Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
I,aboratory cap and well for hanging-drop crystallization methods
FIELD OF THF, INVENTION
The present invention relates to a device for handling molecular and
macromolecular crystallization. More particularly, the device comprises ,t
well and a cap
assembly for growing protein crystals by convcntional vapor diffusiou
tcclmiques. The
present device is particularly advantageous in that it Cacilitates the pre-
filling of the well
with a solution for transport and handlin{; prior to utilization by a
technician.
BACKGROUND OF THE INVENTION
Crystallography is an extremely usefii] tool for scientists, and is therefore
a
field of research attracting a lot of interest. It is a powerful meziis that
provides precise
and detailed description of thc three-dimensional structire of the molecules,
and is of
great help in the understanding of their functions. Crystallography
ofmacromolecules
like proteins is extensively used today, academically as well as industriallv.
Altliough three-diniensional structures of sintple proteins have been obtained
through crystallization nlethods, it is not always casy to obtain crystals
from
20 macromolecules. For esample, the preferred conditions for the
crystallization of a given
molecule can take several hundreds if not thousands of trials. As a result,
means and
methods have been developed to perform a great number of trials
retativel}quickly,
including hanging-drop and sitting-drop metllods. All such rnethods uv.e the
henefit of
vapor diffusion to obtain the crystals.
2 5 $ll8S1Ti1.n $FlEET (RU! E 26)
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The hanging-drop method is currently the most commonly used technique for
scanning various crystallization conditions of maci-omolecules, such as
proteins. It
comprises suspending a droplet ol' approximatel} 2 to 20 l, of solution
c.ontaining the
macromolecule to be crystallized and a precipitating agent, over a
precipitating solution,
such as conventional polyethylene glycol 20% or ammonium sulfate 40'..0,
contained in a
reservoir or well. The system is then sealed hei-rnetically. Aficr a whil(:
vapor diffusion
of the solvent or solvent mixtures betwecn the droplct and the solutioii in
the rescrvoir
reaches equilibrium. The end result is a decrease of water in tlie droplet,
and an increase
of the niacromolecule and precipitating agent concentration therein, thus
causing
crystallization of the macromolecule in optimizcd conditions. "I'hc actual
techni.quc for
the set up of the hanging-drop or sitting-drop experiments is a long and
arduous work and
has to be perfornied by qualified and skilled teclinical personnel.
Conventionally, a commercially available tray niade of an ii-icrt
thermoplastic
'ts material comprising a plurality olreservoirs or wells is prepai-ed, anci
ttic precipitating
solution is placed in each reservoir or well manually. The macromoleculc
solution is then
mixed with a precipitating agent on a glass plate (coverglass) an(i the whole
is inverted
over the wells, thus making the macromoleculc solution overhanging the well.
Prior to
placing a glass plate over a well, the rim of each well is greased to ensure a
proper seal.
z C) Care must be taken when placing the plate over each well, since the
~,rease can easily
contaminate the macromoleculc solutiori. Tlie crystallization process is
{olloweci with the
help of a microscope. After the crystal is ohtained, the glass plate is
ivmoved. Again,
this must be done with great care to prevent contamination of the c1-
ystallized
macromolecule with grease, and/or breaking of the glass plate. On top uf that,
the plates
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are hardly reusable for any experiment because thc grease is hard to remove,
and some of
it remains on the plates.
An advantage of the hanging-cirop and sitting-drop metho<is is that they
providc screening conditions for crystallization, and truly represent a
microcrystallization
technique. The vapor diffusion in the hanging or sitting drop allows scrccning
of a large
range of conditions and necessitates a relatively small amount of
macromolecules.
Further, it allows a relatively clear visualization of the results, and the
eventual crystals
are frce, i.e., they do not adhere or are stuck to any surtace.
-.o
Typically, several hundreds of experiments are recluired to lind appropriate
crystalliziug conditions for the production of high quality crystals.
Accordingly, hanging-
drop and sitting-drop experiments are a vei-y labor-intensive process
demanding skilled
and experienced technical personnel. For example, multiple aspirating and
dispensing
1s steps of components, multiple greasing steps etc. Inust he perl'Ormed in
the experimental
set up. Further-, foi- each well, a sepai-ate coverg(ass niust he manually
inverted. The
number and complexity of steps can therefore produce an undesirable wide
variation in
experimental results.
