C-Kit Ground Pro XRD[X-ray Diffraction Kit]

June. 2024
Confocal Science Inc.

Table of Contents
1.

Introduction ................................................................................................. 3
1.1. Overview ................................................................................................ 3

1.2. Features................................................................................................. 4
2. Crystallization Methods Used in C-Kit Ground Pro XRD ............................. 5
2.1. The counter-diffusion (CD) method ........................................................ 5
2.2. The diffusion-pair osmotic-concentration (DPOC) method..................... 6
3. Experimental Procedure .............................................................................. 8
3.1. Setting up crystallization via the CD method .......................................... 8
3.1.1. Required items ................................................................................ 8
3.1.2. Pre-soaking of the gel tube .............................................................. 9
3.1.3. Preparation of solutions and a marked capillary ............................ 10
3.1.4. Assembling the items to start crystallization .................................. 11
3.2. Setting up crystallization via the DPOC method .................................. 15
3.2.1. Required items .............................................................................. 15
3.2.2. Pre-soaking of the DPOC tube ...................................................... 16
3.2.3. Preparation of solutions ................................................................. 16
3.2.4. Assembling the items to start crystallization .................................. 173.3. Harvesting crystals from the CD capillary ............................................ 20
3.3.1. Desirable harvesting solution......................................................... 20
3.3.2. How to cut the capillary.................................................................. 20
3.3.3. Harvesting crystals ........................................................................ 20
3.4. Harvesting crystals from the DPOC tube ............................................. 22
3.4.1. Desirable harvesting solution......................................................... 22
3.4.2. How to cut the DPOC tube ............................................................ 22
3.4.3. Harvesting crystals ........................................................................ 22
3.5. Cryocooling crystals ............................................................................. 231

4.

Technical Notes ........................................................................................ 25
4.1. Time-dependent diffusion of reagents.................................................. 25
4.1.1. Concentration changes in the CD method sample solution ........... 25
4.1.2. Concentration changes in the DPOC tube solution ....................... 27
4.2. Benefits of the CD and DPOC methods ............................................... 29
4.2.1. Crystals with high resolution (the CD method)............................... 29
4.2.2. Subcomponent elimination from protein sample (the CD method) 29
4.2.3. Small amounts of ligands (the DPOC method) .............................. 30
4.2.4. Easy soaking (the CD method) ...................................................... 30
4.3. Design of crystallization conditions ...................................................... 31
4.3.1. Proteins with unknown crystallization conditions ........................... 31
4.3.2. Proteins with known crystallization conditions ............................... 32

Contents of C-Kit Ground Pro XRD (CRT101-1)
Product name

Qty.

Description

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.

DPOC tube

8

Used in the DPOC method.

C-Cap

16

Silicone tubing (φ1.0 mm × 50 cm)

1

Used for aspirating solution.

Sealing compound

1

Used for sealing capillaries andDPOC tubes.

Round-bottom tube (5 mL)

23

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.
Descriptions in this handbook are subject to change without notice.

2

1. Introduction
1.1. Overview
C-Kit Ground Pro XRD is an equipment for protein crystallization for X-ray
diffraction and developed from the know-how of crystallization experiments at theInternational Space Station (ISS) for decades. This kit provides experimental
tools for two simple methods that do not require large-scale crystallization
screening: the counter-diffusion (CD) method 1) and the diffusion-pair osmoticconcentration (DPOC) method 2). Both methods are used to grow crystals on thebasis of the principle of auto-searching for optimal crystallization conditions (selfsearching mechanism) 3). The newly developed DPOC method enables us tosearch for a wide range of crystallization conditions because the protein samples
and crystallization solutions in the DPOC tube are osmotically concentrated
owing to the osmotic pressure difference between the inside and outside of the
DPOC tube. You can choose the appropriate method according to the sample
characteristics to grow high-quality apo/complex crystals.
The CD equipment included in the kit was jointly developed by Confocal
Science Inc., under commission from JAXA 4), 5). In addition, Confocal Science
Inc. provides C-Kit Ground Pro ND for crystallization for neutron diffraction(http://www.confsci.co.jp/product_e.html) and the C-Kit Space Pro Series forhigh-quality 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) Otálora, F.; García-Ruiz, J.M. Computer model of the diffusion/reaction interplay in thegel acupuncture method. J. Cryst. Growth 1996, 169, 361–367
4) 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 Radiat. 2004, 11, 45–48.
5) JP patent 4354457, US patent 7531037

