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Related Concept Videos

X-ray Crystallography02:18

X-ray Crystallography

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The size of the unit cell and the arrangement of atoms in a crystal may be determined from measurements of the diffraction of X-rays by the crystal, termed X-ray crystallography.
Diffraction
Diffraction is the change in the direction of travel experienced by an electromagnetic wave when it encounters a physical barrier whose dimensions are comparable to those of the wavelength of the light. X-rays are electromagnetic radiation with wavelengths about as long as the distance between neighboring...
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The cell membrane, or plasma membrane, is an ever-changing landscape. It is described as a fluid mosaic where various macromolecules are embedded in the phospholipid bilayer. Among the macromolecules are proteins. The protein content varies across cell types. For example, mitochondrial inner membranes contain ~76% protein content, while myelin contains ~18% protein content. Individual cells contain many types of membrane proteins—red blood cells contain over 50—and different cell...
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Plasma membranes have integral transmembrane proteins involved in facilitated transport. These proteins are collectively referred to as transport proteins, and they function as either channels for the material or as carriers themselves. Channel proteins have hydrophilic domains exposed to the intracellular and extracellular fluids and a hydrophilic channel through their core that provides a hydrated opening for solutes to pass through the membrane layers. Passage through the channel allows...
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Proteins show rotational as well as lateral diffusion across the membrane. The lateral diffusion of proteins was confirmed through the cell fusion experiment where mouse and human cells were fused, resulting in hybrid cells. When the human and mouse cells fused, the specific membrane proteins on human and mouse cells were marked with the red and green-fluorescent markers, respectively. Initially, the red and green fluorescence was located on the respective hemisphere of the cell. As time...
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Ionic crystals consist of two or more different kinds of ions that usually have different sizes. The packing of these ions into a crystal structure is more complex than the packing of metal atoms that are the same size.
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Detergents are used to purify the integral proteins of the membrane. The hydrophobic portion of the detergent can replace membrane phospholipids while solubilizing the membrane proteins. When detergent monomers reach a specific concentration in a solution called critical micelle concentration (CMC), they form micelles. Above CMC, the concentration of the detergent monomers remains in equilibrium with the micelle. The number of detergent monomers present in the CMC varies for each detergent, and...
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Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening
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X-ray transparent microfluidic platforms for membrane protein crystallization with microseeds.

Jeremy M Schieferstein1, Ashtamurthy S Pawate, Michael J Varel

  • 1Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S Mathews Ave, Urbana, IL, USA. ashtamurthy@gmail.com kenis@illinois.edu.

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|February 23, 2018
PubMed
Summary

Microseeding in microfluidic chips simplifies membrane protein crystallization for X-ray crystallography. This method optimizes crystal growth and size by controlling microseed concentration and formulation conditions.

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Area of Science:

  • Structural Biology
  • Biophysics
  • Biochemistry

Background:

  • Crystallization of membrane proteins is essential for determining their atomic-resolution 3-D structures.
  • Understanding membrane protein structure is key to elucidating their function.
  • Microseeding is an effective technique for growing protein crystals, but it is underutilized.

Purpose of the Study:

  • To develop simplified methods for membrane protein crystallization using microseeding in microfluidic chips.
  • To demonstrate microfluidic techniques for introducing microseed dilutions and for microseed screening.
  • To analyze the impact of microseed concentration and crystallization conditions on crystal growth and diffraction quality.

Main Methods:

  • Utilized X-ray transparent microfluidic chips for membrane protein crystallization.
  • Developed a microfluidic method for introducing varying dilutions of microseeds into crystallization experiments.
  • Implemented a microfluidic chip for on-chip formulation and screening of crystallization conditions.
  • Collected data on crystallization composition, crystal size, and diffraction quality.

Main Results:

  • Demonstrated successful microseeding for photoactive yellow protein and cytochrome bo3 oxidase.
  • Observed that decreasing microseed concentration led to fewer, larger crystals.
  • Mapped crystallization outcomes on phase diagrams, identifying distinct regions for crystal growth and diffraction quality.

Conclusions:

  • Microfluidic microseeding offers a simplified and effective approach for membrane protein crystallization.
  • Optimized microseed concentration and crystallization conditions are crucial for obtaining high-quality crystals.
  • This methodology facilitates the structural determination of membrane proteins for structure-function studies.