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Crystal Former 

 

The total volume of the channel is 150nL. While some liquid handling robots are capable of dispensing such small volumes, drop sizes of at least 0.3 µL each for the protein and precipitant are recommended. For total drop volumes of 0.5 µL, the sample consumption for the Crystal Formers is comparable to most automated screening methods.

What protein concentration should I start with for my initial screening?

We recommend starting with the protein concentrations between 60-80% of saturation point. The saturation point can be estimated as the point where your protein begins to precipitate during concentration process. This might correspond to 10-30 mg/ml for reasonably well-behaved proteins, but could be significantly lower for poorly-soluble ones. If you are trying to reproduce the vapor diffusion hit, you we recommend increasing your protein concentration.

What are the recommended sample volumes?

For the 16- and 96-channel Crystal Formers, it is recommended that the protein volume be between 0.3-0.5 µL. The corresponding volume for the crystallization solution typically ranges between 0.3 – 1 µL.    When automation is employed, a volume as low as 0.2 µL for each sample inlet can be utilized. As a rule of thumb, we recommend the use of 0.5 µL for manual pipetting and 0.3 µL for set-up with a liquid handling robot. The microchannels of the Scale-up chip are larger, thus 1.5 µL of protein sample with 0.1-1.5 µL of crystallization solution should be loaded per channel.

How do I know if the channels have filled with protein?

If using colorless protein sample, channels are best visualized on a black background; for colored proteins use white background. If plates are set up manually, channel filling can be confirmed by monitoring the solution movement across the channel immediately after drop application. For most protein samples this takes 1-2 seconds. For samples containing glycerol or other viscous additives, the process might take a bit longer. If the plate is set up using a robot, we recommend pausing the robot after the protein application step, and inspecting the plate visually against the light. Any incompletely-filled channels will be easy to detect at this point. Should incomplete channel filling occur, gently tap the short edge of the plate on the bench to aid the solution to fill any remaining channels. Once you have an idea of how long it takes for a sample of particular viscosity to fill channel, you can insert the appropriate pause in your robotic protocol, if necessary. Please refer to our video demonstration of a manual chip set up 

How do the microchannels vary between the Crystal Former formats?

For all chips, the length of the microchannels is 10 mm. Both the 16- and 96-channel original Crystal Formers have channels that are 150 nL in total volume. In contrast, the microchannels of the Scale-up Crystal Former are 1.1 µL in total volume.

Can I use an alternative sealing method?

Sealing tape is provided in a dual-strip format for sealing only the inlet wells. This design minimizes potential imaging difficulties that arise from air bubbles trapped between the tape and the microchannels when larger tape strips are used. When sealing the Crystal Formers, the user should avoid pressing with excessive force, as this will cause mixing of the reactions and dissipate the gradient. Microlytic’s format for sealing tape thus also avoids placing undue pressure over the microchannels. Any other sealing film regularly utilized for sealing crystallization trials is, in principle, compatible with the Crystal Formers, though it is recommended that the substitute tape be trimmed such that is only covers the sample inlets.

The CF-HT plate has a raised edge making tape application awkward, what do you recommend?

Place the plate with short edge towards yourself and attach the sticky ends of the tape to the tips of your middle fingers. Use your thumbs to place the center of the strip on the center of the row of inlets and rest the ends of the tape taught over the edges of the plate. Turn the plate with the long edge facing you and use one hand to guide the tape exactly over the inlets, while the other hand gently presses the tape over the inlets. Turn the pate 180 degrees and repeat the same process for the other half of the tape. Sealing one tail a time with both hands will give you better control over the direction of the tape, so you can be sure it is applied right over the channels. Once the tape has been laid over the channels, glide the edge of the credit card over the tape to ensure a uniform seal. The tape tails can be folded over the edges of the plate or cut off. Please refer to the video for CF-HT sealing. 

Can the Crystal Formers be used to crystallize my integral membrane protein?

Yes. The materials used to manufacture the Crystal Formers are fully compatible with high concentrations of detergents used in the solubilization of membrane proteins. Keep checking back for the final published reference highlighting the successful crystallization of an integral membrane protein with the Crystal Former.

Can I use organic solvents in the Crystal Former?

Yes. To date, we have not identified any common organic compounds used in protein crystallization that are incompatible with the Crystal Formers.

How should I store the Crystal Formers?

Crystal Formers do not require any special storage conditions. For extended incubation periods, higher humidity storage (>60%) is recommended to minimize evaporation. This can be easily achieved by storing the Crystal Formers in a small container that also includes a moist napkin.   

Can the Crystal Formers be used for in situ testing?

All formats of the Crystal Former are compatible with in situ diffraction testing, providing that a suitable chip mount is available. The 96-channel Crystal Formers are compatible with most experimental set-ups that permit whole plate mounting for in situ screening. The Crystal Formers are also compatible with the PX Scanner from Oxford Diffraction (http://www.microlytic.com//App_Themes/microlytic/ML%20Whitepaper%20PXScanner03%2024.pdf).

Are the Crystal Formers compatible with my UV imaging system?

The materials used in Crystal Former manufacturing are fully compatible with UV imaging. Please refer to our white paper (http://www.microlytic.com//App_Themes/microlytic/pdfs/UV_detection.pdf) for additional information.

Are the Crystal Formers automatable?

Both the 16- and 96-channel Crystal Formers are fully compatible with the Mosquito from TTP Labtech (Cambridge, MA). The plate files are available on request from Microlytic. The Crystal Formers are also compatible with a variety of crystal imaging systems, including the Rock Imager from Formulatrix (Waltham, MA). Please contact our technical support line for additional details regarding product integration.

