MicroED
Cryo-electron microscopy (cryo-EM) methods promise new life to high-throughput macromolecular structure determination. Cryo-EM overcomes the fundamental barrier to X-ray diffraction determination of macromolecular complexes: growing X-ray grade crystals. Fortunately, the emergence of microcrystal electron diffraction (MicroED) facilitates the determination of new protein structures at atomic resolution from vanishingly small crystals.
MicroED exploits the strong interaction between electrons and nano-scale three-dimensional crystals and takes advantage of emerging cryo-EM instrumentation coupled to established crystallographic methods.
Suggested further reading
2021
Martynowycz, Michael W; Clabbers, Max T B; Unge, Johan; Hattne, Johan; Gonen, Tamir
Benchmarking the ideal sample thickness in cryo-EM Journal Article
In: Proc Natl Acad Sci U S A, vol. 118, no. 49, 2021, ISSN: 1091-6490.
Abstract | Links | Altmetric | PlumX
@article{pmid34873060,
title = {Benchmarking the ideal sample thickness in cryo-EM},
author = {Michael W Martynowycz and Max T B Clabbers and Johan Unge and Johan Hattne and Tamir Gonen},
doi = {10.1073/pnas.2108884118},
issn = {1091-6490},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Proc Natl Acad Sci U S A},
volume = {118},
number = {49},
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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
2020
Zhu, Lan; Bu, Guanhong; Jing, Liang; Shi, Dan; Lee, Ming-Yue; Gonen, Tamir; Liu, Wei; Nannenga, Brent L
Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED Journal Article
In: Structure, vol. 28, no. 10, pp. 1149–1159.e4, 2020, ISSN: 1878-4186.
Abstract | Links | Altmetric | PlumX
@article{pmid32735770,
title = {Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED},
author = {Lan Zhu and Guanhong Bu and Liang Jing and Dan Shi and Ming-Yue Lee and Tamir Gonen and Wei Liu and Brent L Nannenga},
doi = {10.1016/j.str.2020.07.006},
issn = {1878-4186},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Structure},
volume = {28},
number = {10},
pages = {1149--1159.e4},
abstract = {The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. However, the crystal size optimization process could be time and resource consuming, if it ever happens. Therefore, improved techniques for structure determination using these small crystals is an important strategy in diffraction technology development. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-transmission electron microscopy to collect electron diffraction data and determine high-resolution structures from very thin micro- and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP converted by 2-methyl-2,4-pentanediol or lipase, including Proteinase K crystals grown in solution, cholesterol crystals, and human adenosine A receptor crystals grown in LCP. These results set the stage for the use of MicroED to analyze microcrystalline samples grown in LCP, especially for those highly challenging membrane protein targets.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. However, the crystal size optimization process could be time and resource consuming, if it ever happens. Therefore, improved techniques for structure determination using these small crystals is an important strategy in diffraction technology development. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-transmission electron microscopy to collect electron diffraction data and determine high-resolution structures from very thin micro- and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP converted by 2-methyl-2,4-pentanediol or lipase, including Proteinase K crystals grown in solution, cholesterol crystals, and human adenosine A receptor crystals grown in LCP. These results set the stage for the use of MicroED to analyze microcrystalline samples grown in LCP, especially for those highly challenging membrane protein targets.
Zhu, Lan; Bu, Guanhong; Jing, Liang; Shi, Dan; Lee, Ming-Yue; Gonen, Tamir; Liu, Wei; Nannenga, Brent L
Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED Journal Article
In: Structure, vol. 28, no. 10, pp. 1149–1159.e4, 2020, ISSN: 1878-4186.
Abstract | Links | Altmetric | PlumX
@article{pmid32735770b,
title = {Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED},
author = {Lan Zhu and Guanhong Bu and Liang Jing and Dan Shi and Ming-Yue Lee and Tamir Gonen and Wei Liu and Brent L Nannenga},
doi = {10.1016/j.str.2020.07.006},
issn = {1878-4186},
year = {2020},
date = {2020-01-01},
journal = {Structure},
volume = {28},
number = {10},
pages = {1149--1159.e4},
abstract = {The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. However, the crystal size optimization process could be time and resource consuming, if it ever happens. Therefore, improved techniques for structure determination using these small crystals is an important strategy in diffraction technology development. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-transmission electron microscopy to collect electron diffraction data and determine high-resolution structures from very thin micro- and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP converted by 2-methyl-2,4-pentanediol or lipase, including Proteinase K crystals grown in solution, cholesterol crystals, and human adenosine A receptor crystals grown in LCP. These results set the stage for the use of MicroED to analyze microcrystalline samples grown in LCP, especially for those highly challenging membrane protein targets.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. However, the crystal size optimization process could be time and resource consuming, if it ever happens. Therefore, improved techniques for structure determination using these small crystals is an important strategy in diffraction technology development. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-transmission electron microscopy to collect electron diffraction data and determine high-resolution structures from very thin micro- and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP converted by 2-methyl-2,4-pentanediol or lipase, including Proteinase K crystals grown in solution, cholesterol crystals, and human adenosine A receptor crystals grown in LCP. These results set the stage for the use of MicroED to analyze microcrystalline samples grown in LCP, especially for those highly challenging membrane protein targets.
