Crystallisation principlesCrystallisation principles

Dr Rupert C. WilmouthDr Rupert C. WilmouthSchool of Biological SciencesSchool of Biological Sciences

NTUNTU

Protein crystalsProtein crystals

Proteins are inherently hard to crystallise and protein Proteins are inherently hard to crystallise and protein crystals are fragile compared to crystals of inorganic crystals are fragile compared to crystals of inorganic compounds. Protein crystals are held together by weak compounds. Protein crystals are held together by weak forces, primarily hydrogen bonds.forces, primarily hydrogen bonds.

Crystals only diffract XCrystals only diffract X--rays when wet. Initial rays when wet. Initial experiments in the early 20experiments in the early 20thth century using dried century using dried protein crystals gave no diffraction. Finally, in 1934, protein crystals gave no diffraction. Finally, in 1934, Bernal and Hodgkin measured diffraction from pepsin Bernal and Hodgkin measured diffraction from pepsin crystals that were still in their mother liquor, crystals that were still in their mother liquor, i.e. weti.e. wet..

Growing crystalsGrowing crystals

In order to grow protein crystals, the protein must be In order to grow protein crystals, the protein must be purepure(usually >95%), (usually >95%), homogeneoushomogeneous, and in , and in high concentrationhigh concentration (> 5 (> 5 mg mLmg mL--11). Since pH greatly affects crystallisation, a ). Since pH greatly affects crystallisation, a bufferbuffer must must be present (e.g. MES, HEPES, TRIS).be present (e.g. MES, HEPES, TRIS).Finally, a Finally, a precipitantprecipitant is also required which causes the protein is also required which causes the protein to precipitate out of solution. Under careful controlled to precipitate out of solution. Under careful controlled conditions (especially conditions (especially temperaturetemperature and and evaporation rateevaporation rate), ), protein solutions will sometimes become supersaturated instead protein solutions will sometimes become supersaturated instead of precipitating. At this critical point, crystals may form.of precipitating. At this critical point, crystals may form.NucleationNucleation, the formation of the first ordered aggregates, is a , the formation of the first ordered aggregates, is a key event. Nucleation conditions are sometimes difficult to key event. Nucleation conditions are sometimes difficult to reproduce, and thus seeding procedures with preformed reproduce, and thus seeding procedures with preformed crystalline material may be invaluable to obtain reproducible crystalline material may be invaluable to obtain reproducible results.results.

Vapour diffusionVapour diffusion

A drop of protein solution is mixed with an approximately equal A drop of protein solution is mixed with an approximately equal amount of amount of crystallisation solution from a reservoir. The reservoir or wellcrystallisation solution from a reservoir. The reservoir or well has a greased has a greased rim and is then sealed with a cover slide (either siliconized glrim and is then sealed with a cover slide (either siliconized glass or plastic).ass or plastic).In the resulting closed system, water vapour diffuses from the hIn the resulting closed system, water vapour diffuses from the hanging anging drop into the reservoir, which contains about twice the precipitdrop into the reservoir, which contains about twice the precipitant ant concentration than the drop. This allows the protein to become mconcentration than the drop. This allows the protein to become more ore concentrated and potentially allows crystallisation to occur.concentrated and potentially allows crystallisation to occur.The drop can either be placed on the underside of the cover slidThe drop can either be placed on the underside of the cover slide (e (hanging hanging dropdrop) or placed on a plastic support above the surface of the reserv) or placed on a plastic support above the surface of the reservoir oir ((sitting dropsitting drop). ).

The method can be used for relatively large drops (2The method can be used for relatively large drops (2--20 20 µµL) suspended L) suspended above 100 above 100 µµL L –– 1 mL of reservoir solution in 241 mL of reservoir solution in 24--well plates. It can also be well plates. It can also be used for microused for micro--drops in small crystallisation strips. The smallest drop size isdrops in small crystallisation strips. The smallest drop size islimited by the evaporation during the time delay from drop applilimited by the evaporation during the time delay from drop application to cation to sealing of the well.sealing of the well.

