Ultrastable gold substrates: Properties of a support for high-resolution electron cryomicroscopy of biological specimens

Electron cryomicroscopy (cryo-EM) allows structure determination of a wide range of biological molecules and specimens. All-gold supports improve cryo-EM images by reducing radiation-induced motion and image blurring. Here we compare the mechanical and electrical properties of all-gold supports to amorphous carbon foils. Gold supports are more conductive, and have suspended foils that are not compressed by differential contraction when cooled to liquid nitrogen temperatures. These measurements show how the choice of support material and geometry can reduce specimen movement by more than an order of magnitude during low-dose imaging. We provide methods for fabrication of all-gold supports and preparation of vitrified specimens. We also analyse illumination geometry for optimal collection of high resolution, low-dose data. Together, the support structures and methods herein can improve the resolution and quality of images from any electron cryomicroscope.

• Publication year

  2016

• Venue

  Journal of Structural Biology

• Publication date

  2016-01-01

• Fields of study

  Biology, Medicine, Materials Science

• Identifiers
  DOI 10.1016/j.jsb.2015.11.006PMID 26592474PMCID 4711342
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Overview and future of single particle electron cryomicroscopy.

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• Advances in Single-Particle Electron Cryomicroscopy Structure Determination applied to Sub-tomogram Averaging

  2015cited by this paper
• Thon rings from amorphous ice and implications of beam-induced Brownian motion in single particle electron cryo-microscopy

  2015cited by this paper
• Single-Particle Cryo-EM at Crystallographic Resolution.

  2015cited by this paper
• Controlling protein adsorption on graphene for cryo-EM using low-energy hydrogen plasmas

  2014influential reference
• Self-assembled monolayers improve protein distribution on holey carbon cryo-EM supports

  2014cited by this paper
• Ultrastable gold substrates for electron cryomicroscopy

  2014influential reference
• The Resolution Revolution

  2014cited by this paper
• Faculty Opinions recommendation of Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM.

  2013cited by this paper
• Electron counting and beam-induced motion correction enable near atomic resolution single particle cryoEM

  2013cited by this paper
• Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles

  2013cited by this paper
• Beam-induced motion of vitrified specimen on holey carbon film.

  2012cited by this paper
• Paraxial charge compensator for electron cryomicroscopy.

  2012cited by this paper
• Theory of Elasticity

  2012cited by this paper
• The graphene-gold interface and its implications for nanoelectronics.

  2011cited by this paper
• Images of paraffin monolayer crystals with perfect contrast: minimization of beam-induced specimen motion.

  2011cited by this paper
• The surface of evaporated carbon films is an insulating, high-bandgap material.

  2011cited by this paper
• Cryomesh™: A New Substrate for Cryo-Electron Microscopy

  2010cited by this paper
• Transmission Electron Microscopy

  2008cited by this paper
• Electron cryo-microscopy of biological specimens on conductive titanium-silicon metal glass films.

  2008cited by this paper
• An Improved Holey Carbon Film for Cryo-Electron Microscopy

  2007cited by this paper
• Principles of physical vapor deposition of thin films

  2006cited by this paper
• Accurate determination of local defocus and specimen tilt in electron microscopy.

  2003cited by this paper
• A Survey of Structure−Property Relationships of Surfaces that Resist the Adsorption of Protein

  2001cited by this paper
• Parameters affecting specimen flatness of two-dimensional crystals for electron crystallography.

  2000cited by this paper
• Nicotinic acetylcholine receptor at 4.6 A resolution: transverse tunnels in the channel wall.

  1999influential reference
• Perforated support foils with pre-defined hole size, shape and arrangement

  1998cited by this paper
• Controlling cell attachment on contoured surfaces with self-assembled monolayers of alkanethiolates on gold.

  1996cited by this paper
• Visualization of beta-sheets and side-chain clusters in two-dimensional periodic arrays of streptavidin on phospholipid monolayers by electron crystallography.

  1996cited by this paper
• Cryo-crinkling: what happens to carbon films on copper grids at low temperature.

  1993cited by this paper
• Specimen flatness of thin crystalline arrays: influence of the substrate.

  1992cited by this paper
• Self-assembled organic monolayers: model systems for studying adsorption of proteins at surfaces

  1991cited by this paper
• Cryo-electron microscopy of vitrified specimens

  1988cited by this paper
• Use of spot-scan procedure for recording low-dose micrographs of beam-sensitive specimens

  1987cited by this paper
• Improvement in high resolution image quality of radiation-sensitive specimens achieved with reduced spot size of the electron beam.

  1986cited by this paper
• Quantitative analysis of image contrast in electron micrographs of beam-sensitive crystals

  1985cited by this paper
• Cryo-electron microscopy of viruses

  1984cited by this paper
• Physics of Semiconductor Devices (2nd edn)

  1982influential reference
• Transmission electron microscopy.

  1973influential reference
• Electron microscopy of tobacco mosaic virus under conditions of minimal beam exposure.

  1970influential reference
• The electric charging of electron-microscope specimens

  1969cited by this paper
• Physics of semiconductor devices

  1969cited by this paper
• Notizen: Zur Defokussierungsabhängigkeit des Phasenkontrastes bei der elektronenmikroskopischen Abbildung

  1966cited by this paper
• Gold : recovery, properties, and applications

  1964cited by this paper
• On the structure of evaporated carbon films

  1960cited by this paper
• Electrical Properties of Arc‐Evaporated Carbon Films

  1957cited by this paper
• The structure of graphitic carbons

  1951cited by this paper

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• Sub-3 Å resolution protein structure determination by single-particle cryo-EM at 100 keV.

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