skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

Abstract

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.

Authors:
ORCiD logo; ; ; ; ; ORCiD logo; ; ORCiD logo; ; ; ; ; ORCiD logo; ORCiD logo; ORCiD logo; ; ; ; ; more »; ; ; ORCiD logo; ; ; ORCiD logo; ; ORCiD logo; ; ORCiD logo; ; ORCiD logo; ORCiD logo; ; ; ; ORCiD logo; ORCiD logo « less
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities Division; National Science Foundation (NSF); National Institutes of Health (NIH)
OSTI Identifier:
1617957
Alternate Identifier(s):
OSTI ID: 1615313; OSTI ID: 1617040
Grant/Contract Number:  
AC02-05CH11231; AC02-76SF00515; STC-1231306; F32 HL129989; GM123159; GM124149; GM117126; LFR-17-476732
Resource Type:
Published Article
Journal Name:
IUCrJ
Additional Journal Information:
Journal Name: IUCrJ Journal Volume: 7 Journal Issue: 2; Journal ID: ISSN 2052-2525
Publisher:
International Union of Crystallography
Country of Publication:
United Kingdom
Language:
English
Subject:
36 MATERIALS SCIENCE; 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; microcrystals; batch crystallization; serial crystallography; MicroED

Citation Formats

Wolff, Alexander M., Young, Iris D., Sierra, Raymond G., Brewster, Aaron S., Martynowycz, Michael W., Nango, Eriko, Sugahara, Michihiro, Nakane, Takanori, Ito, Kazutaka, Aquila, Andrew, Bhowmick, Asmit, Biel, Justin T., Carbajo, Sergio, Cohen, Aina E., Cortez, Saul, Gonzalez, Ana, Hino, Tomoya, Im, Dohyun, Koralek, Jake D., Kubo, Minoru, Lazarou, Tomas S., Nomura, Takashi, Owada, Shigeki, Samelson, Avi J., Tanaka, Tomoyuki, Tanaka, Rie, Thompson, Erin M., van den Bedem, Henry, Woldeyes, Rahel A., Yumoto, Fumiaki, Zhao, Wei, Tono, Kensuke, Boutet, Sebastien, Iwata, So, Gonen, Tamir, Sauter, Nicholas K., Fraser, James S., and Thompson, Michael C. Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals. United Kingdom: N. p., 2020. Web. doi:10.1107/S205225252000072X.
Wolff, Alexander M., Young, Iris D., Sierra, Raymond G., Brewster, Aaron S., Martynowycz, Michael W., Nango, Eriko, Sugahara, Michihiro, Nakane, Takanori, Ito, Kazutaka, Aquila, Andrew, Bhowmick, Asmit, Biel, Justin T., Carbajo, Sergio, Cohen, Aina E., Cortez, Saul, Gonzalez, Ana, Hino, Tomoya, Im, Dohyun, Koralek, Jake D., Kubo, Minoru, Lazarou, Tomas S., Nomura, Takashi, Owada, Shigeki, Samelson, Avi J., Tanaka, Tomoyuki, Tanaka, Rie, Thompson, Erin M., van den Bedem, Henry, Woldeyes, Rahel A., Yumoto, Fumiaki, Zhao, Wei, Tono, Kensuke, Boutet, Sebastien, Iwata, So, Gonen, Tamir, Sauter, Nicholas K., Fraser, James S., & Thompson, Michael C. Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals. United Kingdom. doi:https://doi.org/10.1107/S205225252000072X
Wolff, Alexander M., Young, Iris D., Sierra, Raymond G., Brewster, Aaron S., Martynowycz, Michael W., Nango, Eriko, Sugahara, Michihiro, Nakane, Takanori, Ito, Kazutaka, Aquila, Andrew, Bhowmick, Asmit, Biel, Justin T., Carbajo, Sergio, Cohen, Aina E., Cortez, Saul, Gonzalez, Ana, Hino, Tomoya, Im, Dohyun, Koralek, Jake D., Kubo, Minoru, Lazarou, Tomas S., Nomura, Takashi, Owada, Shigeki, Samelson, Avi J., Tanaka, Tomoyuki, Tanaka, Rie, Thompson, Erin M., van den Bedem, Henry, Woldeyes, Rahel A., Yumoto, Fumiaki, Zhao, Wei, Tono, Kensuke, Boutet, Sebastien, Iwata, So, Gonen, Tamir, Sauter, Nicholas K., Fraser, James S., and Thompson, Michael C. Wed . "Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals". United Kingdom. doi:https://doi.org/10.1107/S205225252000072X.
@article{osti_1617957,
title = {Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals},
author = {Wolff, Alexander M. and Young, Iris D. and Sierra, Raymond G. and Brewster, Aaron S. and Martynowycz, Michael W. and Nango, Eriko and Sugahara, Michihiro and Nakane, Takanori and Ito, Kazutaka and Aquila, Andrew and Bhowmick, Asmit and Biel, Justin T. and Carbajo, Sergio and Cohen, Aina E. and Cortez, Saul and Gonzalez, Ana and Hino, Tomoya and Im, Dohyun and Koralek, Jake D. and Kubo, Minoru and Lazarou, Tomas S. and Nomura, Takashi and Owada, Shigeki and Samelson, Avi J. and Tanaka, Tomoyuki and Tanaka, Rie and Thompson, Erin M. and van den Bedem, Henry and Woldeyes, Rahel A. and Yumoto, Fumiaki and Zhao, Wei and Tono, Kensuke and Boutet, Sebastien and Iwata, So and Gonen, Tamir and Sauter, Nicholas K. and Fraser, James S. and Thompson, Michael C.},
abstractNote = {Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.},
doi = {10.1107/S205225252000072X},
journal = {IUCrJ},
number = 2,
volume = 7,
place = {United Kingdom},
year = {2020},
month = {2}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: https://doi.org/10.1107/S205225252000072X

