The complete collection of eighteen Crystal Growth 101 articles. Introduction, Sample Preparation for Crystallization, Crystallization Screening, Sitting Drop Vapor Diffusion, Hanging Drop Vapor Diffusion, Microbatch Crystallization, Microdialysis Crystallization, Viewing Crystallization Experiments, Salt or Protein Crystals, Optimization, Drop Ratio, Temperature as a Crystallization Variable, Seeding, Buffer Formulation, Phosphate Buffer Dilution Table, Using Volatile Buffers to Adjust Drop pH and Induce Crystallization, Reagent Formulation and Handling, and Halides and Phasing.

The Crystal Growth 101 series prepared by Hampton Research presents an overview of the preparation of the sample, methods, screening, optimization, reagent formulation, and other aspects of protein crystallization. We hope Crystal Growth 101 will prove a useful resource and inspiration in your crystal quest. Best of success with your crystals!

The sample is the single most important variable in the crystallization experiment. Begin with a pure, homogeneous, stable, active sample. The sample should be as pure as possible, 95 to 98%, assayed by Coomassie stained SDS-PAGE. A homogeneous, active sample, free of contaminants, aggregates, and minimal conformational flexibility is desired.

Crystallization screening is the process of evaluating methods, reagents, and other chemical and physical variables with the objective of producing crystals and/or identifying the variables which are positively or negatively associated with crystallization of the sample.

At the time of this writing, up to 40% of samples screened for crystallization will produce some kind of crystalline result and 10% of samples will produce a crystal suitable for X-ray diffraction analysis. About 75% of proteins screened require optimization. Optimization is the systematic manipulation and evaluation of variables which influence the crystallization of the sample.

The complete collection of eighteen Crystal Growth 101 articles. Introduction, Sample Preparation for Crystallization, Crystallization Screening, Sitting Drop Vapor Diffusion, Hanging Drop Vapor Diffusion, Microbatch Crystallization, Microdialysis Crystallization, Viewing Crystallization Experiments, Salt or Protein Crystals, Optimization, Drop Ratio, Temperature as a Crystallization Variable, Seeding, Buffer Formulation, Phosphate Buffer Dilution Table, Using Volatile Buffers to Adjust Drop pH and Induce Crystallization, Reagent Formulation and Handling, and Halides and Phasing.

The sitting drop vapor diffusion technique is another popular method for the crystallization of macromolecules. The principle of vapor diffusion is straightforward. A drop composed of a mixture of sample and reagent is placed in vapor equilibration with a liquid reservoir of reagent. Instructional video for Sitting Drop Vapor Diffusion

The hanging drop vapor diffusion technique is a popular method for the crystallization of macromolecules. The principle of vapor diffusion is straightforward. A drop composed of a mixture of sample and reagent is placed in vapor equilibration with a liquid reservoir of reagent. Typically the drop contains a lower reagent concentration than the reservoir. To achieve equilibrium, water vapor leaves the drop and eventually ends up in the reservoir. As water leaves the drop, the sample undergoes an increase in relative supersaturation. Both the sample and reagent increase in concentration as water leaves the drop for the reservoir. Equilibration is reached when the reagent concentration in the drop is approximately the same as that in the reservoir. Instructional Video for Hanging Drop Vapor Diffusion

Microbatch crystallization is a method where the sample and reagent are combined and sealed in a plate, tube, container, or sealed under a layer of oil.

Oils can also be used as a barrier between the reservoir and the drop in traditional Hanging or Sitting Drop crystallization experiments. This is known as Vapor Diffusion Rate Control.

When the conditions for growing protein crystals by sitting or hanging drop vapor diffusion are known, crystals of comparable or sometimes better quality can usually be produced by microbatch under oil using a general set of guidelines.

Vapor Diffusion to Batch Calculator is an Excel based spreadsheet that allows one to enter vapor diffusion crystallization condition data in order to generate a best guess microbatch condition.  Vapor diffusion crystallization condition data is entered into the yellow fields and best guess microbatch conditions, results, and calculations are displayed in the green fields.