31.2. Features
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A small amount of the protein sample is required: The standard amount is 8
µL (CD method capillary) and 5 µL (DPOC tube).
Slow crystallization methods lead to high-quality crystals, and both CD and
DPOC methods provide mild crystallization conditions by adjusting theconcentrations of the protein sample and crystallization reagent.
Simple protocols: Both CD and DPOC protocols are easy to perform.
High reproducibility and reliability: Both methods have been repeatedly usedin space experiments conducted by JAXA and the Japan Manned Space
Systems Corporation (JAMSS). Thus, their high reproducibility and reliability
have been proven. In particular, the CD method has been applied to the

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◼

◼

crystallization of more than 500 proteins under microgravity since 2002.
Easy soaking in the CD method: Soaking can be performed by simply
transferring crystal-containing capillaries to a container filled with the solution
for post-soaking.
Small amounts of ligand in the DPOC method: Ligands are pre-mixed withthe protein solution loaded into the tube; therefore, a small amount of ligand
is sufficient for co-crystallization.
Long-term stability: The crystals produced with C-Kit Pro XRD are stable fora long period in the CD capillaries with gel tubes and DPOC tubes.
Estimation of optimal conditions: Time-dependent changes in concentrationsof the protein and crystallization reagents in the CD capillaries and DPOC
tubes can be estimated using a one-dimensional diffusion simulation program(available for purchase as C-Kit Pro Advanced Tool [CRT209],
http://www.confsci.co.jp/product_e.html). It helps to determine the optimal
conditions for crystallization.
Please refer to “4.2. Benefits of the CD and DPOC methods” to understandwhich method is better for your experiments.

42. Crystallization Methods Used in C-Kit Ground Pro
XRD
2.1. The counter-diffusion (CD) method
C-Kit Ground Pro XRD provides a set of crystallization equipment for the GT
method, which is one of the CD methods. Fig. 2.1 provides a simple explanationof 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.

5

A feature of this method is that a silicone tube with agarose gel, called a geltube (GT), 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.

2.2. The diffusion-pair osmotic-concentration (DPOC) method
C-Kit Ground Pro XRD includes crystallization tubes for the DPOC method as
another type of crystallization method. Fig. 2.2 provides a simple explanation of
the standard configuration of the DPOC method and mechanism of crystal growth.
The conventional diffusion-pair method of crystallization has been known fora long time: by successively loading a glass capillary with a crystallization reagent
and protein sample to form a diffusion pair and interdiffusion for crystallization 6).
In our DPOC method, the glass capillary is replaced with a water-permeablesilicone rubber tube. The solution in the DPOC tube is osmotically concentrated
owing to the osmotic pressure difference between the reservoir solution outside
and the solution inside. The diffusion phenomenon in the diffusion pair is similar
to that observed in the CD method; however, an equal amount of thecrystallization reagent solution to that of the protein sample is included in the
DPOC tube, which results in a narrower range of crystallization conditions thanthose in the CD method. However, the effect of the osmotic concentration in the
DPOC tube enables this method to search for a wider range of conditions. In
addition, protein samples can be crystallized under concentrated conditions
without prior preparation. This difference from the CD method is explained in
section “4.2. Benefits of the CD and DPOC methods.”
6

Fig. 2.2 DPOC method illustration
Upper left: Configuration of the DPOC method. Upper right: Detailed view of
the DPOC tube. Bottom: Crystals are formed by interdiffusion and osmotic
concentration of sample and crystallization solutions.
6) Salemme, F.R. A free interface diffusion technique for the crystallization of proteinsfor X-ray crystallography. Arch. Biochem. Biophys. 1972, 151, 533–539.

73. Experimental Procedure
3.1. Setting up crystallization via the CD method
3.1.1. 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

A few
µL

Prepare for seeding, if necessary.