Citation Metrics:
Cited by: 1 work
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Hidden alternative structures of proline isomerase essential for catalysis
journal, December 2009

  • Fraser, James S.; Clarkson, Michael W.; Degnan, Sheena C.
  • Nature, Vol. 462, Issue 7273
  • DOI: 10.1038/nature08615

Biomolecular cryocrystallography: Structural changes during flash-cooling
journal, March 2004


Cheetah : software for high-throughput reduction and analysis of serial femtosecond X-ray diffraction data
journal, May 2014

  • Barty, Anton; Kirian, Richard A.; Maia, Filipe R. N. C.
  • Journal of Applied Crystallography, Vol. 47, Issue 3
  • DOI: 10.1107/S1600576714007626

Electron diffraction and three-dimensional crystallography for structural biology
journal, January 2018


Development of an X-ray pixel detector with multi-port charge-coupled device for X-ray free-electron laser experiments
journal, March 2014

  • Kameshima, Takashi; Ono, Shun; Kudo, Togo
  • Review of Scientific Instruments, Vol. 85, Issue 3
  • DOI: 10.1063/1.4867668

Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography
journal, August 2015

  • Mueller, C.; Marx, A.; Epp, S. W.
  • Structural Dynamics, Vol. 2, Issue 5
  • DOI: 10.1063/1.4928706

Overview of the CCP 4 suite and current developments
journal, March 2011

  • Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
  • DOI: 10.1107/S0907444910045749

Grease matrix as a versatile carrier of proteins for serial crystallography
journal, November 2014

  • Sugahara, Michihiro; Mizohata, Eiichi; Nango, Eriko
  • Nature Methods, Vol. 12, Issue 1
  • DOI: 10.1038/nmeth.3172

MeshAndCollect : an automated multi-crystal data-collection workflow for synchrotron macromolecular crystallography beamlines
journal, October 2015

  • Zander, Ulrich; Bourenkov, Gleb; Popov, Alexander N.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 71, Issue 11
  • DOI: 10.1107/S1399004715017927

Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED
journal, February 2017

  • de la Cruz, M. Jason; Hattne, Johan; Shi, Dan
  • Nature Methods, Vol. 14, Issue 4
  • DOI: 10.1038/nmeth.4178

DIALS : implementation and evaluation of a new integration package
journal, February 2018

  • Winter, Graeme; Waterman, David G.; Parkhurst, James M.
  • Acta Crystallographica Section D Structural Biology, Vol. 74, Issue 2
  • DOI: 10.1107/S2059798317017235

ImageJ2: ImageJ for the next generation of scientific image data
journal, November 2017

  • Rueden, Curtis T.; Schindelin, Johannes; Hiner, Mark C.
  • BMC Bioinformatics, Vol. 18, Issue 1
  • DOI: 10.1186/s12859-017-1934-z

Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation
journal, May 2017

  • Martin-Garcia, Jose M.; Conrad, Chelsie E.; Nelson, Garrett
  • IUCrJ, Vol. 4, Issue 4, p. 439-454
  • DOI: 10.1107/S205225251700570X

Femtosecond X-ray protein nanocrystallography
journal, February 2011

  • Chapman, Henry N.; Fromme, Petra; Barty, Anton
  • Nature, Vol. 470, Issue 7332, p. 73-77
  • DOI: 10.1038/nature09750

Electric-field-stimulated protein mechanics
journal, December 2016

  • Hekstra, Doeke R.; White, K. Ian; Socolich, Michael A.
  • Nature, Vol. 540, Issue 7633
  • DOI: 10.1038/nature20571

Towards automated crystallographic structure refinement with phenix.refine
journal, March 2012

  • Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel
  • Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 4
  • DOI: 10.1107/S0907444912001308

Nanosecond pump–probe device for time-resolved serial femtosecond crystallography developed at SACLA
journal, August 2017

  • Kubo, Minoru; Nango, Eriko; Tono, Kensuke
  • Journal of Synchrotron Radiation, Vol. 24, Issue 5, p. 1086-1091
  • DOI: 10.1107/S160057751701030X

Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix
journal, October 2019

  • Liebschner, Dorothee; Afonine, Pavel V.; Baker, Matthew L.
  • Acta Crystallographica Section D Structural Biology, Vol. 75, Issue 10
  • DOI: 10.1107/S2059798319011471

Intrinsic dynamics of an enzyme underlies catalysis
journal, November 2005

  • Eisenmesser, Elan Z.; Millet, Oscar; Labeikovsky, Wladimir
  • Nature, Vol. 438, Issue 7064
  • DOI: 10.1038/nature04105

Electron diffraction data processing with DIALS
journal, May 2018

  • Clabbers, Max T. B.; Gruene, Tim; Parkhurst, James M.
  • Acta Crystallographica Section D Structural Biology, Vol. 74, Issue 6
  • DOI: 10.1107/S2059798318007726

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers
journal, February 2017

  • Fuller, Franklin D.; Gul, Sheraz; Chatterjee, Ruchira
  • Nature Methods, Vol. 14, Issue 4
  • DOI: 10.1038/nmeth.4195

Diverse application platform for hard X-ray diffraction in SACLA (DAPHNIS): application to serial protein crystallography using an X-ray free-electron laser
journal, April 2015

  • Tono, Kensuke; Nango, Eriko; Sugahara, Michihiro
  • Journal of Synchrotron Radiation, Vol. 22, Issue 3
  • DOI: 10.1107/S1600577515004464

Diverse HIV viruses are targeted by a conformationally dynamic antiviral
journal, March 2012

  • Caines, Matthew E. C.; Bichel, Katsiaryna; Price, Amanda J.
  • Nature Structural & Molecular Biology, Vol. 19, Issue 4
  • DOI: 10.1038/nsmb.2253

Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography
journal, September 2017

  • Coquelle, Nicolas; Sliwa, Michel; Woodhouse, Joyce
  • Nature Chemistry, Vol. 10, Issue 1
  • DOI: 10.1038/nchem.2853

