CA2360315C - Laboratory cap and well for hanging-drop crystallization methods - Google Patents

Abstract

The invention relates to a device for molecular and macromolecular crystallization.
More particularly, the device comprises a well and a transparent cap for growing diffrac-tion-duality protein crystals by conventional vapor diffusion techniques. The present de-vice is particularly advantageous in that it al-lows the pre-filling of the well with a solution for transport and handling.

Description

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)

3 I'CT/CA00/00119 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 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

-4-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

5 I'C:T/(:A00/00119 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.

6 PCT/CAOO/00119 --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.

WO 00/47323 1'CT/CA00/00119

-7-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 WO 00/47323 F'CT/CA00/00119

-8-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.

-9-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 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.

-10-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

11 YCT/CA00/00119 --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 hcc., JV. L=UU: ~:3dCtY1 J~Y.~."~T U l.v: lY:1~ ~!l{ dG dJ 'r '='~
~- .. .

-12-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

_ . . - - - =' - = 0 J .. . . . . U.. .

-13-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.,,

Claims (22)

WHAT IS CLAIMED IS:

1. A device for forming crystals by vapor diffusion, comprising a tray having a number of wells, the wells being open at an upper end thereof for receiving a precipitating solution, individual removable caps being provided for separately and independently covering each of a plurality of said wells, each of said individual removable caps having an underside defining a support surface for suspending a droplet of solution containing a molecule or a macromolecule to be crystallized over the precipitating solution contained in an associated one of said plurality of wells, said support surface being at least partly transparent for allowing examination and monitoring of crystal growth, each of said individual caps being further provided with a first integral locking member structurally engageable with a corresponding second locking member associated with each of said plurality of wells to releasably interlock said individual removable caps to said plurality of wells in a sealed manner without the need for an operator to apply an external sealing material between the removable caps and the plurality of wells.

2. A device according to claim 1 wherein the wells and the caps are cylindrical.

3. A device according to claim 1 wherein a rim of each well extends above a top surface of the tray.

4. A device according to claim 3 wherein the cap is tubular and said first locking member comprises locking elements equally spaced apart, each locking element being lockingly insertable into a corresponding opening in a surface of the tray adjacent the rim of the well.

5. A device according to claim 4 wherein each cap comprises a tubular section around an external surface thereof, thereby defining a slot therebetween that is closed at a top thereof, the slot being adapted to receive therein the rim of an associated well, the tubular section comprising the locking elements coupled thereto.

6. A device according to claim 4 or 5 wherein each locking element comprises a tongue extending from top to bottom of the cap, the tongue comprising a portion extending perpendicularly to the outside of the cap external surface.

7. A device according to claim 1 wherein an elastomeric material is inserted between each cap and the top surface of the tray to seal the wells.

8. A device according to claim 5 wherein an elastomeric material is inserted into the slot between the rim of the well and the top of the slot.

9. A device according to claim 1 wherein each cap comprises a cavity in a surface facing bottom surface of the associated well and being adapted to receive a drop of a molecule- or macromolecule-containing solution.

10. A device according to claim 9 wherein the cavity has a flat bottom surface.

11. A device according to claim 10 wherein the cavity comprises a circumferential wall extending perpendicularly to the fat bottom surface.

12. A device according to claim 2 wherein each cap comprises a rim onto an external surface thereof, the rim comprising equally spaced apart slots.

13. A device according to claim 1 further comprising a cover disposed over and around the tray.

14. A device according to claim 1 further comprising equally spaced apart finger grip surfaces on the peripheral wall.

15. A device according to claim 1 wherein the tray and the cap are made of a transparent or translucent material.

16. A device according to claim 15 wherein the material is selected from a group of materials consisting of : polystyrene, polypropylene, polycarbonate, polyacrylate, polymethacrylate, acrylonitrile-styrene copolymers, nitrile-acrylonitrile-styrene copolymers, polyphenyleneoxide, phenoxy resins, and mixtures thereof.

17. A device according to in any one of claims 1 to 16, wherein said first locking member and said second locking member are gradually engageable between a released position and a locked position.

18. A device according to claim 17, wherein said first and second locking members are rotatably engageable with one another between said released position and said locked position.

19. A device according to claim 18, wherein said first end second members includes inter-mating threads respectively on each of said caps and each of said wells.

20. A method for forming molecular or macromolecular crystals, comprising the steps of: dispensing a precipitating solution in a well, providing a droplet of a solution containing a molecule or macromolecule on a drop support having a first locking means engageable with a corresponding second locking means of the well, inverting the drop support over the well so that the droplet is suspended over the precipitating solution contained in the well, and engaging said first and second locking means to simultaneously interlock said drop support on said well and seal the well.

21. A method according to claim 20, wherein the step of engaging includes rotating the first and second locking means relative to one another.

22. A method according to claim 1, comprising the step of pre-filling the well with the precipitating solution and sealing the well before shipment to a purchaser.