2o As stated above, grease is conventionally used to provide a seal bctwecu
thc
well and the coverglass. Other ways fbr sealing the systcm have been
proposecl. For
example, grease can be replaced with irrunersion oil or au adhesive tape. As
witii grease,
these scaling means have serious drawbacks. Grease is uot always casy to
dispense
around the upper rim of the well, and is a tinie consuming operation.
Tecluiiciaiis
215 repeating the operation thousands of times occasionally sul'fci- physical
pain to tlicir
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hands. Other significant problenls and risks arc present when manipulating the
crystal on
a greasy cover slide. The cover slide sometimes breaks during the process,
which may
cause injury to the technicia.n, in addition to loosing the crystals. The
immersion oil is
also problematic. One has to use a determined volume of oil. 'I'oo much oil
leads to
contamination within the well, while not enougil will lead to non-hermetic
seal that niay
result in the evaporation of the precipitating solution. An adhesive tapU
allows quicker
and simpler nianipulations, but all experiniertts are sealed at the end oi'the
set-up, thus
introducing experimental variations between the 1" and the 24"' drop. Further,
crystals
oflen stick to the tape, rendering impossible thc recovery of the crystals,
and the
operations for the recovery of the drop are also prohleniatic.
These conditions promoted the robotization of the procedure. Some automated
crystallization devices already exist. The well-known C.'yberlab-200T "'
apparatus
dispenses solutions in wells, geases the upper rim of each well, pours
clroplets on cover
slides held by a vacuum ann, and places the cover slides over the wells.
Howevei-, such
apparatus still has some drawbacks, namely a complicated experimental set-up,
and the
notable use of grease. Further such apparatus is extremcly expensive.
Relevant references in the field inciude [JS 2,366,886; US 3,107,204; US
3,297,184; t1S 3,537,956; US 3,597,326; lJS 3,649,464; US 3,692,498; US
3,729,382;
US 3,745,091; US 3,907,505; US 4,038,149; US 4,154,795; US 4,495,289; US
4,917,707; US 5,271,795;
It would therefore be highly desirable to develop a device for crystallizing
macromolecules that would overcome the above deficiencies. Sucii device would
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eliminate the requirement of extemal means like grease, oil or an adhesive
tape to seal the
well and the cover, and would pi-eferably be easy to manipulate, either
manually or
autoniatically. Ultimately, the process of experimental set up of the device
would be
greatly facilitated and accelerated, while simultaneously eliminating possible
i-isks of
s contamination of the eventual ci-ystals. Finally, such device should be
usable for various
crystallization processes such as hanging-drop or sG tting-drop processes.
SUMMARY OF THE INVENTION
ln accordance with the pi-esent invention, thcrC is provided a tray
cotnprising a
i o top surface, a bottom, an upright c-ircumferentiLtl wall extending from
the top surftice to
the bottom, and a plurality of wells extending dowuwardly ti-oin, and being
open at, the
top surface, for receiving a precipitating solution; and a cap provided for
eacli well, each
cap comprising locking members fbr locking the cap onto tiic well in a sealed
manner.
Such tray is particularly advantageous tor growing molecular and
macromolecular
15 crystals.
IN THE DRAWINGS
Figure 1 illustrates perspective view of a first cap and wrll assenibly in
accordance with the present invention;
20 Figure 2 illustrates a section view of a preferred embodinient of a cap;
Figure 3 illustrates a perspective view of a tool adapted to install and
remove a
cap from the well;
Figure 4 illustrates a section view of the tool of I~ igure 3; and
Figures 5, 0 and 7 illustrate other enibodiments of the cap and well assembly
zs according to the present invention.
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DETAILED DESCRIPTION OF THE INVENTION
It is an object of the preserit invention to provide a crystal fomning de.vice
using
vapor diffusion method. The device coniprises a well and a transparent cap
designed to
s closc the well and form a sealed volume, the well being seale.d without the
need to add a
sealing material like grease, oil, adhesive tape and the like between the well
and the cap.
The cap is made of transparent material to allow eaamination and rnonitoring
of crystal
growth, as well as manipulation of the crystal under a microscope. The
prese,nt device
therefore represents an iniportant advancc in methods tor growing ci-ystals of
inacroinolecules, especially in the tield of hanging-drop and sitting-drop.