Modelling dynamics in protein crystal structures by ensemble refinement
journal, December 2012


XDS
journal, January 2010

  • Kabsch, Wolfgang
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2
  • DOI: 10.1107/S0907444909047337

Indexing in single-crystal diffractometry with an obstinate list of reflections
journal, April 1992


X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex
journal, April 2016


Serial Femtosecond Crystallography of G Protein-Coupled Receptors
journal, December 2013


Recent developments in CrystFEL
journal, March 2016

  • White, Thomas A.; Mariani, Valerio; Brehm, Wolfgang
  • Journal of Applied Crystallography, Vol. 49, Issue 2
  • DOI: 10.1107/S1600576716004751

Hydroxyethyl cellulose matrix applied to serial crystallography
journal, April 2017

  • Sugahara, Michihiro; Nakane, Takanori; Masuda, Tetsuya
  • Scientific Reports, Vol. 7, Article No. 703
  • DOI: 10.1038/s41598-017-00761-0

Data processing pipeline for serial femtosecond crystallography at SACLA
journal, April 2016

  • Nakane, Takanori; Joti, Yasumasa; Tono, Kensuke
  • Journal of Applied Crystallography, Vol. 49, Issue 3
  • DOI: 10.1107/S1600576716005720

Structure of photosystem II and substrate binding at room temperature
journal, November 2016

  • Young, Iris D.; Ibrahim, Mohamed; Chatterjee, Ruchira
  • Nature, Vol. 540, Issue 7633
  • DOI: 10.1038/nature20161

A compact X-ray free-electron laser emitting in the sub-ångström region
journal, June 2012


The Macromolecular Femtosecond Crystallography Instrument at the Linac Coherent Light Source
journal, February 2019

  • Sierra, Raymond G.; Batyuk, Alexander; Sun, Zhibin
  • Journal of Synchrotron Radiation, Vol. 26, Issue 2
  • DOI: 10.1107/S1600577519001577

Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography
journal, September 2015

  • Keedy, Daniel A.; Kenner, Lillian R.; Warkentin, Matthew
  • eLife, Vol. 4
  • DOI: 10.7554/eLife.07574

Small is beautiful: protein micro-crystallography
journal, August 1998

  • Cusack, Stephen; Belrhali, Hassan; Bram, Andreas
  • Nature Structural Biology, Vol. 5, Issue 8
  • DOI: 10.1038/1325

Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size
journal, March 2019


Structure of the toxic core of α-synuclein from invisible crystals
journal, September 2015

  • Rodriguez, Jose A.; Ivanova, Magdalena I.; Sawaya, Michael R.
  • Nature, Vol. 525, Issue 7570
  • DOI: 10.1038/nature15368

Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography
journal, February 2014

  • Weierstall, Uwe; James, Daniel; Wang, Chong
  • Nature Communications, Vol. 5, Issue 1
  • DOI: 10.1038/ncomms4309

Multiple-beamline operation of SACLA
journal, February 2019

  • Tono, Kensuke; Hara, Toru; Yabashi, Makina
  • Journal of Synchrotron Radiation, Vol. 26, Issue 2
  • DOI: 10.1107/S1600577519001607

Fixed-target protein serial microcrystallography with an x-ray free electron laser
journal, August 2014

  • Hunter, Mark S.; Segelke, Brent; Messerschmidt, Marc
  • Scientific Reports, Vol. 4, Issue 1
  • DOI: 10.1038/srep06026

Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II
journal, November 2015

  • Sierra, Raymond G.; Gati, Cornelius; Laksmono, Hartawan
  • Nature Methods, Vol. 13, Issue 1
  • DOI: 10.1038/nmeth.3667

Features and development of Coot
journal, March 2010

  • Emsley, P.; Lohkamp, B.; Scott, W. G.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444910007493

Modeling truncated pixel values of faint reflections in MicroED images
journal, May 2016

  • Hattne, Johan; Shi, Dan; de la Cruz, M. Jason
  • Journal of Applied Crystallography, Vol. 49, Issue 3
  • DOI: 10.1107/S1600576716007196

