contacts in crystal are reflectedin the space group.
In this study, we obtained three crystal structures for asingle same kind of protein with three different
crystallizing agents. The aim of the present study is toanalyse the behaviour of precipitants in terms of theinteraction with protein molecule in solution and toclarify the influence of precipitants on the manner ofmolecular arrangement of proteins in crystal. The protein
used for this study was influenza virus polymerase acidicsubunit
N-terminal
domainone-loopdeletionrecombinant (PANΔloop), which was handled in ourprevious works.[1, 2] First, we crystallized this proteinusing the three different precipitants; ammonium sulfate,
potassium sodium tartrate, and PEG 8000. Next,
molecular dynamics (MD) simulations were carried out
for the protein solution with each of these precipitants.
Then the relationship between the molecular packing in
crystal and the molecular geometry in simulation wascompared.
2 Experiment
Truncated PAN protein, PANΔloop, from influenza virus
A/Puerto Rico/8/34 (PR8) strain (H1N1) was expressedand purified as follows. The Escherichia coli straintransformed with the pET50b(+) vector containing thePANΔloop gene was cultured in LB medium. The protein
was expressed at 17 °C for 48 h after induction with 0.2
mM IPTG. The protein was purified by a His-affinty
column, followed by the cleavage of 6×His-fused Nus-tagby HRV 3C protease. The cleaved protein was again
purified by Ni-NTA resin. The protein was further
purified by gel filtration with a running buffer of 20 mM

Tris-HCl at pH 8.0 and 100 mM NaCl. Finally, theprotein was concentrated to 9.9 mg/mL.
Three crystal structures for PANΔloop were obtained withthree different crystallizing agents 1, 2, and 3. Crystals of
PANΔloop were grown by the vapor diffusion method withhanging drops consisting of 1.0 µL of 9.9 mg/mL protein
solution containing 4.0 mM MnCl2 and 1.0 µL of
crystallizing agent 1, 2, or 3 at 18°C. The composition ofcrystallizing agent 1 is 100 mM MES at pH 5.8, 1.1 M
ammonium sulfate, 0.1 M potassium chloride and 9%(v/v) trehalose. The crystallizing agent 2 includes 100
mM MES at pH 6.0, 0.8 M potassium sodium tartrate,
and the agent 3 includes 100 mM Tris-HCl at pH 7.5,
28% PEG 8000, 0.2 M calcium acetate, 10% Jeffamine
M-600. The crystals were cryoprotected by brief
immersion into well solution containing 22.5% (v/v)
glycerol, followed by flash-freezing in liquid nitrogen.
X-ray diffraction data for the crystal structures werecollected at 100 K on PF BL-5A or BL-1A beamline. Thediffraction data were indexed, scaled and merged withHKL2000. Intensities were converted into structure
factors and 5% of the reflections were flagged for Rfreecalculations. Atom coordinates for crystal structures weredetermined by the molecular replacement with MOLREP
program. Structure refinement and model building werecarried out using PHENIX and COOT. The PDB codes ofcrystal structures 1, 2, and 3 are 4ZQQ, 5JHT, and 5JHV.
The crystal structure 1 was used as a template forbuilding the models for MD simulation. The template wasplaced in a rectangular periodic boundary box filled with
TIP3P waters, and counter ions were added to neutralize
the model system using the LEaP module in AMBER 14.
The model at this step was used as an initial structure for
MD simulation without any precipitant (simulation 0). To
build the models which include ammonium sulfate as aprecipitant, three water molecules were chosen randomly
in the model for simulation 0 and then two of them werereplaced with ammonium ions and the other one withsulfate ion. This operation was repeated until theconcentration of ammonium sulfate was the same as thatin the crystallizing droplet in experiments. The procedure
for replacing water molecules with ammonium sulfate
was done twice to make two independent initial models
for MD simulations with ammonium sulfate (simulations
1-a and 1-b). Similarly, the water molecules chosenrandomly in the model for simulation 0 were replacedwith either potassium, sodium, or tartrate ion, and theprocedure was duplicated to make two independent initialPhoton Factory Activity Report 2016 #34 (2017)

