MicroED – Three dimensional electron crystallography
We demonstrated 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 1.7Å 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 2.9Å resolution (Figure 9). 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.
In 2014 we further inmproved the MicroED method. Firstly, we developed an improved data collection protocol for MicroED called Continuous rotation. Microcrystals are continuously rotated during data collection yielding improved data, and allowing data processing with the crystallographic software tool MOSFLM, resulting in improved resolution for the model protein lysozyme to 2.5Å resolution. These improvements pave the way for the broad implementation and application of MicroED in structural biology. Current efforts include new phasing methods, automation and program development.
Secondly, we used the improved MicroED protocols for data collection and analysis to determine the structure of catalase. Bovine liver catalase crystals that were only ~160nm thick were used for the structure analysis. A single crystal yielded data to 3.2Å resolution enabling structure determination rapidly.
In 2015 we published the first two previously unknown structures determined by MicroED. The structures of two peptides from the toxic core of a-synuclein of Parkinsons’ Disease. The structures were determined from vanishingly small crystals, only ~200nm thick and wide, and yielded 1.4Å resolution. These structures, which are currently the highest resolution structures determined to date by any cryo EM method, show new and important structural information that could aid in the development of pharmaceuticals against this devastating neurological disease. The study, which was published by Nature also show a number of protons for the very first time.
Raw datasets available for download:
Lysozyme: DOI 10.15785/SBGRID/185 Download here.
Catalase: DOI 10.15785/SBGRID/186 Download here.
a-synuclein G11A: DOI 10.15785/SBGRID/193 Download here.
Stage rotation controller developed at Janelia. Full description can be found here.
All software that we developed for MicroED data processing can be found here.
1. Wisedchaisri G and Gonen T*. (2011) Fragment based phase extension for membrane protein
structure determination by electron crystallography. Structure 19: 976 - 987.
2. Shi D., Nannenga B., Iadanza MG. and Gonen T* (2013). MicroED – Three dimensional electron
crystallography of protein microcrystals. eLife – 2:e01345: 1 - 17.
3. Iadanza MG. and Gonen T*. A suite of software for processing MicroED data of extremely small protein
crystals. Journal of Applied Crystallography. 47: 1140 – 1145.
4.Nannenga BL, Shi D., Leslie AGW. and Gonen T* (2014). High-resolution structure determination by
continuous rotation data collection in MicroED. Nature Methods 11 (9): 927 – 930.
5. Nannenga BL, Shi D., Hattne J., Reyes F. and Gonen T* (2014). Structure of catalase determined by
MicroED. eLife 3:e03600: 1 – 11.
6. Hattne J., Reyes FE., Nannenga BL., Shi D., de la Cruz J., Leslie AGW. And Gonen T*. MicroED data
collection and processing. Acta Crystallographica section A. A71: 353 - 360 (2015).
7. Rodriguez A.J., Ivanova M., Sawaya MR., Cascio D., Reyes F., Shi D., Sangwan S., Guenther EL.,
Johnson L, Zhang M., Jiang L., Arbing M., Nannega B., Hattne J., Whitelegge J., Brewster AS.,
Messerschmidt M., Boutet S., Sauter NK., Gonen T* and Eisenberg D* Structure of the toxic core
of a-synuclein from invisible crystals. Nature 525 (7570): 486 - 490 (2015).
8. Hattne J., Shi D., de la Cruz J., Reyes FE., and Gonen T*. Modeling truncated intensities of faint
reflections in MicroED images. Journal of Applied Crystallography - In Press (2016).
9. Shi D., Nannenga B., de la Cruz J., Jiu L., Guillermo C., Hattne J., Reyes FE., Sawtelle S. and
Gonen T*. The collection of MicroED data for macromolecular crystallography. Nature Protocols 11 (5) : 895 - 904 (2016).
Relevant Reviews and Book Chapters:
1. Wisedchaisri W., Reichow S.L. and Gonen T*. (2011) Advances in structural and functional analysis
of membrane proteins by electron crystallography. Structure. 19:1381-93.
2. Wisedchaisri W. and Gonen T*. (2013) Phasing Electron Diffraction Data by Molecular Replacement:
Strategy for Structure Determination and Refinement. Methods in Molecular Biology 955: 243 – 272.
3. Gonen T*. (2013) The collection of high-resolution electron diffraction data. Methods in Molecular
Biology 955: 153 – 169.
4. Stokes D, Ubarretxena I, Gonen T and Engel A. (2013) High throughout methods in electron
crystallography. Methods in Molecular Biology 955: 273 – 296.
5. Nannenga B, Iadanza M, Vollmar B and Gonen T*. (2013) Electron crystallography of membrane
proteins: crystallization and screening strategies using negative stain electron microscopy.
Current Protocols in Protein Science – 17 (15): 1-11.
6. Nannenga BL. and Gonen T*. Protein structure determination by MicroED. Current Opinion in
Structural Biology. 27: 24 - 31 (2014).
Updated May 15 2016 © Tamir Gonen