REFMAC 5 for the refinement of macromolecular crystal structures
journal, March 2011

  • Murshudov, Garib N.; Skubák, Pavol; Lebedev, Andrey A.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
  • DOI: 10.1107/S0907444911001314

Estimates of internal stress and related mosaicity in solution grown crystals: proteins
journal, January 1999


High-density grids for efficient data collection from multiple crystals
journal, January 2016

  • Baxter, Elizabeth L.; Aguila, Laura; Alonso-Mori, Roberto
  • Acta Crystallographica Section D Structural Biology, Vol. 72, Issue 1, p. 2-11
  • DOI: 10.1107/S2059798315020847

Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL
journal, February 2017

  • Suga, Michihiro; Akita, Fusamichi; Sugahara, Michihiro
  • Nature, Vol. 543, Issue 7643
  • DOI: 10.1038/nature21400

Machining protein microcrystals for structure determination by electron diffraction
journal, August 2018

  • Duyvesteyn, Helen M. E.; Kotecha, Abhay; Ginn, Helen M.
  • Proceedings of the National Academy of Sciences, Vol. 115, Issue 38
  • DOI: 10.1073/pnas.1809978115

Improving signal strength in serial crystallography with DIALS geometry refinement
journal, September 2018

  • Brewster, Aaron S.; Waterman, David G.; Parkhurst, James M.
  • Acta Crystallographica Section D Structural Biology, Vol. 74, Issue 9
  • DOI: 10.1107/S2059798318009191

An introduction to data reduction: space-group determination, scaling and intensity statistics
journal, March 2011

  • Evans, Philip R.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
  • DOI: 10.1107/S090744491003982X

XFEL diffraction: developing processing methods to optimize data quality
journal, January 2015


High-resolution structure determination by continuous-rotation data collection in MicroED
journal, August 2014

  • Nannenga, Brent L.; Shi, Dan; Leslie, Andrew G. W.
  • Nature Methods, Vol. 11, Issue 9
  • DOI: 10.1038/nmeth.3043

The complementarity of serial femtosecond crystallography and MicroED for structure determination from microcrystals
journal, October 2019

  • Zatsepin, Nadia A.; Li, Chufeng; Colasurd, Paige
  • Current Opinion in Structural Biology, Vol. 58
  • DOI: 10.1016/j.sbi.2019.06.004

Improvements in serial femtosecond crystallography of photosystem II by optimizing crystal uniformity using microseeding procedures
journal, July 2015

  • Ibrahim, Mohamed; Chatterjee, Ruchira; Hellmich, Julia
  • Structural Dynamics, Vol. 2, Issue 4
  • DOI: 10.1063/1.4919741

A three-dimensional movie of structural changes in bacteriorhodopsin
journal, December 2016

  • Nango, Eriko; Royant, Antoine; Kubo, Minoru
  • Science, Vol. 354, Issue 6319, p. 1552-1557
  • DOI: 10.1126/science.aah3497

Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED
journal, October 2019


Real-Time Observation of Protein Dense Liquid Cluster Evolution during Nucleation in Protein Crystallization
journal, January 2017

  • Schubert, Robin; Meyer, Arne; Baitan, Daniela
  • Crystal Growth & Design, Vol. 17, Issue 3
  • DOI: 10.1021/acs.cgd.6b01826

Three-dimensional electron crystallography of protein microcrystals
journal, November 2013


Phaser crystallographic software
journal, July 2007

  • McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
  • Journal of Applied Crystallography, Vol. 40, Issue 4
  • DOI: 10.1107/S0021889807021206

The cryo-EM method microcrystal electron diffraction (MicroED)
journal, April 2019


Nanoflow electrospinning serial femtosecond crystallography
journal, October 2012

  • Sierra, Raymond G.; Laksmono, Hartawan; Kern, Jan
  • Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 11, p. 1584-1587
  • DOI: 10.1107/S0907444912038152