models for MD simulations with potassium sodiumtartrate (simulations 2-a and 2-b). Since the length of
PEG 1000 in the linear conformation is almost the sameas that of the longest edge of the rectangular box, therandom generation, as done for the other precipitants, wasnot applicable. Hence, PEG 1000 molecules weregenerated to surround the protein in the model forsimulation 0, and the water molecules which overlappedwith a PEG molecule were deleted. The coordinates of
PEG molecules were changed between two initial models
for MD simulations, by rotating every other PEG by 180°
around the axis perpendicular to the primary molecularaxis (simulations 3-a and 3-b). It should be emphasizedagain that the number of precipitant molecules in eachmodel was set to reflect the initial concentration ofprecipitant in the hanging drop in experiments. Therefore,
the concentration of precipitant in each model is a half ofthat in well solution.
3 Results and Discussion
The crystal structures 1, 2, and 3 were obtained byusing the different precipitants for crystallizing agents(Table 1). The numbers of PANΔloop molecules in an
asymmetric unit were 1, 1, and 2 for the crystal structures
1, 2, and 3, respectively. The structures of these protein
molecules are almost the same among them as shown in
Figure 1a. For the crystal structure 3, the protein moleculeof only chain A is displayed because structural difference
between chain A and B is quite small. The space groupsof the crystal structures 1, 2, and 3 are P41212, P41212,and C2, respectively. According to our previous works, aprecipitant used for crystallization strongly affects thespace group of the crystal. Because the space groups forcrystal structures 1 and 2 are the same, ammonium sulfate
and potassium sodium tartrate, which were used as
precipitants for the crystals, are considered to have
similar influence on the protein. Figure 1b - 1d shows
how protein molecules in multiple asymmetric units arepacked in crystals. Obviously the packing modes in the
crystal structures 1 and 2 are same since they have thesame space group and almost same cell constants. There
are 8 protein molecules per one unit cell in all of the three
crystal structures, while the size of the unit cell of thecrystal structure 3 is much smaller than those of theothers, which means the proteins in the crystal structure 3
are tightly packed.

Fig. 1: Crystal structures 1, 2, and 3. (a) Superimpositionof the three crystal structures 1 (green), 2 (cyan), and 3(yellow, chain A only). (b-d) Crystal packing ofstructures 1 (b), 2 (c), and 3 (d). Unit cells are depicted inlines, and a, b, and c axes are depicted in arrows. In all of
the three crystals, one unit cell contains eight protein
molecules, each of which is depicted in a different color.

Table 1: Crystallographic properties
Crystal structure

1Precipitant

Ammonium sulfate

Space groupa, b, c (Å)α, β, γ ( ° )Resolution (Å)
No. reflections (Rfree set)
Rwork/Rfree2

3

P4121266.33, 66.33, 127.34
90.00, 90.00, 90.00
29.41-1.80
25617 (1316)

Potassium sodium
tartrate
P4121266.86, 66.86, 128.00
90.00, 90.00, 90.00
32.34-1.75
29882 (1519)

C2
64.24, 87.40, 66.50
90.00, 94.31, 90.00
36.49-2.75
9578 (514)0.224/0.274

0.194/0.238

0.197/0.234

To clarify the influences of precipitants on proteinmolecules and crystal structures, we carried out MD
simulations for a PANΔloop molecule without any
precipitant (simulation 0) and with ammonium sulfate(simulations 1-a and 1-b), potassium sodium tartrate(simulations 2-a and 2-b), and PEG 1000 (simulations 3-aand 3-b). PEG 1000 is a substitute for PEG 8000 in
simulation. These models (except for that for simulation