How long does equilibration of the channel take?

Equilibration of ions in the microchannel takes approximately 1 week. Larger polymers and macromolecules will diffuse more slowly and may thus take up to two weeks for full equilibration. Equilibration is not necessary, however, for crystal growth and many crystals appear rapidly (18-72 hours) within the microchannels.

Will I get crystals faster using Crystal Former compared to vapor diffusion?

Liquid-liquid diffusion provides higher crystallization success rate due to more thorough sampling of the protein phase diagram that the vapor diffusion. However, ultimate success is dependent on your target. For challenging targets that have been screened extensively in vapor diffusion and resisted crystallization for months or years, it will likely be necessary to perform more than one screen in Crystal Former.  Same vapor diffusion screens may be utilized, as different mixing kinetics and the power of the concentration gradient may allow for a discovery of new crystallization condition(s) and/or new crystal morphologies.  For new targets, we recommend setting up at least a 48-condition SmartScreen and a 48-condition PurePEG screen in parallel to your regular vapor diffusion screens. You may also use your own vapor diffusion screens in the Crystal Former. For targets that yielded poorly diffracting crystals in the vapor diffusion, we recommend re-screening at least our 96-condition SmartScreen set in the Crystal Former in addition to a standard grid-screen optimization.

I did not get any crystals after my initial screen, what do I do?

Estimate the percentage of the channels that contained any precipitate. If the majority of your channels are completely clear, increase the protein concentration and repeat the screen. If a substantial number of channels contain some precipitate, inspect those channels for precipitation pattern. Heavier protein precipitation is expected next to the precipitant inlet, and lighter-to-no precipitation is expected towards the protein inlet. Absence of any precipitation patterns suggests that the gradient was dissipated during the set-up procedure. This is most often caused by applying too much pressure to the sealing tape. If using your fingertip to seal the inlets, your fingernail should NOT turn lighter from the pressure. Please refer to our video demonstration of a manual chip set up 

How do I harvest crystals from the microchannels?

Crystals are harvested from the back of the Crystal Former. Use a sharp blade to cut the sealing film on either side of the microchannel and peel the film away. To facilitate crystal manipulation, use a loop that is no larger that 100 µm, as this will permit complete submersion of the loop into the channel. The addition of a stabilization solution on exposure of the channel to air is recommended. The stabilization solution can be estimated based on the history of crystal appearance along the channel and the incubation time. As equilibration of the channel typically takes 1 week (for a salt), samples that are at least 1 week old can be stabilized at approximately 50% of the starting crystallization solution. 

Are there any recommended protocols for crystal cryoprotection?

The harvesting and cryoprotection of a protein crystal grown in any given system frequently requires some degree of carefully consideration and optimization. Once crystals have been harvested from the microchannels of the Crystal Former, they may be manipulated using standard methods. As mentioned above, crystals may be stabilized using a solution that approximates the equilibrated condition. Alternatively, the cryoprotectant or cryoprotectant-mother liquor combination may be applied directly over the channel and the crystals flash frozen immediately on extraction. When the conditions for cryoprotection are unclear, mineral oil or paratone oil may be used to cover the exposed microchannel and the crystals again flash frozen after extraction through the oil layer.

The design of the Crystal Former also presents a unique opportunity for the gentle cryoprotection and/or dehydration of crystals. The cryoprotectant can be applied to one of the sample inlets and allowed to diffuse through the microchannel, thus permitting slow diffusion of the cryoprotectant into the crystal channels prior to harvesting.

How do I optimize a crystallization hit? Can crystallization conditions be translated to other crystallization systems?

Many of the crystallization hits can be translated into vapor diffusion systems by systematic grid screening using the initial crystallization condition as a starting point. We also offer the Crystal Former XL, in which the larger channels place less restriction on the maximum growth of the crystals. Many users have successfully optimized their crystals for data collection by altering the ratio of protein to crystallization solution in the Crystal Former. The resultant change in the crystallization gradient has direct impact on the nucleation rate and thus significantly

I’d like to optimize a condition from a PurePeg crystallization condition, how do I go about this?

You can purchase the individual, USP grade PEG components directly from Microlytic. The PEG cocktail is also available for purchase. Optimization of crystallization hits from the PurePeg screen, however, is not necessarily contingent on use of the PEG cocktail.  For many PEG-containing conditions, the identity of the PEG is not absolute for crystal reproducibility. PEG compounds can often be substituted for one another, though a wider range of PEG concentrations should be explored. The major consideration for the reproducibility of crystal hit from the PurePeg screen is the PEG purity. Most PEG compounds utilized for crystallization have not traditionally been USP grade. As such, the presence of contaminants in low PEG grades may hinder the reproducibility of crystals.

Are individual crystallization conditions available for purchase?

Yes, you may order any of the crystallization conditions from the SmartScreen and PurePeg screen.

Why are there only 96 available crystallization conditions? I usually start with many more than that for my initial crystallization trials?

The Crystal Former is designed to enable gently diffusive mixing of protein and precipitant inside microchannels resulting in improved mixing kinetics and significantly higher crystallization hit rates. Liquid-liquid diffusion, harnessed in the Crystal Former, is an orthogonal approach to other methods (e.g. Vapor diffusion, microbatch and dialysis) and samples a significantly different region of the protein phase diagram. When coupled with crystallization conditions that have been optimized for liquid-liquid diffusion approaches, the Crystal Former system provides a robust and efficient method for crystallization screening. The user therefore requires fewer crystallization conditions with significantly increased probability of crystallization success!

How do the SH-1 and SH-2 Crystal Former holders differ from one another?

XZ Dialysis