C-Kit Ground Pro ND[Neutron Diffraction Kit]

Aug. 2024
Confocal Science Inc.

Table of Contents
1.

Introduction ................................................................................................. 4
1.1. Overview ................................................................................................ 4
1.2. Features................................................................................................. 5

2.

Crystallization Methods Used in C-Kit Ground Pro ND ............................... 6
2.1. The counter-diffusion (CD) method ........................................................ 6
2.2. The dialysis method ............................................................................... 8
3. Experimental Procedure .............................................................................. 93.1. Setting up crystallization via the batch method ...................................... 9
3.1.1. Required items ................................................................................ 9
3.1.2. Preparation of the sample solution .................................................. 9
3.1.3. Assembling the items to start crystallization .................................... 9
1. Loading the sample solution ................................................................ 10
3.2. Setting up crystallization via the CD method ........................................ 123.2.1. Required items .............................................................................. 12
3.2.2. Pre-soaking of the gel tube ............................................................ 13
3.2.3. Preparation of solutions and a marked capillary ............................ 14
3.2.4. Assembling the items to start crystallization .................................. 15
3.3. Setting up crystallization via the dialysis method ................................. 19
3.3.1. Required items .............................................................................. 19
3.3.2. Pre-soaking of the LCDM .............................................................. 20
3.3.3. Preparation of solutions ................................................................. 21
3.3.4. Assembling the items to start crystallization .................................. 21
3.4. Harvesting crystals from the LCDM ..................................................... 23
3.4.1. Desirable harvesting solution......................................................... 23
3.4.2. Disassembly of LCDM container ................................................... 23
3.4.3. Harvesting ..................................................................................... 24
4. Technical Notes ........................................................................................ 261

4.1. Time-dependent diffusion of reagents.................................................. 26
4.1.1. Change of reagent concentrations in the CD method capillary ...... 26
4.1.2. Change of reagent concentrations in the LCDM ............................ 27
4.2. How to make a large crystal ................................................................. 28
4.2.1. The amount of required sample protein ......................................... 28
4.2.2. Number of crystals ......................................................................... 28
4.2.3. How to design crystallization experiments for large crystals .......... 30
4.2.4. For crystallization reagents containing multiple components ......... 32
4.2.5. In the cases of inverse concentration dependence ........................ 33
4.2.6. Quality of protein samples ............................................................. 34

2

Contents of C-Kit Ground Pro ND (CRT101-2)
Product name

Qty.

Description

Capillary (φ0.29 mm × 29 mm)

100

Used in the batch method.

Capillary (φ0.5 mm × 47 mm)

15

Gel tube (φ1.0 mm × 1 cm)

18

Used in the CD method. 3 extra
gel tubes included.

LCDM (glass capillary)

6

Used in the dialysis method to

LCDM (quartz capillary)

2

C-Cap

10

grow large crystals. Dialysis
membrane (MWCO 6,000–8000)
is placed in the capillary.

Silicone tubing (φ1.0 mm × 50 cm)

1

Used for aspirating solution.

Ultra-fine gel loading tip

9

Used for loading sample into
LCDM.

Sealing compound

1

Used for sealing capillaries.

Round-bottom tube (5 mL)

30

Vessel for crystallization.

Precautions and Notice◼
◼
◼

This kit is for research purposes only. Please do not use it for any otherpurpose.
When attaching the gel tube to the capillary, please be careful not to injure
yourself.
This product uses US patent 7531037 of the Japan Aerospace Exploration
Agency (JAXA) under license.

3

1. Introduction
1.1. Overview
The C-Kit Ground Pro ND is an equipment for protein crystallization that produces
large crystals for neutron diffraction and has been developed from knowledge ofcrystallization experiments at the International Space Station (ISS) for decades.
This kit provides experimental tools for three simple methods: batch, counterdiffusion (CD) 1), and dialysis 2). First, the crystallization conditions are determinedusing batch method tools; then, the conditions are confirmed using CD method
tools, and large crystals grow in a large-bore capillary with a dialysis membrane(LCDM). These steps enabled the growth of large crystals with small amounts ofprotein.
The CD method tools included in the kit were jointly developed by Confocal
Science, under commission from JAXA 3). This product uses US patent 7531037
of JAXA under license 4). In addition, Confocal Science provides C-Kit Ground
Pro
XRD
for
crystallization
for
X-ray
diffraction(http://www.confsci.co.jp/product_e.html) and C-Kit Space Pro Series for highquality crystallization under microgravity at the ISS.
1) García-Ruiz, J.M.; Moreno, A. Investigations on protein crystal growth by the gel
acupuncture method. Acta Crystallogr., Sect. D 1994, 50 (4), 484–490
2) Takahashi, S.; Koga, M.; Yan, B.; Furubayashi, N.; Kamo, M.; Inaka, K.; Tanaka, H.
JCB-SGT crystallization devices applicable to PCG experiments and their crystallization
conditions. Int. J. Microgravity Sci. Appl. 2019, 36, 360107
3) Tanaka, H.; Inaka, K.; Sugiyama, S.; Takahashi, S.; Sano, S.; Sato, M.; Yoshitomi, S.
A simplified counter diffusion method combined with a 1D simulation program for
optimizing crystallization conditions. J. Synchrotron Rad. 2004, 11, 45–48.
4) JP patent 4354457, US patent 7531037

41.2. Features◼

◼
◼

A small amount of the protein sample is required: The standard amount is 1.3µL (the batch method capillary), 8 µL (the CD method capillary), and 27 µL(the dialysis method capillary).
Simple protocols: all protocols are easy to perform.
High reproducibility and reliability: Both the CD and dialysis methods havebeen used repeatedly in space experiments conducted by JAXA and theJapan Manned Space Systems Corporation (JAMSS). Thus, highreproducibility and reliability were demonstrated. In particular, the CD has
been applied to the crystallization of more than 500 proteins under
microgravity since 2002.