Equipment in the kit
Capillary(φ0.5 mm × 47 mm)

Qty.

Description

1

Mark the upper end of sample before
loading.
8

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.

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 pre-Required equipment
Tube with lid

soaking the gel tube.
Micropipette/Tip
Micropipette/Tip(20 to 100 µL)

1 each1

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 permanent1

It is used for marking the end of the loaded

marker

It is used for preparing solutions.

sample on the capillary.

3.1.2. 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 NaCl
9

solution and about 4 days for the PEG4000 solution to achieve equilibrium in thegel tube.
Fig. 3.1 Simulation results ofinternal NaCl concentrations 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
concentrations. The curves show,fromthebottom,NaClconcentrations after 0.05, 0.1, 0.2,
and 0.5 days.
Fig. 3.2 Simulation results ofinternal PEG4000 concentrationsof a 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
concentrations. The curves show,fromthebottom,PEG4000
concentrations after 0.25, 0.5, 1, 2,
and 4 days.

3.1.3. 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.
10

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. 3.3 Reference lines on the capillary

3.1.4. 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. 3.4 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,
11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. 3.5 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.

12

Fig. 3.6 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. 3.7 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. 3.8 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. 3.9 Cutting the bottom ofcontact with the bottom of the roundthe gel tube
bottom tube.

13

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, in thecapillary with the gel tube side down, thedensity of the solution increases towards
the bottom of the capillary, suppressing
density-driven convection in the capillary.
As a result, we can expect to obtain better
quality crystals owing to the similar effects
ofconvection
suppression
inamicrogravity environment.
Note: The capillary must be placed
horizontally during observation, but this is
not a problem for short periods.

14

Fig. 3.10 Place verticallyand start crystallization

3.2. Setting up crystallization via the DPOC method

3.2.1. Required items(For each crystallization condition)
Solution

AmountDescription

Sample solution

5–7 µLIt contains a protein sample solution forloading into a DPOC tube.
Because the inner diameter of the DPOC
tube is 1.0 mm, the required volume is 4.7µL for a sample length of 6 mm and 6.3 µL
for 8 mm. It is desirable to prepare a little
more solution for loading without failure.

Crystallization solution

5–7 µLIt contains crystallization reagents andbuffers, which may also contain ligands if
required. It interdiffuses with the sample
solution in the DPOC tube to crystallize
the protein. When seeding, it also
contains the seed crystals.

Reservoir solution

ca. 4

It contains the same concentrations ofmL

crystallization reagents and buffers as the
crystallization solution. No ligands orseeds are required. It is used to immerse
the DPOC tube.

Qty.

Description

Equipment in the kit
DPOC tube(φ1.0 mm × 30 mm)

1

It has short capillaries at both ends.

C-Cap

2

It is a stopper to plug the capillaries of the
DPOC tube.

Sealing compound１

It is used to seal the ends of the capillary.

Silicone tubing

1

It is used to load solutions into the DPOC
tube.

Round-bottom tube
(5 mL)

1

A vessel for crystallization.

15

Required equipment
Micropipette/Tip
Micropipette/Tip(20 to 100 µL)
Fine-point permanentmarkerQty.
each 1

Description
It is used for preparing solutions.

1

It is used for loading solutions into DPOC
tubes.

1

It is used for marking the end of theloaded sample on the DPOC tube.

3.2.2. Pre-soaking of the DPOC tube
1. Reservoir solution
This is the solution outside the DPOC tube settled in the round-bottom tube, withthe same concentrations of crystallization reagents and buffer as in the
crystallization solution in the DPOC tube. No ligand components or seeds wererequired.
2. Marking the DPOC tube
Mark a line on the DPOC tube for loading solution by using a permanent marker.
If the sample volume is 4.7 µL (sample length, 6 mm), mark at 9 mm from the endof the container; if the sample volume is 6.3 µL (sample length, 8 mm), mark at 7mm. These loading volumes enable air bubbles to enter both ends of the loadedsolution.

Fig. 3.11 Reference lines on the DPOC tube
3. Pre-soaking the DPOC tube
Immerse the DPOC tube in the reservoir solution for one to several days toremove the air adsorbed on the tube material and allow it to absorb moisture.