Hanging drop v. sitting dropHanging drop v. sitting drop

Hanging dropHanging drop

CheaperCheaper

Easier to setupEasier to setup

Easier to harvest crystalsEasier to harvest crystals

Multiple drops possible on one cover slide

Sitting dropSitting drop

Able to use larger dropsAble to use larger drops

Easier for soakingEasier for soaking

Safer for transportSafer for transport

Multiple drops possible on one cover slide

Crytallisation kinetics Crytallisation kinetics –– phase diagramsphase diagrams

Vapour diffusion Vapour diffusion –– no crystals formedno crystals formed

Crytallisation kinetics Crytallisation kinetics –– phase diagramsphase diagrams

Vapour diffusion Vapour diffusion –– crystals formedcrystals formed

MicrobatchMicrobatch

A 1A 1--2 2 µµL drop of protein solution mixed with an aliquot of crystallisatL drop of protein solution mixed with an aliquot of crystallisation ion solution (containing precipitant, possible additives, buffers, esolution (containing precipitant, possible additives, buffers, etc.) is pipetted tc.) is pipetted onto the surface of a oilonto the surface of a oil--covered microtitre plate well. The drop then sinks to covered microtitre plate well. The drop then sinks to the bottom of the well, and is isolated from the environment. the bottom of the well, and is isolated from the environment. Variations include placing the individual components under oil, Variations include placing the individual components under oil, with either with either protein drop or crystallisation solution first (creating two kinprotein drop or crystallisation solution first (creating two kinetically different etically different scenarios), and using varying ratios of water permeable oils to scenarios), and using varying ratios of water permeable oils to allow water to allow water to diffuse into the environment. It should be noted that alcohols, diffuse into the environment. It should be noted that alcohols, detergents, and detergents, and lipids can diffuse into the oil (and, to a much smaller degree, lipids can diffuse into the oil (and, to a much smaller degree, into the polymer into the polymer of the well material).of the well material).The microThe micro--batch method is well suited for miniaturisation and automation, batch method is well suited for miniaturisation and automation, as as there is no time delay between the application of the drop and tthere is no time delay between the application of the drop and the sealing. The he sealing. The ability to work with minute drops of 1 ability to work with minute drops of 1 µµL or below makes the method very L or below makes the method very suitable for screening. suitable for screening.

Crytallisation kinetics Crytallisation kinetics –– phase diagramsphase diagrams

Microbatch Microbatch –– constant protein concentrationconstant protein concentration

Other crystallisation methodsOther crystallisation methods

Another method used to bring about gradual change in the relativAnother method used to bring about gradual change in the relative e concentrations of the protein and precipitant is concentrations of the protein and precipitant is dialysisdialysis. This ranges from . This ranges from traditional dialysis tubing for milliliter samples to the use oftraditional dialysis tubing for milliliter samples to the use of specialised specialised dialysis buttons which make it easy to dialyse as little as 10 dialysis buttons which make it easy to dialyse as little as 10 µµL of sample. The L of sample. The use of dialysis permits one to increase the concentration of preuse of dialysis permits one to increase the concentration of precipitant while cipitant while essentially holding protein and other component concentrations sessentially holding protein and other component concentrations steady. It is teady. It is also possible to induce crystallisation through a change in pH balso possible to induce crystallisation through a change in pH by dialysing y dialysing with a pH gradient.with a pH gradient.HydrogelsHydrogels can be used for crystal growth with the crystallisation solutiocan be used for crystal growth with the crystallisation solution n soaking a polymeric network. Two common examples are agarose andsoaking a polymeric network. Two common examples are agarose and silica. silica. A silica hydrogel can be formed by reacting sodium silicate withA silica hydrogel can be formed by reacting sodium silicate with acetic acid. acetic acid. Gels can reduce nucleation and allow crystals to grow larger. ThGels can reduce nucleation and allow crystals to grow larger. They can also ey can also provide stability to fragile crystals. The main disadvantage is provide stability to fragile crystals. The main disadvantage is the extra hassle the extra hassle involved and it can also make crystal manipulation more difficulinvolved and it can also make crystal manipulation more difficult.t.

Sparse matrix screensSparse matrix screens

Sparse matrix screens are a common method for initially Sparse matrix screens are a common method for initially determining potential crystallisation conditions. A carefully chdetermining potential crystallisation conditions. A carefully chosen osen wide selection of buffers, pH values, salts and precipitants is wide selection of buffers, pH values, salts and precipitants is screened.screened.The most widely used sparse matrix screen is the one originally The most widely used sparse matrix screen is the one originally sold by Hampton and now copied by Molecular Dimensions and sold by Hampton and now copied by Molecular Dimensions and SigmaSigma--Aldrich (all three are identical).Aldrich (all three are identical).Many other sparse matrix screens have been devised (see the Many other sparse matrix screens have been devised (see the Molecular Dimensions catalogue), and it is worth trying these ifMolecular Dimensions catalogue), and it is worth trying these ifthe the ‘‘HamptonHampton’’ screen does not yield interesting results.screen does not yield interesting results.Remember to make sure that your protein solution is only weakly Remember to make sure that your protein solution is only weakly buffered, otherwise the pH value of the screen will be buffered, otherwise the pH value of the screen will be meaningless. Also, ensure that no other salts or additives have meaningless. Also, ensure that no other salts or additives have been added to your protein solution via the purification procedubeen added to your protein solution via the purification procedure.re.