PEG 80000) were constructed so as to reproduce the concentrationsof the precipitants in the hanging drops at the moment
when they were set up by mixing the protein solution andone of the crystallizing agents. In order to focus on theprecipitants, the other minor chemicals included in thecrystallizing agents were ignored. In simulations 3-a and3-b, simulation time was set to 200 ns, while the others to
100 ns, because the movement of PEG 1000 wasPhoton Factory Activity Report 2016 #34 (2017)

relatively slow and the equilibration enough for the
reliable analysis needed longer simulation time.
The averaged distributions of the precipitant molecules
were analyzed for all of the simulations using thetrajectories of the last 20 ns. Figure 2a shows thedistributions of ammonium ions in dots and sulfate ions inmesh for simulation 1-a. The distributions of these twoions are similar to each other and, to our surprise, thedistributions are not isotropic around the protein. To
clarify what these anisotropic distributions mean, themolecular arrangement in crystal structure was contrasted
to the distribution. In Fig. 1b, each protein molecule has
the same surrounding environment and has direct contacts
with four surrounding protein molecules. One protein
molecule with its four surrounding molecules wereextracted as shown in Fig. 2b. Then the distributions of
the two kinds of ions (Fig. 2a) and the extracted protein
molecules (Fig. 2b) were superimposed (Fig. 2c, backside
views in Fig. 2d). Intriguingly, there were almost no
distributions of precipitants at the areas where the protein
molecule has contacts with other molecules in the crystalstructure. In other words, there was almost no overlap
between the distribution areas of precipitants (result ofthe simulation) and the contact sites between proteins(result of the crystal structure analysis).

Figure 3a shows the averaged distributions of thepotassium and sodium ions in dots (as a sum of the twoions) and the tartrate ion in mesh for the last 20 ns ofsimulation 2-a. The distributions of cations and tartrateions were similar to each other but not isotropic aroundthe protein. In the same manner as ammonium sulfate, aprotein molecule and four protein molecules making
direct contact with the protein were extracted from thecrystal structure 2 (Fig. 3b) and superimposed on thedistributions of cations and tartrate ions in simulations 2a and 2-b (Fig. 3c, backside view in Fig. 3d). Again, there
were no overlap between the distribution areas ofprecipitants and the contact sites of proteins.

Fig. 3: (a) Distributions of cations and tartrate ions insimulation 2-a. The distribution of cations (as a sum of
sodium and potassium ions) is depicted in dots and that oftartrate ions is in mesh. They are colored according to therate of the presence of those ions. Protein molecule isdepicted in cartoon (yellow). (b) Direct contacts ofprotein molecules in the crystal structure 2. A protein
molecule (green) has direct contacts with four otherprotein molecules (red, orange, magenta, and pink). (c, d)
Superimposition of (a) and (b), viewed from two different
angles.
Fig. 2: (a) Distributions of ammonium and sulfate ions insimulation 1-a. The distribution of ammonium ions isdepicted in dots and that of sulfate ions is in mesh. They
are colored according to the rate of the presence of thoseions in each point over the last 20 ns in the simulation(Color scales for both ions are depicted in the bottom.).
Protein molecule is depicted in cartoon (yellow). (b)Direct contacts of protein molecules in the crystal
structure 1. A protein molecule (green) has direct contacts
with four other protein molecules (red, orange, magenta,and pink). (c, d) Superimposition of (a) and (b), viewed
from two different angles.