3.2.3. 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 into the DPOC tube.
2. Crystallization solution
16

A solution that crystallizes proteins by interdiffusion with the sample solution in
the DPOC tube. Add not only crystallization reagents and buffers but also ligands,
if required.
When seeding, prepare micro-seeds or macro-seeds from seed crystals in
advance, and add an adequate amount to the crystallization solution.

3.2.4. Assembling the items to start crystallization
1. Attaching the DPOC tube to a silicone tubing and a micropipette
Pick the pre-soaked DPOC tube from the reservoir solution, and connect its
marked end to a micropipette by using a silicone tubing. If any reservoir solution
remains in the DPOC tube, remove it by pipetting.

Fig. 3.12 Loading solutions into the DPOC
tube
17

2. Loading solutions
Place the other end of the DPOC tube into the crystallization solution, andaspirate 4.7–6.3 µL of the solution. Next, move the end of the DPOC tube into
the sample solution and aspirate the same amount of solution as the
crystallization solution. Then, remove the end of the DPOC tube from the solution,and load air by turning the dial of the micropipette until the surface of thecrystallization solution reaches the marked line on the DPOC tube.
3. Sealing the DPOC tube
Keep the DPOC tube connected to the silicone tubing, and seal the free end of
the DPOC tube with a sealing compound. Then, remove the silicone tubing, andseal the opposite end of the DPOC tube with the sealing compound.

Fig. 3.13 Sealing the DPOC
tube
4. Attaching C-Caps
To protect both outer ends of the DPOC tube from excessive osmotic pressure,plug each outer end of the capillary with C-Caps. Insert the edge of the capillaryin the DPOC tube to the diagonally cut end of the C-Cap. There is a plastic plug
inside the C-Cap, so push it in together. Hold the plug with your finger such thatit does not protrude from the opposite end. The installation is completed when
the plug is pushed to the end of the C-Cap.

18

Fig. 3.14 Attaching C-Caps

5. Starting crystallization
Add about 4 mL of reservoir
solution to the round-bottom tube,
gently place the DPOC tube in itwith the crystallization solution side
down, and cap the round-bottom
tube. Ensure that the entire DPOC
tube is immersed in the reservoir
solution in the round-bottom tube,
and place it vertically at thedesirable temperature. In manycases, the crystallization solution
Fig. 3.15 Place vertically and start
has a higher density than the
crystallization
sample solution. Thus, when the
crystallization solution side is facing
down, the density increases towards the bottom, which suppresses the
movement of the solution in the DPOC tube owing to density-driven convection.
As a result, we can expect suppression of solution convection in a microgravity
environment to yield better-quality crystals.
Note: The DPOC tube must be placed horizontally during the observation;
however, this is not a problem for short periods.

19

3.3. Harvesting crystals from the CD capillary

3.3.1. 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 capillary where the target crystals are located. If the concentrations areinappropriate, the crystals may dissolve or break because of osmotic pressure
differences.
The concentrations of the components in certain parts of the solution in the
CD capillary depend on the position in the capillary and duration of crystallization
reaction. For successful crystal harvesting, we recommend preparing theharvesting solution with estimated component concentrations. Our simulation
program, sold separately, will be helpful for estimating the component
concentrations in CD capillaries (available for purchase as C-Kit Pro Advanced
Tool [CRT209], http://www.confsci.co.jp/product_e.html). If, unfortunately, yourharvested crystal is deteriorated in the solutions, please prepare and try multiple
solutions with component concentrations close to the initial estimated values.

3.3.2. How to cut the capillary
Cut the capillary 3–4 mm apart from either side of the target crystal. Make a sharp
scratch on the capillary glass with a diamond file or capillary cutter, pull both endsof the capillary to break it away from the scratch. The capillary will be cut at theexact position.