Empty drop with some light Empty drop with some light ‘‘skinskin’’

Empty drop with light precipitateEmpty drop with light precipitate

Empty drop with heavy precipitateEmpty drop with heavy precipitate

Heavy precipitate and crystalsHeavy precipitate and crystals

Oil droplets Oil droplets –– phase separationphase separation

Needle clusterNeedle cluster

Single needlesSingle needles

Needles from oil dropletsNeedles from oil droplets

Twinned platesTwinned plates

Rectangular crystalsRectangular crystals

Hexagonal crystals (lysozyme)Hexagonal crystals (lysozyme)

Small thin crystalsSmall thin crystals

Small rectangular crystalsSmall rectangular crystals

Salt crystalsSalt crystals

Dried out PEG dropletDried out PEG droplet

Air bubbleAir bubble

Fungal growthFungal growth

Crystal optimisationCrystal optimisation

Dr Rupert C. WilmouthDr Rupert C. WilmouthSchool of Biological SciencesSchool of Biological Sciences

NTUNTU

Optimisation of conditionsOptimisation of conditions

Grid screensGrid screensTemperature (e.g. 4Temperature (e.g. 4°°C, 10C, 10°°C, 18C, 18°°C)C)AdditivesAdditivesCations (anions)Cations (anions)Substrates/inhibitorsSubstrates/inhibitorsLimited proteolysisLimited proteolysisDifferent protein constructsDifferent protein constructs

Grid screensGrid screens

Grid screens are useful for screening two conditions against eacGrid screens are useful for screening two conditions against each h other. Three dimensional grid screens are also possible by usingother. Three dimensional grid screens are also possible by usingmultiple trays.multiple trays.Grid screens take a fair amount of effort to make up, therefore Grid screens take a fair amount of effort to make up, therefore choose your conditions carefully. Also, take extreme care not tochoose your conditions carefully. Also, take extreme care not tomake errors make errors –– one mistake and you may miss the correct one mistake and you may miss the correct conditions.conditions.In general, screen precipitant conditions more widely than pH. In general, screen precipitant conditions more widely than pH. Most proteins crystallise between pH 6 and pH 8.5. Make sure Most proteins crystallise between pH 6 and pH 8.5. Make sure you use the correct buffer you use the correct buffer (i.e(i.e. within 0.5 pH of the p. within 0.5 pH of the pKKa).a).For example, you find some promising looking precipitate in For example, you find some promising looking precipitate in Hampton condition 36 (0.1 M TRIS pH 8.5, 8% (w/v) PEG Hampton condition 36 (0.1 M TRIS pH 8.5, 8% (w/v) PEG 8000). What grid screen might one use next?8000). What grid screen might one use next?

[PEG 8000] (w/v)4% 6% 8% 12% 16% 20%

0.1 M HEPES pH 7.0

0.1 M HEPES pH 7.5

0.1 M TRIS pH 8.0

0.1 M TRIS pH 8.5

AdditivesAdditives

There are a huge number of additives that can be tried. Usually There are a huge number of additives that can be tried. Usually this is the last thing to be screened. Additives are most usefulthis is the last thing to be screened. Additives are most usefulwhen your crystallisation conditions are when your crystallisation conditions are ‘‘almostalmost’’ correct, for correct, for example, when you want to change the morphology of your example, when you want to change the morphology of your crystals or increase their size.crystals or increase their size.

Examples include cations (e.g. Ca, Fe, Cd, Zn, Ni etc.), Examples include cations (e.g. Ca, Fe, Cd, Zn, Ni etc.), detergents (e.g. ndetergents (e.g. n--octyloctyl--ββ,D,D--glucoside, NDSBglucoside, NDSB--195 etc.), 195 etc.), chaotropic agents (urea, guanidine), sugars (e.g. sucrose, glucochaotropic agents (urea, guanidine), sugars (e.g. sucrose, glucose se etc.), alcohols (e.g. methanol, ethanol, isopropanol, glycerol, etc.), alcohols (e.g. methanol, ethanol, isopropanol, glycerol, 22--methylmethyl--2,42,4--pentanediol etc.).pentanediol etc.).

