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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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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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Related Experiment Video

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Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers
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Tracking Protein Motions using Serial Femtosecond Crystallography with X-Ray Free-Electron Laser.

Eiichi Mizohata1, Eriko Nango2, Takehiko Tosha3

  • 1Graduate School of Engineering, Osaka University, Osaka, Japan.

Current Protocols
|September 23, 2025
PubMed
Summary

Researchers can now visualize protein motion in real-time using serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs). This breakthrough allows for detailed observation of protein dynamics and reaction mechanisms at room temperature.

Keywords:
SACLAmolecular movieprotein motionstructure–function relationshiptime‐resolved analysis

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Biophysics

Background:

  • Understanding protein structure-function relationships is crucial in biochemistry.
  • Visualizing protein motion at high temporal and spatial resolution was previously limited.
  • Advancements in X-ray free-electron lasers (XFELs) enable new structural biology techniques.

Purpose of the Study:

  • To introduce practical methods for visualizing protein motions at room temperature using serial femtosecond crystallography (SFX).
  • To enable the visualization of entire protein reaction mechanisms.
  • To provide a streamlined workflow from sample preparation to capturing dynamic structural snapshots.

Main Methods:

  • Streamlined microcrystallization workflow for hen egg-white lysozyme.
  • Rotational seeding approach for homogeneous microcrystal generation, refined on copper-containing nitrite reductase.
  • Time-resolved SFX strategy using fungal nitric-oxide reductase, photolabile caged substrates, and UV triggering.

Main Results:

  • Demonstrated a practical microcrystallization workflow for SFX.
  • Developed a rotational seeding method yielding high-quality microcrystals for challenging targets.
  • Successfully captured millisecond-timescale catalytic intermediates using time-resolved SFX.

Conclusions:

  • The presented methods facilitate the visualization of protein dynamics using SFX at room temperature.
  • These techniques advance the study of protein reaction mechanisms.
  • Enables researchers to capture dynamic structural snapshots of proteins in action.