3.3.3. Harvesting crystals
Place the prepared harvesting solution in a cavity glass slide. Then, a short pieceof the cut capillary is immersed in the solution and held with forceps. Whileobserving with a stereo microscope, pipette the harvesting solution around the
crystal and pour the crystal out of the capillary into the harvesting solution.
Some of the crystals may adhere to the glass surface of the capillary. In suchcases, 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. The solution
drives the crystals out of the capillary into the harvesting solution. Scoop up the
crystals from the harvesting solution with a cryoloop, immerse in a cryoprotectant
solution if necessary, and then use for cryocooling. The details are provided in“3.5. Cryocooling crystals.”
20

For practice, it is recommended to prepare lysozyme crystals by using the
CD method to familiarize yourself with the procedure for picking out crystals.
Because lysozyme crystals easily adhere to the inner surface of the capillary,
they are suitable for training.

Fig. 3.16 Harvesting crystals
Upper left： Fix the cut capillary with forceps so that you can work with yourhands free while observing with a stereo microscope.
Upper right： Pipette the solution into the capillary and push the crystal gently.
Lower left： If the crystal adheres to the capillary, touch it gently with a gelloading tip and move it a little.
Lower right： The crystal is pushed into the harvesting solution.

21

3.4. Harvesting crystals from the DPOC tube

3.4.1. Desirable harvesting solution
Diffusion of the crystallization reagent inside the DPOC tube is rapid, and eventhe diffusion pair of PEG solutions and sample solution reaches equilibriumaround 10 days. However, the concentration of a sample solution by an external
reservoir solution requires several weeks. The actual concentration in the DPOC
tube can be estimated from the length of the solution in the DPOC tube during
harvesting. Based on this estimation, prepare a harvesting solution by diluting the
crystallization solution to the same concentration as that of the solution aroundthe crystal.

3.4.2. How to cut the DPOC tube
Cut the DPOC tube a few mm apart from either side of the target crystal with asurgical blade (or a sharp knife for delicate work). When cutting, care should betaken not to deform the DPOC tube or crush the crystals.

3.4.3. Harvesting crystals
Add the prepared harvesting solution to a cavity glass slide. Then, immerse ashort piece of the DPOC tube in the solution with forceps. While observing with astereo microscope, pipette the harvesting solution around the crystal and pourthe crystal out of the DPOC tube into the harvesting solution. Scoop up the
crystals from the harvesting solution with a cryoloop, immersed in a
cryoprotectant solution if necessary, and then use for cryocooling. The details areprovided in “3.5. Cryocooling crystals.”
For practice, we also recommend that you prepare lysozyme crystals byusing the DPOC method and familiarize yourself with the procedure for harvesting
crystals.

223.5. Cryocooling crystals
For X-ray diffraction using synchrotron radiation, the crystals must be
cryocooled to protect them from radiation damage. The water around the crystals
needs to freeze into amorphous ice through the freezing process. If not, growing
crystalline ice would damage the protein crystals, and a good X-ray diffraction
image would not be obtained. To prevent the crystalline ice from forming duringfreezing, the crystals must be frozen in a cryoprotectant solution. Scoop up the
crystals from the harvesting solution with a cryoloop, immerse them into acryoprotectant solution for a few seconds to replace the solution around the
crystals with the cryoprotectant, and then cryocool them rapidly. For cryocooling,please use a method that you have already used, such as flash cooling or direct
immersion in liquid nitrogen.
PEG-based crystallization reagents have a cryoprotective effect. Therefore,
if the harvesting solution contains 35–40% PEGs, the crystals can be cryocooled
without the addition of extra cryoprotectants. If the main component of aharvesting solution is PEG-based but at a low concentration, adding the same
type of PEG to the harvesting solution to adjust the PEG concentration toapproximately 40%. It is a reliable method for preparing cryoprotectant solutions.
When salts are used as crystallization reagents, glycerol is the most common
cryoprotectant used as an additive in the harvesting solution. A glycerol
concentration of approximately 40% would suppress the formation of crystalline
ice. Trehalose, ethylene glycol, PEG4000 have also been used as
cryoprotectants.
Before applying a cryoprotectant solution to the target crystals, werecommend you to test whether the solution can suppress crystalline ice
formation. A droplet of a good cryoprotectant solution will freeze into amorphous
ice when scooped with a cryoloop and frozen quickly.
However, cracking of the crystal may sometimes occur, due to the large
osmotic pressure difference between the liquid inside the crystal and the
cryoprotectant solution. This tends to occur when using low-molecular-weight
cryoprotectants such as glycerol. A solution to this problem is to sequentially soak
the crystals in several solutions containing intermediate concentrations of the
cryoprotectant. For example, if the harvesting solution contains 10 mM MgCl2,
1.5 M ammonium sulfate, 50 mM acetate buffer, prepare three solutions: (A) 10
mM MgCl2, 1.5 M ammonium sulfate, 50 mM acetate buffer, 10% glycerol, (B) 10
23