Substrates/inhibitorsSubstrates/inhibitors

If there are substrates, substrate analogs or inhibitors availabIf there are substrates, substrate analogs or inhibitors available, le, then cothen co--crystallisation should always be attempted.crystallisation should always be attempted.The presence of a molecule bound in the active site often makes The presence of a molecule bound in the active site often makes the protein less flexible and hence easier to crystallise.the protein less flexible and hence easier to crystallise.When you solve the structure, having a substrate or inhibitor When you solve the structure, having a substrate or inhibitor bound in the active can give immensely useful mechanistic bound in the active can give immensely useful mechanistic information.information.Be careful when using substrates to ensure they are not turned Be careful when using substrates to ensure they are not turned over by the enzyme. You can use nonover by the enzyme. You can use non--hydrolysable substrate hydrolysable substrate analogs (e.g. ADPNP instead of ATP) or inactive conditions analogs (e.g. ADPNP instead of ATP) or inactive conditions (e.g. low pH).(e.g. low pH).

Different protein constructsDifferent protein constructs

Often it is easier to try a different protein construct Often it is easier to try a different protein construct than trying the thousandth different additive in the than trying the thousandth different additive in the hope of improving your crystal quality.hope of improving your crystal quality.Possible changes include moving the location of the Possible changes include moving the location of the 6His tag, deleting the 6His tag (native purification), 6His tag, deleting the 6His tag (native purification), using a different tag (e.g. GST), performing Nusing a different tag (e.g. GST), performing N--terminal or Cterminal or C--terminal truncations (try deleting terminal truncations (try deleting residues, one at a time).residues, one at a time).Other possibilities may include changing the Other possibilities may include changing the expression system, fungal, insect or mammalian cells expression system, fungal, insect or mammalian cells are alternatives to bacterial expression.are alternatives to bacterial expression.

SeedingSeeding

A seed provides a template for the assembly of molecules to A seed provides a template for the assembly of molecules to form a crystal with the same characteristics as the crystal fromform a crystal with the same characteristics as the crystal fromwhich it originated.which it originated.It is important to differentiate the process of crystal growth fIt is important to differentiate the process of crystal growth from rom nucleation. In general, the degree of supersaturation required fnucleation. In general, the degree of supersaturation required for or nucleation is higher than that required for crystal growth.nucleation is higher than that required for crystal growth.Normally, during aggregation, there is an equilibrium between Normally, during aggregation, there is an equilibrium between the formation of ordered nuclei (reversible) and the formation othe formation of ordered nuclei (reversible) and the formation of f precipitate (irreversible).precipitate (irreversible).When crystal seeds are added, the equilibrium can shift towards When crystal seeds are added, the equilibrium can shift towards crystal formation, and avoids the random nature of spontaneous crystal formation, and avoids the random nature of spontaneous nucleation.nucleation.

SeedingSeeding

There are There are threethree commonly used types of seeding:commonly used types of seeding:

MicroseedingMicroseeding –– adding very small pieces of crushed adding very small pieces of crushed protein crystals to the crystallisation drop.protein crystals to the crystallisation drop.

MacroseedingMacroseeding –– adding an intact, already grown crystal adding an intact, already grown crystal to the crystallisation drop.to the crystallisation drop.

Streak seedingStreak seeding –– similar to microseeding but uses a fine similar to microseeding but uses a fine hair (often a cathair (often a cat’’s whisker is best) to pick up small s whisker is best) to pick up small protein crystal fragments.protein crystal fragments.

MicroseedingMicroseeding

This is generally the easiest and most reliable form of seeding.This is generally the easiest and most reliable form of seeding.A few small crystals, or one large crystal, is broken up to a fiA few small crystals, or one large crystal, is broken up to a fine ne powder using a needle.powder using a needle.A small amount of mother liquor containing this fine powder is A small amount of mother liquor containing this fine powder is added to an aliquot of well solution and vortexed well. Serial added to an aliquot of well solution and vortexed well. Serial dilutions of the microseed solution can then be carried out (e.gdilutions of the microseed solution can then be carried out (e.g. . 1:10, 1:100, 1:1000).1:10, 1:100, 1:1000).Finally, a small quantity (e.g. 0.5 or 1 Finally, a small quantity (e.g. 0.5 or 1 µµL) of the microseed L) of the microseed solution is added to the protein crystallisation drop.solution is added to the protein crystallisation drop.Often the microseed solution is added at the same time that the Often the microseed solution is added at the same time that the crystallisation drop is set up. However, it is important to crystallisation drop is set up. However, it is important to remember that adding the microseed solution to an already remember that adding the microseed solution to an already equilibrated drop can be advantageous.equilibrated drop can be advantageous.