2018
Jones, Christopher G; Martynowycz, Michael W; Hattne, Johan; Fulton, Tyler J; Stoltz, Brian M; Rodriguez, Jose A; Nelson, Hosea M; Gonen, Tamir
The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination Journal Article
In: ACS Cent Sci, vol. 4, no. 11, pp. 1587–1592, 2018, ISSN: 2374-7943.
Abstract | Links | Altmetric | PlumX
@article{pmid30555912,
title = {The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination},
author = {Christopher G Jones and Michael W Martynowycz and Johan Hattne and Tyler J Fulton and Brian M Stoltz and Jose A Rodriguez and Hosea M Nelson and Tamir Gonen},
doi = {10.1021/acscentsci.8b00760},
issn = {2374-7943},
year = {2018},
date = {2018-11-01},
journal = {ACS Cent Sci},
volume = {4},
number = {11},
pages = {1587--1592},
abstract = {In the many scientific endeavors that are driven by organic chemistry, unambiguous identification of small molecules is of paramount importance. Over the past 50 years, NMR and other powerful spectroscopic techniques have been developed to address this challenge. While almost all of these techniques rely on inference of connectivity, the unambiguous determination of a small molecule's structure requires X-ray and/or neutron diffraction studies. In practice, however, X-ray crystallography is rarely applied in routine organic chemistry due to intrinsic limitations of both the analytes and the technique. Here we report the use of the electron cryo-microscopy (cryoEM) method microcrystal electron diffraction (MicroED) to provide routine and unambiguous structural determination of small organic molecules. From simple powders, with minimal sample preparation, we could collect high-quality MicroED data from nanocrystals (∼100 nm, ∼10 g) resulting in atomic resolution (<1 Å) crystal structures in minutes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In the many scientific endeavors that are driven by organic chemistry, unambiguous identification of small molecules is of paramount importance. Over the past 50 years, NMR and other powerful spectroscopic techniques have been developed to address this challenge. While almost all of these techniques rely on inference of connectivity, the unambiguous determination of a small molecule's structure requires X-ray and/or neutron diffraction studies. In practice, however, X-ray crystallography is rarely applied in routine organic chemistry due to intrinsic limitations of both the analytes and the technique. Here we report the use of the electron cryo-microscopy (cryoEM) method microcrystal electron diffraction (MicroED) to provide routine and unambiguous structural determination of small organic molecules. From simple powders, with minimal sample preparation, we could collect high-quality MicroED data from nanocrystals (∼100 nm, ∼10 g) resulting in atomic resolution (<1 Å) crystal structures in minutes.
2013
Shi, Dan; Nannenga, Brent L; Iadanza, Matthew G; Gonen, Tamir
Three-dimensional electron crystallography of protein microcrystals Journal Article
In: Elife, vol. 2, pp. e01345, 2013, ISSN: 2050-084X.
Abstract | Links | Altmetric | PlumX
@article{pmid24252878,
title = {Three-dimensional electron crystallography of protein microcrystals},
author = {Dan Shi and Brent L Nannenga and Matthew G Iadanza and Tamir Gonen},
doi = {10.7554/eLife.01345},
issn = {2050-084X},
year = {2013},
date = {2013-11-01},
urldate = {2013-11-01},
journal = {Elife},
volume = {2},
pages = {e01345},
abstract = {We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1-1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name 'MicroED', that may have wide applicability in structural biology. DOI: http://dx.doi.org/10.7554/eLife.01345.001. },
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1-1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name 'MicroED', that may have wide applicability in structural biology. DOI: http://dx.doi.org/10.7554/eLife.01345.001.
Suggested reviews
2021
Danelius, Emma; Gonen, Tamir
Protein and Small Molecule Structure Determination by the Cryo-EM Method MicroED Journal Article
In: Methods Mol Biol, vol. 2305, pp. 323–342, 2021, ISSN: 1940-6029.