◼
◼

Long-term stability: The crystals produced using the C-Kit Pro ND were stable
for a long period in the capillaries.
Estimation of optimal conditions: Time-dependent changes in protein andcrystallization reagent concentrations in the CD capillaries and LCDM were
estimated using a one-dimensional diffusion simulation program (available
forpurchase
as
C-Kit
Pro
Advanced
Tool[CRT209],
http://www.confsci.co.jp/product_e.html). This helps to determine the optimal
conditions for crystallization.

5

2. Crystallization Methods Used in C-Kit Ground Pro
ND
This kit provides experimental tools for three methods: batch, counter-diffusion(CD), and dialysis. The principle of batch crystallization is simple; therefore, we
explain the other two methods.

2.1. The counter-diffusion (CD) method
C-Kit Ground Pro ND provides a set of crystallization equipment for the gel tube
(GT) method, which is one of the CD methods. Figure 2.1 provides a simple
explanation of the standard configuration of the method and mechanism of crystal
growth.

Fig. 2.1 GT (CD) method illustration.
Upper left: Configuration of the GT method. Upper right: Detailed view of acapillary with a gel tube. Bottom: Crystallization mechanism of the GT method.
6

A feature of this method is that a silicone tube with agarose gel, called a gel tube,
is connected to the end of the capillary loaded with the protein sample. The
reservoir solution containing the crystallization reagents gradually diffuses into
the capillary through the gel tube, forming a concentration gradient of thecomponents of the reservoir solution inside the capillary. The protein samples
and other coexisting components in the sample solution also gradually diffuse outof the capillary. The diffusion results in a continuous and wide range ofconcentration combinations of the crystallization reagent and sample solution
within the capillary. Consequently, crystallization is triggered when theconcentration combination is suitable for crystallization. This phenomenon is
known as “the self-searching mechanism.” Understanding the diffusion timecourse of the crystallization reagent and protein sample is important for thiscrystallization method 4). See “4.1. Time-dependent diffusion of reagents” formore details.

72.2. The dialysis method
The C-Kit Ground Pro ND includes crystallization tools for the dialysis method as
another type of crystallization method for large crystals. Figure 2.2 provides asimple explanation of the standard configuration of the dialysis method using
LCDM and the crystal growth mechanism.

Crystals are formedby diffusion of crystallization
solutions into the LCDM.

Fig. 2.2 LCDM illustration
Upper left: Configuration of the dialysis method. Upper right: Detailed view of
the LCDM. Bottom: The crystal growth mechanism.
A large bore capillary loaded with the sample solution is connected to a gel tube
via a dialysis membrane. The crystallization solution diffuses into the large-bore
capillary through the gel tube and crystallizes when its concentration increases
to an appropriate level. Understanding the diffusion time course of thecrystallization reagents is important for this method. See “4.1. Time-dependent
diffusion of reagents” for more details.

83. Experimental Procedure
3.1. Setting up crystallization via the batch method
3.1.1. Required items(For each crystallization condition)
Solution

AmountDescription

Sample solution

3–5 µLIt contains a protein sample solution andcrystallization reagents and is loaded intoa capillary.
Because the inner diameter of thecapillary is 0.29 mm, the required volume
is 1.3 µL for a sample length of 20 mm. It
is desirable to prepare a little more
solution for loading without failure.

Equipment in the kit

Qty.

Description

Capillary(φ 0.29 mm × 29 mm)

1

Sealing compound１

It is used for sealing both ends of thecapillary.

Silicone tubing

1

It is used if suction is required whenloading solutions into the capillary.

Round-bottom tube
(5 mL)

1

A vessel for crystallization.

3.1.2. Preparation of the sample solution
Solutions containing protein samples and appropriate concentrations ofcrystallization reagents (and ligands, if necessary) are prepared for loading.

3.1.3. Assembling the items to start crystallization

91. Loading the sample solution
When the tip of the capillary is immersed in the sample solution, the solution issucked into the capillary via capillary action. If this is difficult, place the capillaryon its side to make it easier (Fig. 3.1).

The solution is sucked
into the capillaryby capillary action.

If this is difficult, lay thecapillary on its side.

Fig. 3.1 Loading the sample
solution by capillary action
When the solution does not enter by capillary action use a micropipette forquantitative loading as follows: Attach a silicone tubing to a 20 µL micropipettetip, and attach its other end to the top of the capillary. Adjust the micropipette to
the filling volume, and insert the bottom end of the capillary in the sample solution
for aspiration. Alternatively, turn the micropipette dial to aspirate the solution
quantitatively into the capillary. After loading the required amount, remove thesilicone tubing gently, taking care not to change the amount of liquid in thecapillary.

