Use of transmission electron microscopy to identify nanocrystals of challenging protein targets

The current practice for identifying crystal hits for X-ray crystallography relies on optical microscopy techniques that are limited to detecting crystals no smaller than 5 μm. Because of these limitations, nanometer-sized protein crystals cannot be distinguished from common amorphous precipitates,...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2014-06, Vol.111 (23), p.8470-8475
Main Authors: Stevenson, Hilary P., Makhov, Alexander M., Calero, Monica, Edwards, Andrea L., Zeldin, Oliver B., Mathews, Irimpan I., Lin, Guowu, Barnes, Christopher O., Santamaria, Hugo, Ross, Ted M., Soltis, S. Michael, Khosla, Chaitan, Nagarajan, V., Conway, James F., Cohen, Aina E., Calero, Guillermo
Format: Article
Language:English
Subjects:
Animals
Biological Sciences
Crystal lattices
Crystal structure
Crystallization
Crystallography
Crystallography, X-Ray
Crystals
Diffraction
Escherichia coli - genetics
Fluorescence
Imaging
Microscopy
Microscopy, Electron, Transmission - methods
Nanocrystals
Nanoparticles - chemistry
Nanoparticles - ultrastructure
Proteins
Proteins - chemistry
Proteins - genetics
Proteins - ultrastructure
Recombinant Proteins - chemistry
Recombinant Proteins - ultrastructure
Reproducibility of Results
Sf9 Cells
Transmission electron microscopy
Wave diffraction
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Summary:The current practice for identifying crystal hits for X-ray crystallography relies on optical microscopy techniques that are limited to detecting crystals no smaller than 5 μm. Because of these limitations, nanometer-sized protein crystals cannot be distinguished from common amorphous precipitates, and therefore go unnoticed during screening. These crystals would be ideal candidates for further optimization or for femtosecond X-ray protein nanocrystallography. The latter technique offers the possibility to solve high-resolution structures using submicron crystals. Transmission electron microscopy (TEM) was used to visualize nanocrystals (NCs) found in crystallization drops that would classically not be considered as “hits.” We found that protein NCs were readily detected in all samples tested, including multiprotein complexes and membrane proteins. NC quality was evaluated by TEM visualization of lattices, and diffraction quality was validated by experiments in an X-ray free electron laser.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1400240111