Abstract | Links | Altmetric | PlumX
@article{pmid33950397,
title = {Protein and Small Molecule Structure Determination by the Cryo-EM Method MicroED},
author = {Emma Danelius and Tamir Gonen},
doi = {10.1007/978-1-0716-1406-8_16},
issn = {1940-6029},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Methods Mol Biol},
volume = {2305},
pages = {323--342},
abstract = {Microcrystal Electron Diffraction (MicroED) is the newest cryo-electron microscopy (cryo-EM) method, with over 70 protein, peptide, and several small organic molecule structures already determined. In MicroED, micro- or nanocrystalline samples in solution are deposited on electron microscopy grids and examined in a cryo-electron microscope, ideally under cryogenic conditions. Continuous rotation diffraction data are collected and then processed using conventional X-ray crystallography programs. The protocol outlined here details how to obtain and identify the nanocrystals, how to set up the microscope for screening and for MicroED data collection, and how to collect and process data to complete high-resolution structures. For well-behaving crystals with high-resolution diffraction in cryo-EM, the entire process can be achieved in less than an hour.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microcrystal Electron Diffraction (MicroED) is the newest cryo-electron microscopy (cryo-EM) method, with over 70 protein, peptide, and several small organic molecule structures already determined. In MicroED, micro- or nanocrystalline samples in solution are deposited on electron microscopy grids and examined in a cryo-electron microscope, ideally under cryogenic conditions. Continuous rotation diffraction data are collected and then processed using conventional X-ray crystallography programs. The protocol outlined here details how to obtain and identify the nanocrystals, how to set up the microscope for screening and for MicroED data collection, and how to collect and process data to complete high-resolution structures. For well-behaving crystals with high-resolution diffraction in cryo-EM, the entire process can be achieved in less than an hour.
Martynowycz, Michael W; Gonen, Tamir
Studying Membrane Protein Structures by MicroED Journal Article
In: Methods Mol Biol, vol. 2302, pp. 137–151, 2021, ISSN: 1940-6029.
Abstract | Links | Altmetric | PlumX
@article{pmid33877626,
title = {Studying Membrane Protein Structures by MicroED},
author = {Michael W Martynowycz and Tamir Gonen},
doi = {10.1007/978-1-0716-1394-8_8},
issn = {1940-6029},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Methods Mol Biol},
volume = {2302},
pages = {137--151},
abstract = {Microcrystal electron diffraction (MicroED) enables atomic resolution structures to be determined from vanishingly small crystals. Soluble proteins typically grow crystals that are tens to hundreds of microns in size for X-ray crystallography. But membrane protein crystals often grow crystals that are too small for X-ray diffraction and yet too large for MicroED. These crystals are often formed in thick, viscous media that challenge traditional cryoEM grid preparation. Here, we describe two approaches for preparing membrane protein crystals for MicroED data collection: application of a crystal slurry directly to EM grids, and focused ion beam milling in a Scanning Electron Microscope (FIB-SEM). We summarize the case of preparing an ion channel, NaK, and the workflow of focused ion-beam milling. By milling away the excess media and crystalline material, crystals of any size may be prepared for MicroED. Finally, an energy filter may be used to help minimize inelastic scattering leading to lower noise on recorded images.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microcrystal electron diffraction (MicroED) enables atomic resolution structures to be determined from vanishingly small crystals. Soluble proteins typically grow crystals that are tens to hundreds of microns in size for X-ray crystallography. But membrane protein crystals often grow crystals that are too small for X-ray diffraction and yet too large for MicroED. These crystals are often formed in thick, viscous media that challenge traditional cryoEM grid preparation. Here, we describe two approaches for preparing membrane protein crystals for MicroED data collection: application of a crystal slurry directly to EM grids, and focused ion beam milling in a Scanning Electron Microscope (FIB-SEM). We summarize the case of preparing an ion channel, NaK, and the workflow of focused ion-beam milling. By milling away the excess media and crystalline material, crystals of any size may be prepared for MicroED. Finally, an energy filter may be used to help minimize inelastic scattering leading to lower noise on recorded images.
Mu, Xuelang; Gillman, Cody; Nguyen, Chi; Gonen, Tamir
An Overview of Microcrystal Electron Diffraction (MicroED) Journal Article
In: Annu Rev Biochem, vol. 90, pp. 431–450, 2021, ISSN: 1545-4509.