mM MgCl2, 1.5 M ammonium sulfate, 50 mM acetate buffer, 20% glycerol and(C) 10 mM MgCl2, 1.5 M ammonium sulfate, 50 mM acetate buffer, 30% glycerol.
Then, soak the crystal in the solutions in the order of (A), (B) and then (C) togradually increase the cryoprotectant concentration around the crystal. Pleaserefer to the later section, “4.2.4. Easy soaking” for tips.

24

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. To obtain
crystals, we recommend that understanding the time course of concentration
changes in the protein solution and crystallization reagents in the CD method
capillary and DPOC tube and optimizing concentrations of the solutions to occur
suitable combinations.

4.1.1. Concentration changes in the CD method sample solution
A one-dimensional diffusion simulation can be used to estimate the time courseof crystallization reagent concentrations 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 reagents such as NaCl and PEG4000 are shown below.
Example 1
Sample solution： 40 mm length in a capillary
Reservoir solution： 1 M NaCl
Fig. 4.1 Estimation of NaCl
concentration 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, so the diffusion time course is almost the
same.
25

Example 2
Sample solution： 40 mm length in a capillary
Reservoir solution： 25% PEG4000
Fig. 4.2 Estimation of PEG 4000
concentrations in a capillary.
, ■, ▲, x, and Ж indicate PEG
4000 concentrations 1, 4, 12, 32,
and 64 days after loading,respectively. The horizontal axis
shows the distance from the endof the gel tube, and the verticalaxis indicates the PEG 4000
concentrations.
Even after 64 days, the PEG4000 concentration in the capillary increased toapproximately half of the reservoir solution concentration at the distal end of thegel tube. To obtain crystals within a short period, either a higher concentration of
PEG4000 in the reservoir solution or premixing an appropriate concentration of
PEG4000 with the protein sample in the capillary is recommended. Because thediffusion coefficient of PEGs is approximately inversely proportional to the 1/2
power of their molecular weight 7), equilibration of the PEG concentration is muchslower when using higher-molecular-weight PEGs.
7) Luo, Z.; Zhang, G. Scaling for sedimentation and diffusion of poly(ethylene glycol) in
water. J. Phys. Chem. B. 2009, 113, 12462–12465

Example 3
Sample solution： containing 12.5% PEG 4000, 40 mm length in a capillary
Reservoir solution：25% PEG4000

Fig. 4.3 Estimation of PEG4000
concentrations in a capillary.
Symbols are the same as in Fig. 4.2.

26

PEG4000 was added to the sample solution in the capillary, as shown in Fig. 4.3;
thus, the PEG concentration increased faster than that shown in Fig. 4.2.

4.1.2. Concentration changes in the DPOC tube solution
The time-dependent interdiffusion of the protein with the crystallization solution in
the DPOC tube can also be estimated using one-dimensional diffusion simulation.
Example 4
Sample solution (8 mm) and 1 M NaCl solution (8 mm) in a DPOC tube
Reservoir solution： 1 M NaCl
Fig. 4.4 Estimation of NaCl
concentrations in the DPOC
tube.
and
■ indicate NaCl
concentrations 0.25 and 1 day
after loading, respectively. The
horizontal axis shows the
distance from the end of thetube, and the vertical axis
indicates
theNaClconcentrations at the location.
The NaCl concentration in the capillary reaches near equilibrium in only 1 day.
Example 5
Sample solution (8 mm) and 25% PEG4000 solution (8 mm) in a DPOC tube
Reservoir solution： 25% PEG4000
Fig. 4.5 Estimation of PEG4000
concentrations in a DPOC tube.
, ■, ▲, x, and Ж indicate PEG4000
concentrations 0.25, 1, 3, 8, and 16
days after loading, respectively. The
horizontal axis shows the distancefrom the end of the tube, and thevertical axis indicates the PEG4000
concentrations at the location.
PEG4000 in the capillary reaches equilibrium 8 days after loading.
27