Fig. 3.2 Loading sample solutions
using a micropipette

10When the solution comes in contact with the sealing compound at thecapillary ends, crystals sometimes grow. To prevent unwanted crystallization, thecapillary should be held in the hand and slowly tilted to move the solution toward
its center.
2. Sealing both ends of the capillary
Seal the ends of the capillary with thesealing compound. Turn the sealing
compound container upside down, and push
it onto the upper end of the capillary until thecapillary reaches the bottom plate of thesealing compound container. Turn thecapillary left and right 2–3 times and thenFig. 3.3 Sealing the capillarypull it out (Fig. 3.3). Repeat this process until
approximately 2 mm of the sealing compound is packed into the top of thecapillary.
3. Starting crystallization
The capillary is then gently placed in a round-bottom tube and capped. It is thenplaced vertically at an appropriate temperature.

11

3.2. Setting up crystallization via the CD method
3.2.1. Required items(For each crystallization condition)
Solution

AmountDescription

Sample solution

6–8 µLIt contains a protein sample solution,
which may also contain a ligand or
crystallization reagents, and is loaded intoa capillary.
Because the inner diameter of thecapillary is 0.5 mm, the required volume is
6 µL for a sample length of 30 mm and 8µL for 40 mm. It is desirable to prepare alittle more solution for loading without
failure.

Reservoir solution

ca. 1
mL

It contains crystallization reagents andbuffer solution, which may also containligand components. It diffuses into thecapillary through a gel tube to crystallize
the protein.

Gel-tube soaking
solution

ca. 4
mL

It is a solution for immersing the gel tube
before the start of crystallization,
containing only the buffer component of
the reservoir solution or its complete
components. A sufficient amount of liquid
to immerse the gel tube (10 mm in length)
is required.

Seed crystal

１–2 µL

Prepare for seeding, if necessary.

Equipment in the kit

Qty.

Description

Capillary(φ0.5 mm × 47 mm)

1Mark the upper end of sample before
loading.

Gel tube(φ1.0 mm × 1 cm)

1

Gel tubes in C-Kit Ground Pro are
immersed in 0.04% NaN3 solution.

Sealing compound1

It is used for sealing the upper end of thecapillary.
12

Silicone tubing

1

It is used if suction is required whenloading solutions into the capillary.

Round-bottom tube
(5 mL)

1

A vessel for crystallization.

Qty.

Description

1

A tube with a height of 40 mm and acapacity of about 5 mL. It is used for presoaking the gel tube.

Required equipment
Tube with lid

Micropipette/Tip
Micropipette/Tip(20 to 100 µL)

1 eachIt is used for preparing solutions.

1

It is used for loading solutions into thecapillary.

Surgical blade(or a cutting blade)1

It is used for cutting the lower end of thegel tube. The cutting blade must be sharp.

Fine-point permanentmarker1

It is used for marking the end of the loaded
sample on the capillary.

3.2.2. Pre-soaking of the gel tube
1. Preparation of the gel-tube soaking solution
The crystallization reagent requires time to diffuse into the gel tube. Therefore, if
you want the crystals to grow faster, the gel-tube soaking solution should havethe same composition as that of the reservoir solution. For crystallization under
mild conditions, either the buffer component alone or that containing the required
amount of the crystallization reagent is available for the solution. Add about 4 mLof the solution to the tube with a lid and immerse the gel tubes for at least a few
days before use. Caution: Always keep your gel tubes immersed in the liquid toprevent them from drying.
2. Time required for soaking the gel tube
According to the simulation results of the solute concentration changes in the geltube immersed in the gel soaking solution, it takes about 0.5 days for the NaClsolution and about 4 days for the PEG4000 solution to achieve equilibrium in thegel tube.

13

Fig. 3.4 Simulation results ofinternal NaCl concentration of agel tube immersed in 2 M NaClsolution.
The horizontal axis is the distancefrom the end of the gel tube, and thevertical
axis
is
the
NaCl
concentration. The curves show,fromthebottom,NaClconcentrations after 0.05, 0.1, 0.2,
and 0.5 days.
Fig. 3.5 Simulation results ofinternal PEG4000 concentration ofa gel tube immersed in 20%
PEG4000 solution.
The horizontal axis is the distancefrom the end of the gel tube, and thevertical axis is the PEG4000
concentration. The curves show,fromthebottom,PEG4000
concentrations after 0.25, 0.5, 1, 2,
and 4 days.

3.2.3. Preparation of solutions and a marked capillary
1. Sample solution
Prepare the required amount of protein sample solution for loading. If it isnecessary to mix the crystallization reagents, ligands, etc., mix them before
loading them into the capillary.
2. Seed solution
If you are planning to perform micro-seeding or macro-seeding, prepare seed
crystals in a suitable solution.

14

3. Reservoir solution
Prepare the required amount of reservoir solution for filling a 5 mL round-bottom
tube.
4. Capillary with a marked line
Use a permanent marker to mark the end of the loaded sample on the capillary.
Place a mark at 40 mm from the bottom edge when loading the 8 µL sample
solution, and mark two lines at 35 mm and 40 mm when seeding.

Fig. 3.6 Reference lines on the capillary

3.2.4. Assembling the items to start crystallization
1. Loading the sample solution to the marked line
When the tip of the capillary is immersed in the sample solution, the solution issucked into the capillary by capillary-action. If this is difficult, lay the capillary on
its side to make it easier. Ensure that you do not lift the capillary out of the sample
solution during loading to avoid air bubbles in the capillary.

