Deciding how to optimally distribute electrons across the sample during data collection requires a good understanding of the
sample’s susceptibility to damage and the camera’s response to
electrons. Ideally, this information can be obtained under minimal
exposure of the sample to the beam. Even then, all strategies reflect
trade-offs because information in MicroED can only be gained by
exposing a crystal to electrons which invariably will deposit some of
their energy to the sample and damage it.
In practice, a certain amount of radiation damage will have to
be accepted, the merged data will exhibit some degree of incompleteness, and the highest-resolution observations will not necessarily contribute to a better atomic interpretation of the data
[43]. The data may contain over, as well as underexposed reflections, as it is generally impossible to optimize the exposure such
that all reciprocal space is measured at its most suitable exposure.
Instead of the perfect dataset, the experimenter is forced to seek the
optimal compromise.
Acknowledgments
Gerd Rosenbaum (Advanced Photon Source, Argonne, IL) is
acknowledged for helpful discussions on dose in MicroED. Michael
Martynowycz (UCLA, Los Angeles, CA) critically read the manuscript and provided insightful comments.
References
1. Shi D, Nannenga BL, Iadanza MG, Gonen T
(2013) Three-dimensional electron crystallography of protein microcrystals. Elife [Internet]
2:e01345.
http://www.ncbi.nlm.nih.gov/
pubmed/24252878
2. Nannenga BL, Gonen T (2019) The cryo-EM
method microcrystal electron diffraction
(MicroED). Nat Methods [Internet] 16
(5):369–379.
http://www.nature.com/arti
cles/s41592-019-0395-x
3. Martynowycz MW, Gonen T (2018) From
electron crystallography of 2D crystals to
MicroED of 3D crystals. Curr Opin Colloid
Interface Sci [Internet] 34:9–16. https://
linkinghub.elsevier.com/retrieve/pii/
S1359029417301589
4. Baker LA, Rubinstein JL (2010) Radiation
damage in electron cryomicroscopy. In: Methods in enzymology [Internet]. Elsevier, Masson SAS, pp 371–388. https://linkinghub.
elsevier.com/retrieve/pii/
S0076687910810158
5. Henderson R (1995) The potential and limitations of neutrons, electrons and X-rays for
atomic resolution microscopy of unstained
biological molecules. Q Rev Biophys [Internet]
28(2):171–193. https://www.cambridge.org/
core/product/identifier/
S003358350000305X/type/journal_article
6. Langmore JP, Smith MF (1992) Quantitative
energy-filtered electron microscopy of
biological molecules in ice. Ultramicroscopy
[Internet]
46(1–4):349–373.
https://
linkinghub.elsevier.com/retrieve/pii/
030439919290024E
7. Jones GDDD, Lea JS, Symons MCRR, Taiwo
FA (1987) Structure and mobility of electron
gain and loss centres in proteins. Nature [Internet] 330(6150):772–773. https://doi.org/
10.1038/330772a0
8. Garman EF (2010) Radiation damage in macromolecular crystallography: what is it and why
should we care? Acta Crystallogr Sect D Biol
Crystallogr [Internet] 66(4):339–351. http://
scripts.iucr.org/cgi-bin/paper?
S0907444910008656
Low-Dose MicroED
317
sample’s susceptibility to damage and the camera’s response to
electrons. Ideally, this information can be obtained under minimal
exposure of the sample to the beam. Even then, all strategies reflect
trade-offs because information in MicroED can only be gained by
exposing a crystal to electrons which invariably will deposit some of
their energy to the sample and damage it.
In practice, a certain amount of radiation damage will have to
be accepted, the merged data will exhibit some degree of incompleteness, and the highest-resolution observations will not necessarily contribute to a better atomic interpretation of the data
[43]. The data may contain over, as well as underexposed reflections, as it is generally impossible to optimize the exposure such
that all reciprocal space is measured at its most suitable exposure.
Instead of the perfect dataset, the experimenter is forced to seek the
optimal compromise.
Acknowledgments
Gerd Rosenbaum (Advanced Photon Source, Argonne, IL) is
acknowledged for helpful discussions on dose in MicroED. Michael
Martynowycz (UCLA, Los Angeles, CA) critically read the manuscript and provided insightful comments.
References
1. Shi D, Nannenga BL, Iadanza MG, Gonen T
(2013) Three-dimensional electron crystallography of protein microcrystals. Elife [Internet]
2:e01345.
http://www.ncbi.nlm.nih.gov/
pubmed/24252878
2. Nannenga BL, Gonen T (2019) The cryo-EM
method microcrystal electron diffraction
(MicroED). Nat Methods [Internet] 16
(5):369–379.
http://www.nature.com/arti
cles/s41592-019-0395-x
3. Martynowycz MW, Gonen T (2018) From
electron crystallography of 2D crystals to
MicroED of 3D crystals. Curr Opin Colloid
Interface Sci [Internet] 34:9–16. https://
linkinghub.elsevier.com/retrieve/pii/
S1359029417301589
4. Baker LA, Rubinstein JL (2010) Radiation
damage in electron cryomicroscopy. In: Methods in enzymology [Internet]. Elsevier, Masson SAS, pp 371–388. https://linkinghub.
elsevier.com/retrieve/pii/
S0076687910810158
5. Henderson R (1995) The potential and limitations of neutrons, electrons and X-rays for
atomic resolution microscopy of unstained
biological molecules. Q Rev Biophys [Internet]
28(2):171–193. https://www.cambridge.org/
core/product/identifier/
S003358350000305X/type/journal_article
6. Langmore JP, Smith MF (1992) Quantitative
energy-filtered electron microscopy of
biological molecules in ice. Ultramicroscopy
[Internet]
46(1–4):349–373.
https://
linkinghub.elsevier.com/retrieve/pii/
030439919290024E
7. Jones GDDD, Lea JS, Symons MCRR, Taiwo
FA (1987) Structure and mobility of electron
gain and loss centres in proteins. Nature [Internet] 330(6150):772–773. https://doi.org/
10.1038/330772a0
8. Garman EF (2010) Radiation damage in macromolecular crystallography: what is it and why
should we care? Acta Crystallogr Sect D Biol
Crystallogr [Internet] 66(4):339–351. http://
scripts.iucr.org/cgi-bin/paper?
S0907444910008656
Low-Dose MicroED
317