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Related Concept Videos

Crystal Growth: Principles of Crystallization01:25

Crystal Growth: Principles of Crystallization

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Crystallization is a phase transformation process in which crystals are precipitated from a supersaturated solution or formed from other sources. During crystallization, atoms or molecules arrange themselves into a well-defined, rigid crystal lattice to minimize energy.
Initiating crystallization involves manipulating the concentration of the solute and the temperature of the solution. Since crystal growth occurs when the ratio of concentration and solubility of the solute in the solvent...
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Recrystallization: Solid–Solution Equilibria01:10

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Recrystallization is a purification technique used to separate impurities from solid compounds. In this technique, no chemical reactions occur. Instead, it exploits physical properties only, specifically, the solubility differences between the desired compound and impurities, either at a single temperature or at different temperatures, and under other selected conditions. The solid-solution equilibrium (solubility equilibrium) of each component in the solution represents a binary phase...
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Precipitation Processes

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The experimental conditions in a gravimetric analysis should be optimized to maximize the particle size and purity of the obtained precipitate. Ideally, the concentration of the precipitating reagent should be low with effective stirring to maintain low relative supersaturation for the growth of large crystals. In homogeneous precipitation, the precipitant is slowly generated by a chemical reaction in the solution to avoid local reagent excesses. For example, urea decomposes gradually to...
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Washing, Drying, and Ignition of Precipitates00:52

Washing, Drying, and Ignition of Precipitates

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After filtration, the precipitate is washed to remove coprecipitated impurities and any remaining mother liquor. Colloidal precipitates, such as silver chloride, are washed with an electrolyte (such as dilute nitric acid) to prevent the peptization of the precipitate. In the case of slightly soluble precipitates, the wash solution contains a common ion to reduce solubility. Lead sulfate, which is slightly soluble in water, is washed with dilute sulfuric acid. Similarly, wash solutions may be...
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Colloidal precipitates01:09

Colloidal precipitates

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The high insolubility of some precipitates can result in an unfavorable relative supersaturation. This can lead to colloidal particles with a large surface-to-mass ratio, where adsorption is promoted. For instance, in the precipitation of silver chloride, silver ions are adsorbed on the surface of the colloidal particles, forming a primary layer. This layer attracts ions of opposite charge (such as nitrate ions), forming a diffuse secondary layer of adsorbed ions. This electric double layer...
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Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering
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Design and implementation of suspended drop crystallization.

Cody Gillman1,2, William J Nicolas1,3, Michael W Martynowycz1

  • 1Departments of Biological Chemistry and Physiology, University of California, Los Angeles CA, USA.

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|April 10, 2023
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Summary

We introduce suspended drop crystallization, a new method for growing protein crystals directly on electron microscopy grids. This technique simplifies sample preparation for advanced structural analysis techniques like MicroED.

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Area of Science:

  • Structural Biology
  • Biophysics
  • Materials Science

Background:

  • Traditional protein crystallization methods often involve complex steps and can be challenging for certain sample types.
  • Sample preparation for advanced imaging techniques like cryo-electron microscopy (cryo-EM) and MicroED requires high-quality crystals and can be a bottleneck.

Approach:

  • Developed a novel suspended drop crystallization method where protein and precipitant are mixed directly on an electron microscopy grid.
  • Designed a specialized crystallization chamber allowing for dual-sided vapor diffusion and in-situ monitoring via various microscopy techniques.
  • Demonstrated the method by crystallizing proteinase K and determining its structure using MicroED after focused ion beam/scanning electron microscopy (FIB/SEM) milling.

Key Points:

  • Suspended drop crystallization eliminates the need for support layers on the EM grid.
  • Allows for direct transfer of crystals from the grid to X-ray crystallography or MicroED.
  • Facilitates crystal growth monitoring using light, UV, or fluorescence microscopy.
  • Overcomes challenges with viscous media, mechanical stress sensitivity, and preferred orientation.

Conclusions:

  • Suspended drop crystallization offers a streamlined and versatile workflow for protein crystal growth and structural analysis.
  • This method provides a valuable alternative for preparing challenging samples for MicroED and other structural biology techniques.