intelligent solutions for protein
Molecular
Dimensions
crystallization plates
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Introduction
The CrystalHarpTM plate is designed for crystallization based on capillary diffusion
and can be used for crystallization screening and optimization.
Capillary diffusion achieves a much broader screening of variables in one single
experiment. The CrystalHarpTM contains 48 capillaries in total and is in the SBS
standard format to facilitate handling and imaging.
Addition of cryoprotectants or derivatives for phasing studies can be easily added
after crystal appearance. The SBS format enables the usage of the plate directly
on beamline robots or alternatively single capillaries can be easily mounted to a
standard magnetic base, enabling in-situ diffraction analysis.
The unique capillary material allows data collection at room temperature. Flashcooling in a liquid nitrogen stream (with or without the use of cryoprotectants) is
also feasible. The formation of ice-rings is kept to a manageable minimum.
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Thingsyouwillneedbeforeyoubegin:
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Microscope
MultiͲchannelpipetteP100
PipetteP100
Pipettetips
MineralOil(5mL)*(MD2Ͳ71)
2xMagneticcapswithCryoPins*
Scissorsorcuttingtool*(MD7Ͳ520)
CounterͲdiffusioncrystallizationscreen*
(MultiXtal,MD1Ͳ65)
x RubberͲtippedtweezers*(MD7Ͳ521)
*theseitemscanbepurchasedseparatelyfromMolecular
Dimensions.
1
Orient the CrystalHarpTM as
shown in Step1. Pipette 22-23
µL of protein solution into one
of the protein loading wells, a
or b (as shown in Figure 1).
The capillary is open at both
ends.
Apply a slight vacuum onto the
opposite empty protein loading
well with the 2 mL syringe
provided. The protein runs
quickly through the capillary
system. This step is time
dependent and is related to
the viscosity of your protein
sample.
2
3
Monitor the filling of the capillary
with a microscope or by holding
plate up to the light.
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If the protein has run too far
through the capillary (seen as
air bubbles near original protein
loading well) simply apply
vacuum to where the protein
was loaded initially. You can
recover excess protein sample
from the protein loading well
with a pipette tip.
4
Break all capillaries in the first
left hand sealing trough using
the 2 mL syringe with the flat
green nozzle.
5
Put the sealing paste (shown
with brown tip) using the
syringe needle into the first left
hand sealing trough. Ensure
the sealing paste completely
encloses each broken end of
the capillaries without any
entrapped air bubble. Fill the
entire trough to the top with
sealing paste.
Repeat step 4 and 5 for the
three remaining troughs, one
at a time.
6
Pipette 20 µL per well of
crystallization condition into
the precipitant wells* using a
multi-channel pipette.
NB. The first tip enters the
well #1 and the second will be
in #3. A second pipetting
operation is used to fill wells
2, 4, 6 etc.
Use
the
CrystalHarpTM
scoring sheet to register the
location of each condition to
the respective location.
*Ensure no air bubbles are trapped on the bottom of the well.
7
Break the capillary in the
48 precipitant wells - using
a twisting motion - with the
flat ended green syringe,
one by one. The breaking
is audible and can be
checked
with
a
microscope.
To
avoid
crosscontamination clean
the syringe tip after
each cut with tissue
wipe.
8
Drop of
mineral oil
The precipitant wells need to be sealed to avoid evaporation
with a drop of mineral oil to each well. The CrystalHarpTM plate
is ready for incubation.
Imaging and X-ray analysis
Manual inspection of crystal growth can be performed using a
microscope. Alternatively the SBS formatted CrystalHarpTM
plate allows for the usage of commercial automatic incubation
and imaging systems.
X-ray Diffraction Analysis on single capillaries –
cutting of capillaries for data collection experiments.
9
For
mounting
the
capillary segment a
CryoPin (ID 0.2mm)
that has been premounted to a standard
magnetic
base
is
required. (Shown in
9,(left).
Samples
of
these are provided in
the Starter Kit).
9b
The desired capillary segment is removed from the plate using
the cutting tool (provided in the Starter Pack) or a pair of small
scissors. It is recommended to first cut the end near the
precipitant well to avoid uncontrolled movement of the liquid in
the capillary.
9c
For room temperature studies- the whole capillary can be used,
but for flash-cooled experiments, capillaries should be cut
anywhere along the 3 cm length. For example, crystals shown in
the following capillaries can be cut in the places indicated (dotted
line).
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10
Mounting of capillaries under flash-cooled conditions
For flash-cooled capillaries, you need to have at least 5-7 mm of
capillary left outside of the CryoPin when mounting *(shown
below). This length is short enough to avoid the problem of being
only partially immersed in the focused cryostream.
To hold the capillary in place in
the pre-mounted CryoPin - dip the
CryoPin into the sealing paste
prior to adding the capillary.
The cut capillary segment is then
stuck into the metal capillary
without the addition of any glue or
sealing the top end.
Capillary
With 5-7
mm left
outside of
CryoPin.
10 contd.
The mounted capillary can now be flash-cooled in liquid nitrogen in
the customary way, i.e. for freezing a loop.
For placing the mounted capillary onto the goniometer (shown above,
it is recommended to use a cryo tong designed for large pin heights
(24mm).
Mounting of capillaries at ambient conditions
NB. For this method you do not need to have the 5 - 7
mm of capillary exposed. It can be any length.
11
If the capillary is not flash-cooled in liquid nitrogen (i.e. for
transport or storage) we recommend sealing one end of the
capillary segment with wax. The unsealed end is placed into the
pre-mounted CryoPin and a drop of e.g. glue or sealing paste
applied to fix the capillary segment containing the crystals.
CrystalHarp Capillary
CryoPin