The solution is sucked
into the capillaryby capillary action.

If this is difficult, lay thecapillary on its side.

Fig. 3.7 Loading the sample
solution by capillary action
For seeding, fill the sample solution up to the first reference line, move thecapillary into the seed solution, and fill the seed solution until it reaches thesecond reference line.
Depending on the properties of the sample solution, the solution does notenter by capillary action (e.g., in case of a highly viscous solution); conversely,15

the solution immediately fills the capillary (in case of solutions that include
detergents and organic solvents). In these cases, use a micropipette forquantitative loading as follows:
Attach a silicone tubing to a 20 µL micropipette tip, and attach its other end
to the top of the capillary. Adjust the micropipette to the filling volume, andimmerse the bottom end of the capillary in the sample solution for aspiration.
Alternatively, turn the micropipette dial to aspirate the solution quantitatively into
the capillary. After loading the required amount, remove the silicone tubing gently,
taking care not to affect the amount of liquid in the capillary.

Fig. 3.8 Loading sample solutions
using a micropipette

For seeding, fill the sample solution up to the first marked line, move thecapillary into the seed solution, and turn the dial of the micropipette so that the
sample solution reaches the second line.
2. Sealing the capillary
Seal the upper end of the capillary withthe sealing compound. Turn the sealing
compound container upside down, andpush it onto the upper end of the capillary
until the capillary reaches the bottom
plate of the sealing compound container.

16

Fig. 3.9 Sealing the capillary

Air bubbles sometimes enter the
lower end of the capillary duringloading. Air bubbles prevent thereservoir solution from diffusing in, so
stuff the sealing compound repeatedly
from the top of the capillary to push out
the air from the bottom. Allow thedroplet of sample solution to beobserved from at bottom of theFig. 3.10 Tips to avoid air
bubbles at the bottom endof the capillary

capillary.
3. Attaching the gel tube
Drip a small amount of gelsoaking solution at thecapillary end of the gel tube
before attaching the capillary
to prevent air from flowing
between the capillary and geltube. Then, attach the gel tube
to the bottom of the capillary.
A small amount of gel will be
Fig. 3.11 Attaching the gel tube
pushed out from the bottom of
the gel tube after theconnection.
Next, cut the bottom of the geltube diagonally with a sharp blade.
This enables the reservoir solution toeasily diffuse into the capillary, even if
the tip of the gel tube is in close
Fig. 3.12 Cutting the bottom ofcontact with the bottom of the roundthe gel tube
bottom tube.

17

4. Starting crystallization
Gently place the capillary into the round-bottom tube containing the reservoir
solution and cap it. Place it vertically with the gel tube facing down at theappropriate temperature. In many cases, the reservoir solution containing the
crystallization reagent has a higher density than the sample solution. Thus, when
the crystallization reagent enters the geltube by diffusion into the capillary with thegel tube side down, the density of thesolution increases towards the bottom of thecapillary,
suppressing
density-driven
convection in the capillary. As a result, we
can expect to obtain better quality crystals
owing to the similar effects of convection
suppression in a microgravity environment.
Note: The capillary must be placed
horizontally during observation, but this is
not a problem for short periods.

18

Fig. 3.13 Place verticallyand start crystallization

3.3. Setting up crystallization via the dialysis method

3.3.1. Required items(For each crystallization condition)
Solution

AmountDescription

Sample solution

ca. 27µL

It contains a protein sample solution forloading into an LCDM tube.
Because the inner diameter of the LCDM
tube is 1.95 mm, the required volume is 27µL for a sample length of 9 mm. It is
desirable to prepare a little more solution
for loading without failure.

Reservoir solution

ca. 1
mL

It interdiffuses with the sample solution in
the LCDM to crystallize the protein. It
contains crystallization reagents andbuffers, which may also contain ligands if
required.

Equipment in the kit

Qty.

Description

LCDM tube(φ1.95 mm × 29 mm)

1

It has a gel tube at one end.

C-Cap

1

It is a stopper to plug the capillaries of the
LCDM.

Sealing compound１

It is used to seal the end of LCDM.

Silicone tubing

1

It is used to load solutions into the LCDM.

Round-bottom tube
(5 mL)

1

A vessel for crystallization.

Qty.

Description

Required equipment
Micropipette/Tip
Micropipette/Tip(20 µL)each 1

It is used for preparing solutions.

1

It is used for loading solutions into an
LCDM.

Surgical blade(or a cutting blade)1

It is used for cutting the lower end of thegel tube. The cutting blade must be
sharp.
19

3.3.2. Pre-soaking of the LCDM
1. Pre-soaking solution
LCDMs in the C-Kit Ground Pro are immersed in a 0.04% NaN3 solution. Protein
samples may be damaged by rapid changes in salt concentration if LCDM with
low ionic conditions is used directly for protein crystallization without pre-soaking.
The crystallization reagent requires time to diffuse into the LCDM. Therefore, thecrystals grow faster, when the LCDM has been presoaked in a solution with thesame composition as that of the reservoir solution. For crystallization under mild
conditions, either the buffer component alone or that containing the required
amount of the crystallization reagent is available for the pre-soaking solution. Add
approximately 4 mL of the solution to the tube with a lid and immerse the LCDM
for at least a few days before use. The entire LCDM, not only the gel tube, shouldbe immersed to equilibrate at the upper end of the LCDM.
2. Time required for soaking LCDMs
According to the simulation results of the solute concentration changes in the
LCDM immersed in the soaking solution, it takes approximately 1 day for the NaClsolution and approximately 2 weeks for the PEG4000 solution to achieve
equilibrium in the LCDM.
Fig. 3.14 Estimation of NaCl
concentrations in an LCDM.
The horizontal axis is the distancefrom the end of the gel tube, and adialysis membrane is placed at 14mm. The vertical axis is the NaCl
concentrations. The curves show,fromthebottom,NaClconcentrations 0.5, 1, and 2 days
after soaking in 1M NaCl.

