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 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.T he emergence of X-ray free electron laser (X-FEL)-based serial femtosecond crystallography holds the promise of solving the 3D structure of proteins that can only crystallize as "nanocrystals" (NCs) or are highly sensitive to radiation damage (1-5). NCs appropriate for X-FEL experiments are considered to be 200 nm to 2 μm in size (6). This size is constrained primarily by the requirements of the NC delivery system to the X-FEL beam. In addition to allowing for structure resolution of NCs by X-FEL experiments, they provide the advantage of requiring no crystal cryoprotection because these experiments are performed at room temperature (3, 7). Given the opportunities that X-FELs offer to the field of crystallography, efficient methodologies to detect NCs from single crystallography drops and to optimize these identified conditions yielding NCs will be essential for future developments in structural biology. Current methods to detect the presence of NCs include dynamic light scattering (DLS), bright-field microscopy, birefringence microscopy, and intrinsic tryptophan UV fluorescence imaging, as well as technologies that rely upon second harmonic generation, such as second order nonlinear imaging of chiral crystals (SONICC) (8, 9) and X-ray powder diffraction. However, limitations of these imaging techniques include (i) ineffective detection of crystals smaller than 5 μm (8, 10), (ii) false-positive conditions as a result of interference from precipitate backgrounds (8, 10), and (iii) false-negative conditions resulting from the lack of tryptophan residues in the case of UV fluorescence and from the lack of chiral centers in the case of SONICC (11). Although DLS can accurately measure the size distribution of nanometer-sized protein aggregates, it is unable to distinguish unambiguously between amorphous and crystalline (12). Finally, X-ray powder diffraction, a method that has been applied to evaluate samples for the presence and concentration of NCs, requires more material than is produced in a single crystallization screening d...

Serial femtosecond crystallography: A revolution in structural biology

“…This is illustrated in Figure 3 where a batch approach for macro- and nanocrystals differs only by starting point within the phase space 67 . It has been reported in some cases that large crystals grown by traditional methods can also be mechanically crushed to obtain the smaller crystals needed for SFX 6,68 . This is, however, not generally applicable and may lead to loss of quality or destruction of fragile crystals, such as those common amongst membrane proteins.…”

“…It is also important to include buffer in the capillary to avoid drying out of the small crystals which can affect diffraction quality. While powder diffraction data can be collected from nanocrystals at low flux X-ray home sources, it requires several microliters of dense crystal sample which is more than is produced from commercial screens 68 . It also depends highly on the sample quality and density so establishing conditions for powder diffraction typically requires synchrotron radiation to produce measurable diffraction for un-optimized samples 83 .…”

“…While this technique can provide insightful characterization, it requires elaborate sample preparation and often involves negative staining which can affect electron diffraction and is only suitable for very thin nanocrystals (< 200 nm) 83,84 . Large crystals can be mechanically crushed in order to achieve dimensions suitable for TEM 68 .…”

Goniometer-based femtosecond crystallography with X-ray free electron lasers

“…As NCs may be a ubiquitous but generally overlooked outcome of commercial crystallization screens that fail to produce larger crystals (18), FX may open up many systems to crystallographic analysis. However, to develop FX into a generally applicable method, a number of challenges in the areas of sample preparation, data collection, and data processing must be overcome.…”

Transmission electron microscopy as a tool for nanocrystal characterization pre- and post-injector

“…This is a green-coloured protein with a large crystal lattice readily identifiable by TEM with crystal sizes ranging from 1 to 5 mm. The crystallization conditions for these systems were determined through direct examination of commercial crystallization screening drops using a novel selection protocol that combines (i) brightfield microscopy, UV fluorescence microscopy and dynamic light scattering as screening methods to detect crystallization drops containing NCs and (ii) TEM to accurately identify protein NCs and determine NC quality [17]. Lysozyme NCs were also tested as a soluble protein control.…”