Furthermore, the solution in the DPOC tube was slowly concentrated by theosmotic concentration caused by the concentration difference between thesolution in the DPOC tube and the reservoir solution; therefore, the concentration
process was measured as shown in Fig. 4.6.
Example 6
Sample solution (4 mm) and 20% PEG4000 solution (4 mm) in a DPOC tube
Reservoir solution： 20% PEG4000 containing 600 mM NaCl
Fig. 4.6 Time-dependent solution
concentration measured by thesolution length in a DPOC tube.
After 22 days, the solution volume in
the DPOC tube is concentrated byhalf and reaches near equilibrium.

Fig. 4.6 shows that the time to reach equilibrium is approximately 3 weeks.

28

4.2. Benefits of the CD and DPOC methods
The advantages and limitations of the CD and DPOC methods have been
provided below. These differences should be considered when selecting the
optimal crystallization method.

4.2.1. Crystals with high resolution (the CD method)
In general, higher the concentration of the main crystallization reagent (e.g.,
PEGs or ammonium sulfate), higher is the diffraction resolution. In theconventional vapor diffusion method, crystallization under these conditions tends
to cause clustering or precipitation. In contrast, in the CD method, a higher
concentration of crystallization reagents can be used to obtain crystals thatdiffract X-rays at a high resolution, since concentration of the reagents in the CD
capillary increases slowly.

4.2.2. Subcomponent elimination from protein sample (the CD
method)Protein samples usually contain low-molecular-weight compounds such as salts,
EDTA, or buffers from the purification process. Some of these compounds may
inhibit crystallization. A typical component that inhibits crystallization is highconcentrations of NaCl. Contaminating NaCl may cause protein precipitation
when a crystallization reagent is added.
The CD method is suitable for the crystallization of such protein samples. In
the CD method with a gel tube, the concentration of the crystallization reagent in
the capillary increases over time, as shown in the previous section.
Simultaneously, low-molecular-weight components in the capillary diffuse out,
and their diffusion rates are faster than those of the protein samples.
Consequently, the low-molecular-weight components in the capillary decrease,allowing crystallization without the effects of components that inhibit
crystallization.
However, the osmotic concentration in the DPOC method increases theconcentrations of inhibitory components in the protein samples, making theircrystallization more difficult.

29

4.2.3. Small amounts of ligands (the DPOC method)
During co-crystallization with the ligand, the added free compound diffuses out of
the capillary in the CD method. Therefore, preparation of complex crystals with aligand whose KD value is greater than M requires the addition of the ligand to
the reservoir solution.
In contrast, in the DPOC method, loss of ligands from the sample solution
would not occur, and the ligand solution would be concentrated during the
crystallization period. Thus, only a small amount of ligand added to the sample
solution is required for the DPOC method, which enables you to obtain complex
crystals with low-affinity ligands.

4.2.4. Easy soaking (the CD method)
Soaking crystals with ligands using the CD method is very easy. After the crystals
are obtained, the CD capillary is simply transferred to a fresh reservoir solution
containing the ligand and left for a period of time for the ligand to diffuse in.
You can also apply this procedure to solution exchange for cryoprotection of
the crystals. Crystals may deteriorate sometimes when they are dipped in a
cryoprotectant solution, since concentration of the cryoprotectant component
around the crystals increases too rapidly. To avoid the rapid change, soak the
crystals in cryoprotectants, such as high concentrations of PEGs, ethylene glycol,
and glycerol. It enables slowly exchange of the solution around the crystals. For
example, if the reservoir solution contains 10 mM MgCl2, 1.5 M ammonium sulfate,
50 mM acetate buffer, prepare a protectant solution containing 10 mM MgCl2, 1.5
M ammonium sulfate, 50 mM acetate buffer, 30% glycerol, and transfer the CD
capillary containing the crystals into it. After the solutions inside and outside thecapillary reach equilibrium, the crystals are harvested from the capillary using thisprotectant solution and frozen.
However, in the DPOC method, the crystals must be removed from the tube
and transferred to a soaking solution.