Because of its simplicity, the operations of Glling the well with the
precipitating solutioti, placing a drop of the macromolecule solution onto the
cap and
sealing the well hy putting the cap in position ovcr thc well can he
accomplished by any
1.5 competent technician, and not only skilled personnel.
In a preferred embodiment of the inve.ntion, a plurality of wells are molded
together, for example in a tray comprising 4 rows of 6 wells cacli, witil
corresponding
transparent caps are provided thereon. The resulting tray and caps may also bc
optionally
treated with a hydrophobic agent such as a silicouing agent.
Because of the transparency of the cap and of the bottom surtace of the well,
crystallization can be followed with mininzal hanaling, and without disturbing
the vapor
equilibriunl within each well. Further, visualization of the results under thu
microscope
are siinple because the cap is made of a transparent or translucent (ciear)
niaterial.
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I'referably, the material of the tray an(l the cap arc thc sam,, ancl comprise
materials that can be nlolded easily at a reasonable cost. hhe material
shottl.d he stable 16r
extended periocls of time towards the various chemicai ;)roducts present in
the well and
onto the cap. 'I,he material should also prelcrahly not absorb water, and he-
goc>d optical
quality to facilitate work and obsmation under a nncroscopC. F.xatnple
suitable materials
includc various thermoplastics such as polystvrenc. polypropylene.
polycarbonate.
polyaerylate. polynicthaerylate, acrylonitrile-styrc;nc copolymers, niUile
acryle~nitrile
styrene copolymers, polyphenyleneoxidc, phenoxy re.sins, etc.. the most
pretcrrcd material
~U being polystyrenc.
It is another object of the present invention to provide a crystal -furmint-,
device
that allows the manipulation of the growtll crystals undcr thc microsciqpc
without any
transfer froni the cap, wliere solutions can be acldC(i dirOctly without an\'
transior ot'the
11-ctystals, in a greaseless environment.
Anotlier major advantage of the device otf thc= prescnt invention is that once
a
series of experiinents is completecl. the tray is rea lily re.usable, simply
b~\talcing another
series of caps containing a cirop of a si>lutiotr containinb a ntacrotnolecule
to he
20 crystallized, and reinstalling the caps ()ver the wells. Further, ai!.iven
cap ntay he
removecl fronl its original well and locl;ed ont~) another one contait;in", <,
clifferent
precipitatinp solution.
Tlie invention is also concerned with ~i mcthocl lur lormiuk, crystals of a
~_ macrotnolecule, the method comprisin" the steps ~>f~ dispensint.Y a
precipit,itin~1 s()lutioi1 in
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a well; forming a droplet in a cap comprising locking members to lock the cap
over ttle
well; and locking and scaling the well. In a preferred embodinlent, a ring
made of an
elastonleric material like polypropylene, an ethylene-propylenc copolyrner,
"I'e11onTM etc.,
is preferably provided between the cap and the weli. In a furthet- prefert-ed
einbocliment,
the well can be filled in advance and tightly sealcd, so that they a tray is
provided to a
technician in a "ready-to-use" manner.
Because of the ergonoinics of the present invention, the cap is eugaged easily
so ttiat there is no need for special manual dexterity comparativchto the usc
of
Zo conventional thin, fragile, microscope eoverl;lasses. The presence of a
cavity in the
surface of the cap facing the bottoin surface of the well allows the addition
of liquid
directly over the drop, after placing the cap upside down on a table, without
the need to
transfer the crystals to another well, thus limiting the riianipulations tha:
miglrt ruin the
fragile crystals.
~s
The use of the cap and well assembly o+' the prescnt inve.ntion can be
automated in a straightforward manner by providing the extreniity of an
automated arni
with a simple grip element having an cnd provide(i with a structure adapted to
releasably
grip the cap. There is no need for the application of grease oi- the
manipulation of fragile
2 o coverglass pieces. The grip element inay also be manipulated manuall~~ by
a technician,
as described hereinbelow.
The cap and well assembly of the present invention also finds applications in
the field of cell cultures, molecular oi- cellular biology etc.