Streak seedingStreak seeding

This is a quick method of seeding, and can be used to seed a This is a quick method of seeding, and can be used to seed a series of drops rapidly.series of drops rapidly.The main difficulty is to find a suitable fibre to use, catThe main difficulty is to find a suitable fibre to use, cat’’s s whiskers are excellent (if you can find a willing cat!).whiskers are excellent (if you can find a willing cat!).For ease of use, superglue the whisker onto a plastic rod. For ease of use, superglue the whisker onto a plastic rod. Degrease the whisker with ethanol or methanol, and then rinse Degrease the whisker with ethanol or methanol, and then rinse with distilled water.with distilled water.Either, touch an existing crystal with the fibre to dislodge a fEither, touch an existing crystal with the fibre to dislodge a few ew seeds, or stroke the fibre through a microcrystalline precipitatseeds, or stroke the fibre through a microcrystalline precipitate.e.Then introduce the seeds into a fresh drop by stroking the fibreThen introduce the seeds into a fresh drop by stroking the fibrein a straight line through the drop.in a straight line through the drop.

MacroseedingMacroseeding

This is perhaps the most difficult seeding technique to use This is perhaps the most difficult seeding technique to use successfully.successfully.A single crystal, free from twinning or other crystalline A single crystal, free from twinning or other crystalline deformities, is selected.deformities, is selected.This crystal is then washed in a slightly dissolving solution toThis crystal is then washed in a slightly dissolving solution toremove the top layer which may contain invisible defects. The remove the top layer which may contain invisible defects. The solution must not cause excessive etching or cracking.solution must not cause excessive etching or cracking.After washing several times, the crystal is then transferred to After washing several times, the crystal is then transferred to fresh drop, preferably prefresh drop, preferably pre--equilibrated.equilibrated.Extreme care has to be taken when handling the crystal to Extreme care has to be taken when handling the crystal to avoid damage and the generation of microseeds. Needles are avoid damage and the generation of microseeds. Needles are particularly difficult to handle.particularly difficult to handle.

CryoCryo--conditionsconditions

These days, mounting of most protein crystals on an XThese days, mounting of most protein crystals on an X--ray diffractometer is ray diffractometer is done at cryogenic temperatures, usually around 100K.done at cryogenic temperatures, usually around 100K.

A small nylon loop is used to pick up a crystal, immerse it a crA small nylon loop is used to pick up a crystal, immerse it a cryoprotectant yoprotectant solution for a short period of time, and then either plunge it isolution for a short period of time, and then either plunge it in liquid nitrogen n liquid nitrogen (or sometimes liquid propane), or place it directly on the gonio(or sometimes liquid propane), or place it directly on the goniometer head in a meter head in a stream of cold nitrogen gas.stream of cold nitrogen gas.

The choice of cryoprotectant is critical. It must prevent ice foThe choice of cryoprotectant is critical. It must prevent ice formation and it rmation and it must not damage the crystal. Always screen several cryoprotectanmust not damage the crystal. Always screen several cryoprotectants (e.g. ts (e.g. glycerol, ethylene glycol, MPD, PEG 400, sucrose etc.) at severaglycerol, ethylene glycol, MPD, PEG 400, sucrose etc.) at several different l different concentrations, and at several different soaking durations.concentrations, and at several different soaking durations.

Do not forget the possibility of mounting your crystal at room tDo not forget the possibility of mounting your crystal at room temperature in emperature in a quartz capillary. This is a very difficult technique to mastera quartz capillary. This is a very difficult technique to master, but remains the , but remains the best way of checking the diffraction of your crystals.best way of checking the diffraction of your crystals.

CryoCryo--annealing and dehydrationannealing and dehydration

These are last ditch attempts to improve the diffraction These are last ditch attempts to improve the diffraction of your crystals.of your crystals.CryoCryo--annealing is where your frozen crystal is (briefly) annealing is where your frozen crystal is (briefly) defrosted before being refrozen. Occasionally the defrosted before being refrozen. Occasionally the mosaicity (and less often the resolution) can mosaicity (and less often the resolution) can dramatically improve.dramatically improve.Dehydration is the intentional drying out of your Dehydration is the intentional drying out of your crystal. This can reduce the water content of your crystal. This can reduce the water content of your crystal, and hence perhaps improve the resolution. crystal, and hence perhaps improve the resolution. Two possibilities: stepwise increase of your precipitant Two possibilities: stepwise increase of your precipitant concentration, or use a hanging drop suspended above concentration, or use a hanging drop suspended above a humectant solution (e.g. 50% or 75% glycerol).a humectant solution (e.g. 50% or 75% glycerol).