20

Fig. 3.15 Estimation of PEG 4000
concentrations in an LCDM.
The curves show, from the bottom,
PEG 4000 concentrations 1, 2, 4, 8,
and 16 days after soaking in 20%
PEG 4000. Others are same as inFig. 3.14.

3.3.3. Preparation of solutions
1. Sample solution
Prepare the required amount of protein sample solution for loading. If it isnecessary to mix ligands, mix them before loading them into the LCDM.
2. Crystallization solution
Solution that crystallizes proteins by interdiffusion with the sample solution in the
LCDM, contains crystallization reagents and buffers, which may also containligands if required.

3.3.4. Assembling the items to start crystallization
1. LCDM
Remove the LCDM from the soaking
solution and aspirate the liquid insideusing an ultrafine gel-loading tip.

Do not block
the top end
with the gelloading tip.

2. Sample loading to LCDM
Load the sample solution into the LCDM
using a 20 µL micropipette with an ultrafine gel-loading tip. Load approximately
27 µL sample solution and leave at least
1 mm of air at the top of the LCDM.
Fig. 3.16 Loading
sample into LCDM

21

3. Sealing the LCDM
Seal the upper end of the LCDM with thesealing compound. Then, attach the CCap to protect the sealing from theosmotic pressure. Insert the top of LCDM
into the diagonal cut side of the C-Cap.
Push the plastic plug inside the C-Cap
while covering the other end of the C-Cap
with your finger to prevent the plug frompopping out. When the plastic plug ispushed to the end of the other side of the C-cap, installation is complete.
4. Cutting the bottom of LCDM
The bottom of each gel tube is then cut diagonally using a sharp blade. This
enables the reservoir solution to easily diffuse into the LCDM, even if the tip of
the gel tube is in close contact
with the bottom of the roundbottom tube.
5. Starting crystallization
Approximately 4 mL of the reservoir solution
is added to the round-bottom tube, the LCDM
is gently placed with the gel tube side down,
and the round-bottom tube is capped. It isplaced vertically at the desired temperature.
In many cases, the crystallization solution has
a higher density than the sample solution.
Therefore, when the crystallization reagent
diffuses into the LCDM with the gel tube side
down, the density of the lower part of theFig. 3.19 Place verticallyand start crystallization

LCDM increases, which suppresses thesolution movement in the LCDM due to density-driven convection.
Note: The LCDM must be placed horizontally during the observation period;
however, this is not a problem for short periods.

22

3.4. Harvesting crystals from the LCDM
If there is a single crystal in a suitable location in the quartz capillary, it is possible
to remove the solution from the capillary with a gel-loading tip and directly performa neutron diffraction experiment. However, in some cases, harvesting the crystals
may be necessary. The following is an explanation of how to achieve this:If you would like to remove a crystal from a CD method capillary, please referto the description in the instruction manual for the C-Kit Ground Pro XRD,
CRT101-1, "3.3. Harvesting crystals from a CD method capillary"(http://www.confsci.co.jp/images/Ckit%20Ground%20Pro%20XRD%20Manual%20Eng%20240613.pdf).

3.4.1. Desirable harvesting solution
To harvest crystals while maintaining good quality, the harvested crystals should
be stored in a harvesting solution with the same composition as the solution in
the LCDM, where the target crystals are located. The solution composition in the
LCDM does not vary significantly depending on its location in the LCDM; however,it does vary depending on the duration of the crystallization reaction. The
crystallization reagent concentration in the LCDM takes about one month to reachequilibrium if salts are diffused, and several months or more if PEGs are diffused("4.1. Time-dependent diffusion of reagents"). Therefore, if one plans to harvest
crystals before equilibrium is reached, one must predict and prepare a harvesting
solution containing crystallization reagent components at approximately the same
concentration as the crystallization reagent in the LCDM. If the concentrations
are inappropriate, the crystals may dissolve or break because of osmoticpressure differences.
For the prediction of the concentration, please refer to the description in "4.1.
Time-dependent diffusion of reagents" In addition, to ensure reliable crystal
harvesting, we recommend preparing and testing several solutions withconcentrations above and below the predicted concentrations. The preparedharvesting solutions are placed on the depressions of cavity glass slides.

3.4.2. Disassembly of LCDM container
The thin capillary on the C-Cap side of the LCDM can be removed by pulling itout, taking care not to apply excessive force. The thin capillary on the gel tube
23

side cannot be removed because it has an attached dialysis membrane; therefore,
the end of the LCDM container is cut (broken) with a diamond file.