Abstract | Links | Altmetric | PlumX
@article{pmid34153215,
title = {An Overview of Microcrystal Electron Diffraction (MicroED)},
author = {Xuelang Mu and Cody Gillman and Chi Nguyen and Tamir Gonen},
doi = {10.1146/annurev-biochem-081720-020121},
issn = {1545-4509},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Annu Rev Biochem},
volume = {90},
pages = {431--450},
abstract = {The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds, peptides, and proteins. The development of new structure characterization tools, particularly those that fill critical gaps in existing methods, presents important steps forward for structural biology and drug discovery. The emergence of microcrystal electron diffraction (MicroED) expands the application of cryo-electron microscopy to include samples ranging from small molecules and membrane proteins to even large protein complexes using crystals that are one-billionth the size of those required for X-ray crystallography. This review outlines the conception, achievements, and exciting future trajectories for MicroED, an important addition to the existing biophysical toolkit.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds, peptides, and proteins. The development of new structure characterization tools, particularly those that fill critical gaps in existing methods, presents important steps forward for structural biology and drug discovery. The emergence of microcrystal electron diffraction (MicroED) expands the application of cryo-electron microscopy to include samples ranging from small molecules and membrane proteins to even large protein complexes using crystals that are one-billionth the size of those required for X-ray crystallography. This review outlines the conception, achievements, and exciting future trajectories for MicroED, an important addition to the existing biophysical toolkit.
2017
Liu, Shian; Hattne, Johan; Reyes, Francis E; Sanchez-Martinez, Silvia; de la Cruz, M Jason; Shi, Dan; Gonen, Tamir
Atomic resolution structure determination by the cryo-EM method MicroED Journal Article
In: Protein Sci, vol. 26, no. 1, pp. 8–15, 2017, ISSN: 1469-896X.
Abstract | Links | Altmetric | PlumX
@article{pmid27452773,
title = {Atomic resolution structure determination by the cryo-EM method MicroED},
author = {Shian Liu and Johan Hattne and Francis E Reyes and Silvia Sanchez-Martinez and M Jason de la Cruz and Dan Shi and Tamir Gonen},
doi = {10.1002/pro.2989},
issn = {1469-896X},
year = {2017},
date = {2017-01-01},
urldate = {2017-01-01},
journal = {Protein Sci},
volume = {26},
number = {1},
pages = {8--15},
abstract = {The electron cryo-microscopy (cryoEM) method MicroED has been rapidly developing. In this review we highlight some of the key steps in MicroED from crystal analysis to structure determination. We compare and contrast MicroED and the latest X-ray based diffraction method the X-ray free-electron laser (XFEL). Strengths and shortcomings of both MicroED and XFEL are discussed. Finally, all current MicroED structures are tabulated with a view to the future.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The electron cryo-microscopy (cryoEM) method MicroED has been rapidly developing. In this review we highlight some of the key steps in MicroED from crystal analysis to structure determination. We compare and contrast MicroED and the latest X-ray based diffraction method the X-ray free-electron laser (XFEL). Strengths and shortcomings of both MicroED and XFEL are discussed. Finally, all current MicroED structures are tabulated with a view to the future.
2016
Nannenga, Brent L; Gonen, Tamir
MicroED opens a new era for biological structure determination Journal Article
In: Curr Opin Struct Biol, vol. 40, pp. 128–135, 2016, ISSN: 1879-033X.
Abstract | Links | Altmetric | PlumX
@article{pmid27701014,
title = {MicroED opens a new era for biological structure determination},
author = {Brent L Nannenga and Tamir Gonen},
doi = {10.1016/j.sbi.2016.09.007},
issn = {1879-033X},
year = {2016},
date = {2016-01-01},
urldate = {2016-01-01},
journal = {Curr Opin Struct Biol},
volume = {40},
pages = {128--135},
abstract = {In 2013 we unveiled the cryo-electron microscopy (CryoEM) method of MicroED, or three-dimensional (3D) electron diffraction of microscopic crystals. Here tiny 3D crystals of biological material are used in an electron microscope for diffraction data collection under cryogenic conditions. The data is indexed, integrated, merged and scaled using standard X-ray crystallography software to determine structures at atomic resolution. In this review we provide an overview of the MicroED method and compare it with other CryoEM methods.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In 2013 we unveiled the cryo-electron microscopy (CryoEM) method of MicroED, or three-dimensional (3D) electron diffraction of microscopic crystals. Here tiny 3D crystals of biological material are used in an electron microscope for diffraction data collection under cryogenic conditions. The data is indexed, integrated, merged and scaled using standard X-ray crystallography software to determine structures at atomic resolution. In this review we provide an overview of the MicroED method and compare it with other CryoEM methods.
2014
Nannenga, Brent L; Shi, Dan; Leslie, Andrew G W; Gonen, Tamir
High-resolution structure determination by continuous-rotation data collection in MicroED Journal Article
In: Nat Methods, vol. 11, no. 9, pp. 927–930, 2014, ISSN: 1548-7105.