In the microdialysis crystallization method the sample is separated from the crystallization reagent by a semi-permeable membrane. The semi-permeable membrane allows small molecules, such as salts, additives, and other crystallization reagents to pass, but prevents biological macromolecules from crossing the membrane. Crystallization of the sample takes place due to the diffusion of crystallization reagent out of, or into the sample, at constant sample concentration.

A stereomicroscope with zoom (8 to 12.5:1) and 10 to 100x magnification, LED illuminators to minimize temperature change during observation, polarizing optics to discern birefringence, camera attachment, and a viewing platform large enough to support a crystallization plate when viewed from any and all wells is a good starting point for observing the experiment. Automated imaging systems are also available, as well as imaging systems with ultraviolet and other capabilities.

In viewing crystallization experiments, when crystals are observed, one must determine if the crystals are of the target biological macromolecule (protein) or salt (inorganic, small molecule) crystals. Before deciding on how to distinguish whether the crystal is salt or protein,take a picture of the crystal for documentation before you destroy the crystal or make it disappear.

Optimization is the manipulation and evaluation of biochemical, chemical, and physical crystallization variables, towards producing a crystal, or crystals with the specific desired characteristics.

Varying the drop ratio is a simple method to utilize during crystallization screening and optimization. Drop ratios allow one to explore varying levels of initial and final protein and reagent concentration, explore different equilibration paths, and cover a wider range of relative supersaturation. All by simply changing the amount of sample and reagent added to the drop.

Drop Ratio Calculator is an Excel based spreadsheet that will display the effects of varying the drop ratio of sample and crystallization reagent in a vapor diffusion experiment. Protein Concentration and Reagent concentration can be entered into the gray fields and the results displayed above in the gray fields. Custom Protein Concentration and Custom Reagent Concentration can be entered into the blue fields, along with Custom Protein Volume and Custom Reagent Volume and the results displayed above in the blue fields.

Temperature can be a significant variable in the crystallization of biological macromolecules (proteins). Temperature often influences nucleation and crystal growth by manipulating the solubility and supersaturation of the sample. Temperature has also been shown to be an important variable with phase separation in detergent solutions during membrane protein crystallization.

Seeding allows one to grow crystals in the Metastable Zone, where spontaneous homogeneous nucleation cannot occur, but crystal growth from seeds can occur. Why would one want to do this? For control, reproducibility, and to improve the likelihood of a successful crystallization experiment. In the Metastable Zone crystals can grow from seeds but cannot spontaneously nucleate. By placing a seed or solution of seeds in a drop which is saturated to the Metastable Zone one can use the seeds to grow larger single crystals. By controlling the number of seeds introduced into the Metastable Zone drop one can control the number of crystals grown. It is not practically possible to measure and know the number of seeds introduced to a drop, but by performing serial dilutions from a concentrated seed stock one can control the number of crystals grown in the Metastable Drop.

Crystallization buffer table in order of useful pH range.

Details about the formulation of Hampton Research buffers, including how to titrate, useful pH range, pKa and more.

Use this table to easily create Sodium potassium phosphate buffers and reagents between 0.2 and 4.0 M while varying the pH between 5 and 8.2.

Volatile buffers, when added only to the reagent reservoir of a vapor diffusion experiment, can alter the pH of the crystallization drop by vapor diffusion of the volatile acid or base component from reservoir into the drop. This may be particularly useful when the sample is known to have pH dependent solubility and may be used to induce crystallization.

Good crystallization results depend on being able to replicate experiments, and experiments cannot be replicated unless the reagents are made correctly and consistently. Be consistent with sources, methods, and techniques. Maintain accurate and detailed records; your future self and perhaps others, will thank you later.

Solubility data for formulating a saturated solution of the salts listed at the designated temperature.

Bromide and iodide can diffuse into protein crystals when soaked with the appropriate solution and can successfully be used for phasing.

24 well plate format scoring sheet for screening and optimization.

PEG Stability: A Look at pH and Conductivity Changes over Time in Polyethylene Glycols

Hampton Research Catalog and Crystal Growth 101 - Complete

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