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In a most preferred embodiment of the invention, the well is f,lled bcforehand
and sealed with the cap. The technician thcrefore reccives a"ready-tc~-use"
assemblv,
thus eliminating the time-consuming operation of filling each well with the
appropriate
precipitating solution. The buyer may therefore order as many assemblies as
desired with
s the sanic or different precipitating solutions. For shipping purposes, the
cap may be
replaced on the assenibly with a f-ilm to prevent contact of'the precipitating
solution with
the cap. Such eontact would necessitate the cleanin4 of the cap prior to its
LISC. One may
also use a cap for shipping purposes, and a (lit'ferent cap to cai-iy out the
experiments. It is
important that the well bc sealed to avoid evaporation and spilling of thc
precipitatin;=
ia solution, eithei- during shipment of the pre-filled wells, or during the
experiments.
Referring to the drawings which illustrate preferred embodiments of the
invention, Figure 1 illustrates a cap and well assembly 10 which crnnprises a
tray or base
plate 12 provided with a plurality of wells 14 anu corresponding caps 10.
Asstmbly 10
ls may also include a cover 18 usecl for shipping or storage purposes. "The
preferred form of
cover 18 comprises with an insertion (not shown) at each comer that allows
retention of
the cover over the caps without touching them. Cover 18 further allo~tis the
storage of
several trays of experiments one on top of the other. Tray 12 comprises a rinl
20
extending about the four side walls thereoi; and is provided with finger grip
surfaces 22
2 o such as those described in US 4,038,149, on two opposed side walls for
rasier handling of
the tray by the technician. Fingei- grip surfaces 22 are provided to avoid
mishaps, and
greatly facilitate handling of covered and uncovered trays. Cover 18 comprises
a section
24 adapted to engage around finger grip surfaces 22 for proper titting ove1-
assembly 10.
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Figure 2 illustrates a section view of cap 16. As it can be seen, cap 16
comprises a cylindrical slot 26 into which is inserted an 0-ring element 28
made of a
resilient material. Such material, although optional, is provided to ensure an
appropriate seal when cap 16 is fitted over upper rim 30 of well 14. The inner
surface
32 of slot 26 has a portion or ridge 34 extending passed the planar surface 36
thereby
forming a cavity 38. Surface 36 may be concave or convex, but the planar
configuration illustrated on Figure 2 is much preferred. As stated above, the
material of
cap 16 is such that it is sufficiently transparent or translucent so that cap
16 can be
placed directly under a microscope for observation and/or manipulation of the
crystals.
Each cap 16 comprises a pair of locking element 40 diametrically opposed to
each other and comprising a ridge portion 41. Cap 16 also comprises a further
rim 46
provided with a series of spacer 45 underneath. Once the precipitating
solution is
poured into well 14, the technician puts cap 16 upside down on a flat surface
and
places a drop of the macromolecule-containing solution onto surface 36. Cap 16
is
then flipped over cautiously, and each locking element 40 is inserted into a
corresponding opening 42 provided onto the upper surface 44 of tray 12 until
the
abutment of upper rim 30 of well 14 with the 0-ring element 28 inside slot 26
is
achieved. Each opening 42 acts as a second locking member for cooperating with
the
first locking member 40 provided on the cap 16. Cap 16 is then rotated so that
locking elements 40 slide each into a slot 43 having a width smaller than that
of
opening 42 and extending on a portion of the periphery of well 14 until the
upper
surface of portion 41 is entirely under upper surface 44, thereby efficiently
scaling
well and maintaining cap 16 in place. In a most preferred embodiment, a
section 47
of portion 41 is tapered to facilitate sliding under upper surface 44. To
ensure even
better locking and maintenance of the cap in position, a small bump (not
shown) is
provided onto section 49
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that is adapted to fit into a corresponding recess (not snown) present under
surface 44
after completc insertion of portion 41 under surface 44.
To put cap 16 in place onto well 14, or for renioval therefrom, a tool 48 may
he
used, as illustrated ui Figures 3 and 4. 'lool 48 comprisc;s a body 50 divided
in a portion
52 shaped in a nlanner such as to facilitate holding by the tecliniciitn or
ai7 autonlated arm;
a cylindrical portion 54 with an external surface 57 having a cireumtcreric:;
slighily big~cr
than that of rim 46, and an internal surface 59 having a circurnference
slightly smaller
than that of rin- 46. T'ool 48 further comprises two diametrically opposed cap
gripping
Ao elements 60 eacti provided with a gripping finger 62. 't'hc gripping elc-
ment 60 can be
provided onto intet'tlal surface 59_ directly on rim 66, or onto external
surlace 57. In
operation, tool 48 is placed over cap 16 so that each finger 62 is inserted
into a slot 64 cttt
into rini 46 until at least a pot-tion 65 of eacli element 60 is abutted onto
tinl 46. Tool 48
is then rotated until gripping iingers 62 are completely engagecl under rim
46, and the
is rotation is maintained until the locking -nember5 40 arc aligned with slots
42. C'ap 16 is
then simply pulled up. To reintroduce the cap in position, the procedurc is
carried out in
an opposite masmer. The extern.al surface 53 of portion 52 should he planar,
so that it can
be laid on a table or under a nlicroscope in a stable manner, and allow the
technician to
observe and/or work on tiie crystals. 'To be able to work under a microscope
directlv,
20 surface 53 must comprises an opening preferably corresponding to the
internal diameter
of cylindrical portion 54 (see Figure 4).
