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Related Concept Videos

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An applied magnetic field causes loosely bound π-electrons in organic molecules to circulate, producing a local or induced diamagnetic field over a large spatial volume. As the molecules tumble in solution, the field generated by π-electrons in spherical substituents results in a zero net field. However, the net field generated by π-electrons in non-spherical substituents is not zero. The effect of this induced field depends on the orientation of the molecule with respect to B0,...
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The wavelengths of visible light ultimately limit the maximum theoretical resolution of images created by light microscopes. Most light microscopes can only magnify 1000X, and a few can magnify up to 1500X. Electrons, like electromagnetic radiation, can behave like waves, but with wavelengths of 0.005 nm, they produce significantly greater resolution up to 0.05 nm as compared to 500 nm for visible light. An electron microscope (EM) can create a sharp image that is magnified up to 2,000,000X.
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A scanning electron microscope (SEM) is used to study the surface features of a sample by using an electron beam that scans the sample surface in a two-dimensional manner. Typically, areas between ~1 centimeter to 5 micrometers in width can be imaged. SEM can be used to image bacteria, viruses, tissues as well as larger samples like insects. Conventional SEM gives a magnification ranging from 20X to 30,000X and spatial resolution of 50 to 100 nanometers.
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High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE
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High resolution single particle cryo-electron microscopy using beam-image shift.

Anchi Cheng1, Edward T Eng2, Lambertus Alink3

  • 1Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027, USA; National Resource for Automated Molecular Microscopy, New York Structural Biology Center, New York, NY 10027, USA.

Journal of Structural Biology
|July 29, 2018
PubMed
Summary
This summary is machine-generated.

Automated data acquisition in cryo-electron microscopy (cryo-EM) uses beam-image shift for efficiency. This study shows that beam-image shift, despite causing phase errors, does not limit high-resolution 3D reconstructions.

Keywords:
AutomationComa free alignmentCryo-EMPhase error

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Area of Science:

  • Structural biology
  • Microscopy techniques

Background:

  • Automated data acquisition is crucial for single-particle reconstruction in cryo-electron microscopy (cryo-EM) due to low signal-to-noise ratios.
  • Beam-image shift is a fast and accurate method for automated data acquisition, contrasting with less efficient stage movement methods.
  • Beam-image shift is known to induce beam-tilt, potentially causing phase errors that theoretically limit high-resolution data collection.

Purpose of the Study:

  • To investigate the impact of beam-image shift-induced phase errors on high-resolution cryo-EM single-particle reconstructions.
  • To reconcile the theoretical phase error limit with practical observations of high-resolution data obtained using beam-image shift.

Main Methods:

  • Performed cryo-electron microscopy (cryo-EM) single-particle reconstructions on a T20S proteasome sample.
  • Applied beam-image shifts corresponding to beam tilts ranging from 0 to 10 mrad.
  • Evaluated reconstruction quality by comparing Fourier Shell Correlation (FSC) values and analyzing water density peaks in the 3D map.

Main Results:

  • 3D reconstructions were successfully achieved using beam-image shifts that induced phase errors beyond the theoretical π/4 limit.
  • Fourier Shell Correlation (FSC) values and water density peak analysis indicated high-quality reconstructions.
  • The practical results contradicted the theoretical assumption that phase errors limit resolution.

Conclusions:

  • The phase error induced by beam-image shift does not limit the validity of 3D reconstructions in single-particle averaging beyond the π/4 resolution limit.
  • Beam-image shift can be reliably used for high-resolution cryo-EM data collection.
  • This finding supports the continued use of beam-image shift for efficient and high-quality cryo-EM structural studies.