3.4.3. Harvesting
The prepared harvesting solution is placed on a cavity glass slide. A short pieceof the cut LCDM is then immersed in the solution and held with forceps. It can beeasily held by using the stand with reverse action tweezers (sold separately,(http://www.confsci.co.jp/images/CKit%20Pro%20Advanced%20Tool_2208_Eng.pdf). While observing under astereomicroscope, pour the harvesting solution into the fixed LCDM from the CCap side. Pipette the solution around the crystal to force it out of the capillary andinto the harvesting solution. It is recommended to pour the crystals out throughthe broken side with the larger opening.
Some crystals may adhere to the glass surface of the LCDM. In such cases,
carefully touch and move them with a thin, blunt tool, such as a gel-loading tip(QSP 124-R204). Pipette the solution again around the crystal; it drives the
crystals out of the capillary into the harvesting solution.
In practice, it is recommended to prepare lysozyme crystals using the CD
method to familiarize oneself with the procedure for harvesting crystals. As
lysozyme crystals easily adhere to the inner surface of the capillary, they aresuitable for training.

Fig. 3.20 Harvesting24Upper left: Fix the cut LCDM with forceps so that you can work with your hands
freely while observing under a stereomicroscope.
Upper right: Pipette the solution into the LCDM and push the crystal gently.
Lower left: If the crystal adheres to the LCDM, touch it gently with a gel-loading
tip and move it a little. The broken side with the larger opening is easier to touch.
Lower right: The crystal is pushed into the harvesting solution.

25

4. Technical Notes
4.1. Time-dependent diffusion of reagentsProtein crystallization can be expected when the concentrations of the protein
sample and crystallization reagent are in a suitable combination. Therefore, it isimportant to understand time-dependent diffusion of the proteins andcrystallization reagents in the CD method capillary and LCDM.

4.1.1. Change of reagent concentrations in the CD method capillary
A one-dimensional diffusion simulation can be used to estimate the time courseof crystallization reagent concentration in the CD capillary. A simulation program(available for purchase as C-Kit Pro Advanced Tool, CRT209,
http://www.confsci.co.jp/product_e.html) makes it easy to estimate the diffusion
profiles under various conditions. Examples of diffusion simulations for commonlyused crystallization reagent, NaCl are shown below.
Example 1
Sample solution： 40 mm length in a capillary
Reservoir solution： 1 M NaCl
Fig. 4.1 Estimation of NaCl
concentrations in a capillary.
, ■, ▲, x, and Ж show NaCl
concentrations 0.25, 1, 3, 8, and 16
days after loading, respectively.
The horizontal axis shows the
distance from the end of the geltube, and the vertical axis indicatesthe NaCl concentrations at thelocation.
The NaCl concentration in the capillary reaches near equilibrium in 16 days.
Ammonium sulfate, which is often used as a crystallization reagent, has almost
the same diffusion coefficient as NaCl. Thus, its diffusion time course is almost
the same.

264.1.2. Change of reagent concentrations in the LCDM
Example 2
Sample solution: 9 mm length (27 L) in the LCDM
Reservoir solution： 1 M NaCl
Fig. 4.2 Estimation of NaCl
concentrations in the LCDM.
, ■, ▲, x, and Ж show NaCl
concentrations 4, 8, 16, 32,
and 64 days after loading,respectively. The horizontal
axis shows the distance fromthe end of the gel tube, and adialysis membrane is placed at
14 mm. The vertical axis
indicates
theNaClconcentrations at the location.
The NaCl concentration in LCDM reaches equilibrium in 32 days. Ammonium
sulfate, which is often used as a salt, has a diffusion coefficient similar to that ofNaCl, so the time lapse is similar.
The dialysis membrane in LCDM has a MWCO of 6,000-8,000, but our actual
measurements show that even PEG4000, which has a lower molecular weight,
permeates very slowly. PEGs, which has a large molecular weight and highviscosity, are useful as crystallization reagents. Although, when using it as a
crystallization solution of the dialysis method, it takes a long time, such as several
months, to grow crystals. In that case, we recommend that a component thatcannot permeate the dialysis membrane (such as high molecular weight PEGs)
should be added to the protein sample in advance.

27

4.2. How to make a large crystal

4.2.1. The amount of required sample protein
Large amounts of proteins are required to create large crystals. The amount canbe estimated as follows:
For simplicity, the crystal is assumed as a cube with each side measuring “amm.” The following number of protein molecules will be present in this cube.
(Vm is the Matthews Coefficient, and M is the molecular weight).
𝑎31000 × 𝑉𝑚 × 𝑀 × 10−24(1)Number of moles of the protein present is:𝑎3
𝑉𝑚 × 𝑀 × 6.02 × 102(2)

The mass (g) of the protein present is:𝑎3
𝑉𝑚 × 6.02 × 102(3)

If the concentration of the protein sample is C mg/mL, and the solubility (protein
concentration at which crystals no longer grow) is Ce mg/mL, the required volumeof the protein solution (L) is as follows:
𝑎3 × 1660
𝑉𝑚 × (𝐶 − 𝐶𝑒 )(4)

For example, for lysozyme (PDB: 3IJV), Vm is 1.84. Assuming a crystal sizeof 1 mm3, protein concentration of 35 mg/mL, and solubility of 4 mg/mL, therequired solution volume is approximately 29.1 μL. In the case of α-amylase(PDB: 6TAA), Vm is 2.18; assuming that other conditions are the same, therequired volume is 24.6 μL.