Abstract | Links | Altmetric | PlumX
@article{pmid25086503,
title = {High-resolution structure determination by continuous-rotation data collection in MicroED},
author = {Brent L Nannenga and Dan Shi and Andrew G W Leslie and Tamir Gonen},
doi = {10.1038/nmeth.3043},
issn = {1548-7105},
year = {2014},
date = {2014-09-01},
urldate = {2014-09-01},
journal = {Nat Methods},
volume = {11},
number = {9},
pages = {927--930},
abstract = {MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology. },
keywords = {},
pubstate = {published},
tppubtype = {article}
}
MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology.
Protocols
2021
Martynowycz, Michael W; Gonen, Tamir
Protocol for the use of focused ion-beam milling to prepare crystalline lamellae for microcrystal electron diffraction (MicroED) Journal Article
In: STAR Protoc, vol. 2, no. 3, pp. 100686, 2021, ISSN: 2666-1667.
Abstract | Links | Altmetric | PlumX
@article{pmid34382014,
title = {Protocol for the use of focused ion-beam milling to prepare crystalline lamellae for microcrystal electron diffraction (MicroED)},
author = {Michael W Martynowycz and Tamir Gonen},
doi = {10.1016/j.xpro.2021.100686},
issn = {2666-1667},
year = {2021},
date = {2021-09-01},
urldate = {2021-09-01},
journal = {STAR Protoc},
volume = {2},
number = {3},
pages = {100686},
abstract = {We present an in-depth protocol to reproducibly prepare crystalline lamellae from protein crystals for subsequent microcrystal electron diffraction (MicroED) experiments. This protocol covers typical soluble proteins and membrane proteins embedded in dense media. Following these steps will allow the user to prepare crystalline lamellae for protein structure determination by MicroED. For complete details on the use and execution of this protocol, please refer to Martynowycz et al. (2019a, 2020a).},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We present an in-depth protocol to reproducibly prepare crystalline lamellae from protein crystals for subsequent microcrystal electron diffraction (MicroED) experiments. This protocol covers typical soluble proteins and membrane proteins embedded in dense media. Following these steps will allow the user to prepare crystalline lamellae for protein structure determination by MicroED. For complete details on the use and execution of this protocol, please refer to Martynowycz et al. (2019a, 2020a).
2017
de la Cruz, M Jason; Hattne, Johan; Shi, Dan; Seidler, Paul; Rodriguez, Jose; Reyes, Francis E; Sawaya, Michael R; Cascio, Duilio; Weiss, Simon C; Kim, Sun Kyung; Hinck, Cynthia S; Hinck, Andrew P; Calero, Guillermo; Eisenberg, David; Gonen, Tamir
Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED Journal Article
In: Nat Methods, vol. 14, no. 4, pp. 399–402, 2017, ISSN: 1548-7105.
Abstract | Links | Altmetric | PlumX
@article{pmid28192420,
title = {Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED},
author = {M Jason de la Cruz and Johan Hattne and Dan Shi and Paul Seidler and Jose Rodriguez and Francis E Reyes and Michael R Sawaya and Duilio Cascio and Simon C Weiss and Sun Kyung Kim and Cynthia S Hinck and Andrew P Hinck and Guillermo Calero and David Eisenberg and Tamir Gonen},
doi = {10.1038/nmeth.4178},
issn = {1548-7105},
year = {2017},
date = {2017-02-01},
journal = {Nat Methods},
volume = {14},
number = {4},
pages = {399--402},
abstract = {Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.
2016
Shi, Dan; Nannenga, Brent L; de la Cruz, M Jason; Liu, Jinyang; Sawtelle, Steven; Calero, Guillermo; Reyes, Francis E; Hattne, Johan; Gonen, Tamir
The collection of MicroED data for macromolecular crystallography Journal Article
In: Nat Protoc, vol. 11, no. 5, pp. 895–904, 2016, ISSN: 1750-2799.
Abstract | Links | Altmetric | PlumX
@article{pmid27077331,
title = {The collection of MicroED data for macromolecular crystallography},
author = {Dan Shi and Brent L Nannenga and M Jason de la Cruz and Jinyang Liu and Steven Sawtelle and Guillermo Calero and Francis E Reyes and Johan Hattne and Tamir Gonen},
doi = {10.1038/nprot.2016.046},
issn = {1750-2799},
year = {2016},
date = {2016-05-01},
journal = {Nat Protoc},
volume = {11},
number = {5},
pages = {895--904},
abstract = {The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals. },
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.