30

4.3. Design of crystallization conditions

4.3.1. Proteins with unknown crystallization conditions
Commerciallyavailable
screening
kit
reagents(e.g.,
https://hamptonresearch.com/cat-1.html) are typically used to screen
crystallization conditions. In these experiments, the self-searching mechanism of
the CD and DPOC methods allows a wider range of combinations ofconcentration conditions to be searched in a single capillary/tube than thecommonly used batch and vapor diffusion methods.

1.

Easy protocol for screening crystallization condition is as follows:
Choose 2 groups of screening kit reagents: one group containing salts(e.g., ammonium sulfate) and the other containing PEGs (e.g., PEG4000) as themain crystallization reagent. Calculate the pI value of your protein sample. Then,
select 2 reagent bottles from each group: one with a preset pH above the pI (+1
to +2) and the other below the pI (-1 to -2) of the protein sample.
2.
First, perform crystallization experiments by using the CD method.
Prepare 2 sample solutions for each reagent: one containing only the protein
sample and the other containing the protein sample mixed 1:1 with the selected
reagents. Use the corresponding 4 reagents as reservoir solutions. Then, test theprotein crystallization with the 8 crystallization conditions.
3.
Crystallization experiments using the DPOC method are recommended
using the 4 selected screening kit reagents as both crystallization and reservoir
solutions.
4.
When no crystals are obtained after a maximum of 1 month, the possible
reasons and countermeasures may be as follows:
CD method

DPOC
methodCountermeasure

No change

The protein molecule may have a large
charge. Retest with a reservoir solution
containing an additional 100–150 mM NaCl.

Gradual precipitation

Close to the conditions for obtaining crystals.
Retest using a reservoir solution with
reduced concentration of one component of31

the kit solution. Reduce the concentration ofthe
supporting
componentofthecrystallization reagent.
Crystals growonce on the geltube side of thecapillary, butdisappear
later.

－

Sample solution may contain something
required for crystallization. Retest the CD
method by adding components to thereservoir solution that are found only in thesample solution.

5.
If crystals are not obtained even after these countermeasures, try several
other reagents with a similar pH in the screening kit.

4.3.2. Proteins with known crystallization conditions
Even in the case of protein samples for which crystallization has been reported,
we often observe that the crystals cannot be reproduced. A possible cause is theslight difference in the salt and buffer concentrations included in your protein
preparation. Therefore, the CD method is preferable in these cases because it isless affected by these molecules in protein.
In both CD and DPOC methods, the self-searching mechanism allows awider range of combinations of concentration conditions to be searched in asingle capillary/tube than in the batch or vapor diffusion method, so that you canobtain crystals more efficiently from your own preparation.
Easy protocols to screen crystals are as follows:1.
Select several crystallization conditions reported in papers or PDB.
2.
Then, crystalize with the CD method by using the reported crystallization
solution as the reservoir solution. Prepare 2 sample solutions: one is the reported
protein solution and the other is the protein solution mixed 1:1 with the reported
crystallization solution.
3.
Setup crystallization experiment by using the DPOC method.
4.
If no crystals are obtained after a maximum of 1 month, please refer tothe previous section (“4.3.1. Proteins with unknown crystallization conditions”) for
countermeasures.

32

Furthermore, on-line technical support (paid) is available via email or video
meeting from experts in both methods: C-Kit Ground Pro e-mail support(CRT101-3 http://www.confsci.co.jp/product_e.html), C-Kit Ground Pro video
support (CRT101-4 http://www.confsci.co.jp/product_e.html) will surely help
you to achieve crystallization.
© 2024 Confocal Science Inc.

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TEL: +81-3-5809-1561 FAX: +81-3-6411-6261
E-mail: info@confsci.co.jp

33

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