Figure 5 illustrates another emhodimeiu ot'the present invention. The cap and
well assembly 100 which comprises a tray ot- base plate (not shown ) provided
with a
25 plurality of wells 112 and corresponding cap 114, which coniprises a
cylindrical slot 116
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into which is inserted an 0-ring elemcnt 118 made of a resi]ient material. As
for the
previous orribodlment iIlustrated, the 0-ning, although optional, is provided
to mnsure an
appropriate seal.
s Each cV 114
comprises a pair of locking eleaneat 120 diamctrically opposed
to each other and comprising a ridge portion 122. Cap 114 is locked into
position on the
tray by inserting each loclcing eiement 120 into a coaesponding opening 124
provided
onto the uppar surface 126 of the tray until the lower surface 128 of cap 114
lies flat onto
the tray upper surface 126. Cap 114 is then rotated so that locldng elaaonts ]
20 slida inw
a slot 130 having a width smaller than that of opcning 124 and extending on a
portion of
the periphery of well 112 until the upper surface of ridge portion 122 is
entirely under
upper surface 126, thereby ef3cieatly sealing well and maintaining cap 114 in
place.
Figure 6 illustrates aaother simple vaustion of the present invention, wheroin
is the upper surface 152 of a well 150 comprises a slot 154 along its
circumference and
adapted to receive an 0-ring element 156 coupled to a cap 158. The section of
slot 154 is
such that is slightly smaller than that of eletnaat 156, so that upon
insertion into the slot, a
tight seal is formed by the locking of cap 158 to well 150 without the need of
any
adhesive or grcase.
Figure 7 illustrates yet another embod'unent of the invention, wherein the cap
170 is screwed on the well 172. This consOruCtion as wcll as the one
ilhistrated in Figs. 1,
2 and 5 provides for gradual engagemcnt of the loe1ang mechanisin of the cap
with the
wc11 for movement between a released position and a locked position.
AMENDED SHEET
EMPFANGSZEIT 1. MAI. 0:36 Au.>uRl1CKS1FiT I p,iat n.A I
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The prescnt cap and well assembly is particularly suitable for both hanging-
drop or sitting-drop crystallization methods. With respect to the sitting-drop
nnethod,
although not specifieally iIlustrated in the drawings, anyone skilled in the
art will readity
appreciate that any conventional drop support can be insated or molded into
the well.
Examples of such sitting-drop support include the Micro-BridgesTM or the glass
sitting
drop rods manufactured and sold by Hampton Research (Laguna Mls, California).
Each well is carefully filled with a selected equilibrating solution.
Subsequently, a selected protein drop is deposited on the cap. The shape and
the texture
of the lower surface can be varied to obtain optimunn reaults for a particular
protein
solution being crystallized, for exatnple, when lower surface tension
solutions, inaluding
protein solutions conbining detergents, are used. The addition of the
equilibYating
solution and the protein drops to the device may be carried out either
manually or through
coznmercial automatod plpetting apparatus, and the sealing of the cap over the
solution
is may also be carried out manually or in an automau xl menner.
While the invention has been descnbed in connection = with specific
embodi.ments thereoA it will be andarstood that it is capable of fiuthar
nraodifications and
this epplication is intended to cover any variations, uses or adaptations of
the invention
following, in general, the principles of the invention and including such
departures from
the pre.cart desaription as come within known or customary practice within the
art to
which the invention pertains, and as may be appliod to the esseahal features
herainbefore
set forth, and as follows in the scope of the appended claims.
AMENDED SHEET
tMPFANGSZE IT 1. MAI. 0:36 ancnA+Wre7r I r AJA , n.,,