4.2.2. Number of crystals
The amount of the protein sample estimated in the previous section is for one
crystal. If three crystals are formed, the size of each crystal may be reduced by
one-third.
The probability of protein crystal nucleation can be estimated using thefollowing two equations 5).

28

𝑑𝐿(𝑡)= 𝐴1 (𝐶(𝑡) − 𝐶𝑒 ).
𝑑𝑡
𝐼(𝑡) = 𝐶(𝑡)𝐴2 𝑒𝑥𝑝 −

(5)
𝐴3
2

𝐶(𝑡)
𝑙𝑛 ( 𝐶 )
(
)
𝑒

(6)

where C(t) and L(t) are the concentration of the protein and the characteristic
length of the crystal, respectively, assumed to be a cube at time t. I(t) is thenucleation probability at time t. Ce, A1, A2, A3 are constants experimentally
determined for each crystallization.
In our previous result 5), the four constants were estimated by applying theexperimental results, the dependence of the crystal growth rate, and the numberof nuclei formed on the protein concentration. The nucleation probability I(t)(number of nucleation per unit time and unit volume) at time t was then calculated,
as shown in Fig. 4.3. This indicates that the probability increases approximately
linearly above a certain protein concentration. According to the results, thenumber of lysozyme crystals after 24 h in the experiment with the 30 L
crystallization vessel is 5.9, 32.8, 62.5, and 91.9, for protein concentrations of 10,
15, 20, and 25 mg/mL, respectively. This analysis shows that the protein
concentration has a significant effect on the number of crystals obtained.
Fig. 4.3 Nucleation probability
on protein concentration 5).
Lysozyme was crystallized in
0.4 M NaCl, 15% PEG4000, 50
mM acetate buffer (pH 4.5) at
20°C.
Based
on
the
experimental results of crystal
formation, parameters were
estimated, and the protein
concentration dependence ofthe nucleation probability wascalculated
based ontheequation (6).

In principle, it is possible to estimate the specific protein concentration
required to grow one crystal: the first crystal nucleates and grows to reduce theprotein concentration in the vessel; thus, the probability of nucleation is reduced,
making it impossible to grow the second crystal. However, this type of29crystallization requires long incubation times using large volumes of lowconcentration samples, and estimating the protein sample concentration is quitedifficult.
5) Nakamura, H., Takahashi S., Inaka K., Tanaka H. Semi-empirical model to estimate
ideal conditions for the growth of large protein crystals. Acta Cryst. 2020, D76, 1174–
1183.

4.2.3. How to design crystallization experiments for large crystals
We recommend the following procedure for determining conditions for growing
large crystals.
1. Understanding the overview using the batch method
Set up crystallization using the batch method. The sample solutions contain aconstant protein concentration and varying concentrations of crystallization
reagents. Observe for approximately 1–2 weeks. Identify the highest
concentration of crystallization reagent at which crystals are not obtained (CL)and the lowest concentration at which crystals are obtained (CH). For example, inthe experiment shown in Fig. 4.3, batch crystallization with NaCl concentrationsin the range of 100–1000 mM, in increments of 100 mM, is recommended.
2. Understanding the dependence of nucleation probability on crystallization
reagent concentration
Prepare the crystallization reagents at several concentrations between CL–CH(determined in the previous section) and conduct various batch crystallization
reactions. In general, the number of growing crystals increases over time,
eventually reaching a constant value. In an actual experiment, it is difficult toobserve the time course in detail; therefore, it is sufficient to observe theapproximate time and number. The probability of nucleation at each time point
during the crystallization process is calculated by dividing the number of newly
appeared crystals per unit time by the volume of the solution.
For example, a crystallization experiment was conducted under the following
conditions (Fig. 4.4): 20 mg/mL lysozyme, 50 mM acetate buffer (pH 4.5), 10%
PEG4000, and NaCl concentrations of 200–400 mM (in 20 mM intervals),
temperature 4°C.

30

Probability of nucleation (h·L)-1

100.0000

y = 4.91E-28x1.06E+01
R² = 7.69E-01

10.0000

1.00000.10000.01000.00100.0001
100

200

300

400

500

600

NaCl (mM)
Fig. 4.4 Analysis of probability of nucleation
Based on these results, we will attempt to obtain a large crystal. It is
important not to grow crystals one after another, so we will determine theconditions to obtain the first crystal approximately 10 days after starting
crystallization.
When the solution volume is 30 L, the nucleation probability for obtaining
the first crystal on the 10th day is 1/240 h/30 L = 0.00014. The NaCl
concentration is estimated to be 163 mM using the approximate formula shownin the graph above.
3. Confirmation by counter diffusion (CD) method
Confirm the estimated conditions on a small scale using the CD method. Set up
crystallization using the CD method with a crystallization reagent concentration(e.g., 180 mM) slightly higher than that estimated in the previous section (163mM) and observe the progress. If the crystals grow faster and in greater numbers
than expected, change the concentration of the crystallization reagent slightly
lower. If the opposite is true, set it slightly higher.

31

4. Crystallization by the dialysis method
If crystallization proceeds over a favorable time course using the CD method,
attempt crystallization using LCDM. As discussed in "4.1. Time-dependent
diffusion of reagents", the increase in the concentration of crystallization reagentsin the LCDM, is approximately half than that in the CD method capillary. First, use
the dialysis method under the same conditions as the CD method.

