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Benchmarking the ideal sample thickness in cryo-EM.

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Martynowycz MW, Clabbers MTB, Unge J, Hattne J, Gonen T
OpenAccess logo Proc Natl Acad Sci U S A 118 (2021)
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Cited: 15 times
EuropePMC logo PMID: 34873060

Abstract

The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.

Reviews citing this publication (5)

  1. Bringing Structure to Cell Biology with Cryo-Electron Tomography. Young LN, Villa E. Annu Rev Biophys 52 573-595 (2023)
  2. Electron Diffraction of 3D Molecular Crystals. Saha A, Nia SS, Rodríguez JA. Chem Rev 122 13883-13914 (2022)
  3. MicroED: conception, practice and future opportunities. Clabbers MTB, Shiriaeva A, Gonen T. IUCrJ 9 169-179 (2022)
  4. Cryo-electron tomography related radiation-damage parameters for individual-molecule 3D structure determination. Xue H, Zhang M, Liu J, Wang J, Ren G. Front Chem 10 889203 (2022)
  5. Analysis and comparison of electron radiation damage assessments in Cryo-EM by single particle analysis and micro-crystal electron diffraction. Shi D, Huang R. Front Mol Biosci 9 988928 (2022)

Articles citing this publication (10)

  1. Ab initio phasing macromolecular structures using electron-counted MicroED data. Martynowycz MW, Clabbers MTB, Hattne J, Gonen T. Nat Methods 19 724-729 (2022)
  2. A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals. Martynowycz MW, Shiriaeva A, Clabbers MTB, Nicolas WJ, Weaver SJ, Hattne J, Gonen T. Nat Commun 14 1086 (2023)
  3. Hydrogens and hydrogen-bond networks in macromolecular MicroED data. Clabbers MTB, Martynowycz MW, Hattne J, Gonen T. J Struct Biol X 6 100078 (2022)
  4. Electron-counting MicroED data with the K2 and K3 direct electron detectors. Clabbers MTB, Martynowycz MW, Hattne J, Nannenga BL, Gonen T. J Struct Biol 214 107886 (2022)
  5. Investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae. Parkhurst JM, Crawshaw AD, Siebert CA, Dumoux M, Owen CD, Nunes P, Waterman D, Glen T, Stuart DI, Naismith JH, Evans G. IUCrJ 10 270-287 (2023)
  6. Measuring the effects of ice thickness on resolution in single particle cryo-EM. Neselu K, Wang B, Rice WJ, Potter CS, Carragher B, Chua EYD. J Struct Biol X 7 100085 (2023)
  7. Electron counting with direct electron detectors in MicroED. Hattne J, Clabbers MTB, Martynowycz MW, Gonen T. Structure 31 1504-1509.e1 (2023)
  8. Electron crystallography and dedicated electron-diffraction instrumentation. Simoncic P, Romeijn E, Hovestreydt E, Steinfeld G, Santiso-Quiñones G, Merkelbach J. Acta Crystallogr E Crystallogr Commun 79 410-422 (2023)
  9. Overcoming resolution attenuation during tilted cryo-EM data collection. Aiyer S, Baldwin PR, Tan SM, Shan Z, Oh J, Mehrani A, Bowman ME, Louie G, Passos DO, Đorđević-Marquardt S, Mietzsch M, Hull JA, Hoshika S, Barad BA, Grotjahn DA, McKenna R, Agbandje-McKenna M, Benner SA, Noel JAP, Wang D, Tan YZ, Lyumkis D. Nat Commun 15 389 (2024)
  10. Previously uncharacterized rectangular bacterial structures in the dolphin mouth. Dudek NK, Galaz-Montoya JG, Shi H, Mayer M, Danita C, Celis AI, Viehboeck T, Wu GH, Behr B, Bulgheresi S, Huang KC, Chiu W, Relman DA. Nat Commun 14 2098 (2023)


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