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


ABSTRACT: 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 ?m. 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.

SUBMITTER: Stevenson HP 

PROVIDER: S-EPMC4060711 | biostudies-other | 2014 Jun

REPOSITORIES: biostudies-other

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

Stevenson Hilary P HP   Makhov Alexander M AM   Calero Monica M   Edwards Andrea L AL   Zeldin Oliver B OB   Mathews Irimpan I II   Lin Guowu G   Barnes Christopher O CO   Santamaria Hugo H   Ross Ted M TM   Soltis S Michael SM   Khosla Chaitan C   Nagarajan V V   Conway James F JF   Cohen Aina E AE   Calero Guillermo G  

Proceedings of the National Academy of Sciences of the United States of America 20140528 23


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 pos  ...[more]

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