4.2.4. For crystallization reagents containing multiple components
In most cases, crystallization reagents used for protein samples consist of (1) ahigh concentration of the main crystallization reagent, (2) a low concentration of
the secondary crystallization reagent, and (3) a buffer. Examples of (1) include
several molars of ammonium sulfate and PEGs at concentrations of several tensof percent. Examples of (2) include tens to hundreds of millimolars of NaCl. The
nucleation probability described in the previous section depends not only on theconcentration of the main crystallization reagent but also on the concentration of
the secondary crystallization reagent.
The crystallization conditions can be easily controlled by changing theconcentration of the crystallization reagent component, which has a milder effecton the nucleation probability. Therefore, we examine how the concentrations of
the main (e.g., ammonium sulfate) and secondary (e.g., NaCl) crystallization
reagents affect the number of crystals formed using the batch crystallization. The
results will determine which crystallization reagent component is the key
component that has a milder effect on the number of growing crystals. The
concentration of this key component is then analyzed, as shown in Fig. 4.4, to
determine the appropriate concentration for growing a single crystal.
Next, crystallization is performed using the CD method under the following
condition: 1) the crystallization reagent component with fixed concentration isadded to both the protein sample and reservoir solution, 2) the tested key
component is further added to the reservoir solution at a concentration slightly
higher than that estimated in the previous test, and then 3) its crystallization
progress is observed. After the desired crystallization process is confirmed using
the CD method, it is scaled up for crystallization in the LCDM.

32

Care must be taken when PEG is the main crystallization reagent. PEGs,
which have large molecular weights, do not pass through the dialysis membraneof the LCDM (see "4.1. Time-dependent diffusion of reagents"). Therefore, toachieve the desired crystallization, it is essential to add PEGs to the protein
sample in advance and adjust the concentration of the secondary crystallization
reagent as a key component.

4.2.5. In the cases of inverse concentration dependence
In some cases, increasing the concentration of the crystallization reagent may
decrease the probability of nucleation without any precipitation. For protein
crystallization with such crystal-formation characteristics, it is also possible todetermine conditions for growing large crystals.
Here, we explain the case where PEG is the main crystallization reagent,and many crystals are obtained with additional 50 mM NaCl but not at 150 mM.
Using the batch method, the number of crystals formed is measured using several
concentrations of the crystallization reagent, ranging from the concentration atwhich crystals can be obtained (e.g., NaCl 50 mM) to the concentration at which
crystals are difficult to obtain (e.g., NaCl 150 mM), and a graph of theconcentration dependence of the crystal formation probability (similar to Fig. 4.4)
is created. Next, from the graph, the crystallization reagent concentration at which
one crystal can be obtained in approximately 1–2 weeks is estimated. For the
purposes of the following explanation, we will assume that the concentration isestimated to be 75 mM from the graph.
In the CD experiment, a protein sample is mixed with PEG, a buffer solution,and the high concentration of NaCl (150 mM NaCl in the example), and thenloaded in a capillary. The reservoir solution contains the same concentration of
PEG, buffer, and 75 mM NaCl. Under these conditions, crystallization is
confirmed using the CD method, and then that for large crystals is attemptedusing the dialysis method.
As in the case, crystallization, in which the concentration of a specificcomponent is gradually reduced to crystallization, is unique to the CD and dialysis
methods.

33

4.2.6. Quality of protein samples
To optimize crystallization conditions using the experiments described so far, it isimportant that the nucleation probability shows a simple behavior with respect toconcentration change of crystallization reagents, and reproducibility for the
specific crystallization conditions. For the desired nucleation probability, theprotein molecules should be well dispersed in the solution as monomolecules ina uniform state. If the sample is slightly denatured over time and forms multimers,
these will act as nuclei and promote crystal formation, making it difficult to control
the nucleation probability.
The following indicators can be used to determine whether a protein sample
is appropriate.
A single band of the desired molecular weight is detected by SDS-PAGE.
The single peak in ion exchange chromatography, and the concentration of the
NaCl gradient during elution roughly is consistent with the calculated charge
density.
The single band in native-PAGE and its migration distance roughly matches
the number of charges.
Dynamic light scattering shows monodisperse signals with reasonably narrow
peak widths, indicating that the particle size is an integer multiple of the molecularweight.
Note that the nucleation probability may vary depending on the sample
preparation lot. Therefore, it is desirable to do everything from examining theconditions using the batch method to crystallization in LCDM using the same lot
sample. Thus, we recommend the preparation of a sufficient amount of protein
samples.

Furthermore, online technical support (paid) is available via email or video
meeting from experts in both methods: C-Kit Ground Pro e-mail support(CRT101-3),
C-Kit
GroundPro
video
support(CRT101-4,(http://www.confsci.co.jp/images/C-kit%20Ground%20Pro_220908E.pdf)
surely help you to achieve highly challenging protein crystallization.

will© 2024 Confocal Science Inc.

34

Confocal Science Inc.
5-14-15 Fukasawa, Setagaya-ku, Tokyo 158-0081
TEL: +81-3-5809-1561 FAX: +81-3-6411-6261
E-mail: info@confsci.co.jp

http://www.confsci.co.jp/

35