Based on the findings in this study, we propose themechanism of the protein crystallization under the firsttwo precipitants as follows. First, precipitant molecules
are attracted to the protein surface due to the electrostatic
potential and are distributed heterogeneously around theprotein. This causes the electrostatic screening of theprotein surface charge, which leads to the suppression of
the electrostatic repulsion of protein molecules, and thestabilization of the conformational fluctuation of protein
molecules, both of which make the protein solubility
lower. Then protein molecules are promoted to makecontacts with each other. Precipitant molecules coveringsome sites of the protein surface prevent the other proteinPhoton Factory Activity Report 2016 #34 (2017)

molecules from accessing to the covered sites. Hence, thecontact sites on the proteins are restricted, which helpsprotein molecules to form regularly ordered clusters, thatis, to crystallize with a specific space group.
Figure 4a shows the averaged distributions of PEG
molecules for the last 20 ns of simulations 3-a. PEGs
cover most parts of the protein surface, which isconsiderably different from the results of the other two
precipitants. In the crystal structure 3, each protein
molecule has direct contacts with other 10 molecules (Fig.
4b). The protein molecule of chain A in the crystalstructure was used as a center molecule in these figures(colored green) since the two molecules in an asymmetricunit are almost identical in structure and contact geometry.
In a similar manner to the other two precipitants, thedistributions of PEG molecules (Fig. 4a) and the extracted
crystal structure (Fig. 4b) were superimposed andcompared (Fig. 4c, d). Although many parts of the protein
surface are covered by PEGs or surrounding protein
molecules, only the sites that are not covered by PEGs in
the simulations have no contact with other protein
molecules in the crystal structure (Those sites areindicated by red arrows in Fig. 4c, d).

mixture of protein solution and crystallizing agents fordroplet, PEGs are bound to some specific sites of proteinmolecules. The molecular contacts of proteins are formed
with the progress of crystallization, accompanying therelease of PEG molecules that were originally bound tothe contact sites. The release of PEG molecules leads tothe increase of the entropy of the system, whichefficiently drives the crystallization. This can be regarded
as a kind of depletion forces, in which the decrease of thedepletion zone by the increase of contacts ofmacromolecules is promoted by the entropic gain of PEG
molecules in solution. Accordingly PEGs play a role ofguiding protein molecules for making adequate contacts
and packing, thereby leading to a specific space group incrystal.
In conclusion, three kinds of crystals were obtainedwith three different precipitants for a single same kind ofprotein. The space group of one of the crystals wasdifferent from that of the others. The relationship between
the precipitants used in the protein crystallization and themolecular packing in the crystals was investigated by
combining crystallographic structure analysis and MD
simulation. It was found that precipitant molecules were
not randomly distributed around the protein and, instead,the distribution showed a strong anisotropy. MD
simulations with precipitants in a high-concentrationequivalent to the crystallizing condition enabled us toclarify molecular interactions of precipitants with theprotein. Our calculation suggested that heterogeneous
distribution of precipitants played a role in restricting thecontact sites of the protein, thereby causing a specificmolecular packing in crystal and leading to the growth in
the corresponding space group. Accordingly, theprecipitant that can sufficiently restrict the contact sites ofprotein molecules will be one of the critical elements forsuccessful crystallization.
Acknowledgement
Calculations were performed at Research Center for
Computational Science, Okazaki, Japan and atInformation Technology Center of the University ofTokyo. This work has been performed under the approvalof the Photon Factory Program Advisory Committee(proposal no. 2014G563, 2015G554).

Fig. 4: (a) Distribution of PEGs in simulation. The
distribution is shown in mesh. Mesh is colored according
to the rate of PEG atom presence at each point over thelast 20 ns in the simulation. Protein molecule is depicted
in cartoon (yellow). (b) Direct contacts of protein
molecules in the crystal structure 3. A protein molecule(green) has direct contacts with 10 other protein
molecules (10 different colors). (c, d) Superimposition of(a) and (b), viewed from two different angles.
Based on the findings that protein molecules
preferentially make contacts with each other at the sites
covered by PEGs in simulation, we propose thecrystallization process using PEG as a precipitant asfollows. In the initial stage of crystallization just after theReferences[1] S. Fudo et al., Bioorg. Med. Chem. 23, 5466 (2015).
[2] S. Fudo et al., Biochemistry 55, 2646 (2016).
* hoshino@chiba-u.jp