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

Area of Science:

• Structural biology
• Biophysics
• Materials science

Background:

• Advancements in X-ray free-electron laser (XFEL) sources enable serial femtosecond diffraction with nanometre-sized crystals (NCs).
• This technique is vital for determining structures of proteins, especially membrane proteins, that yield only submicrometre crystals.
• Efficient use of limited XFEL beamtime necessitates thorough pre-characterization of NCs and sample delivery methods.

Purpose of the Study:

• To demonstrate the utility of transmission electron microscopy (TEM) for evaluating nanometre-sized crystals (NCs) in liquid injector solutions.
• To assess the impact of sample delivery processes on NC concentration and diffraction quality.
• To provide a method for pre-characterizing NCs for XFEL serial femtosecond diffraction experiments.

Main Methods:

• Utilized transmission electron microscopy (TEM) to examine nanometre-sized crystals (NCs) in liquid injector carrier solutions.
• Evaluated crystal lattice and calculated fast Fourier transforms (FFTs) from TEM images to assess diffraction quality.
• Analyzed both injector reservoir solutions and post-injection solutions for three protein NC types: PTHR1, Pol II-GFP, and lysozyme.

Main Results:

• TEM successfully detected and evaluated nanometre-sized crystals (NCs) within liquid injector solutions.
• The concentration and diffraction quality of NCs can be affected by the sample delivery process.
• High solvent content and mechanically sensitive NCs showed particular susceptibility to changes during delivery.

Conclusions:

• Transmission electron microscopy (TEM) is a valuable tool for pre-characterizing nanometre-sized crystals (NCs) for XFEL serial femtosecond diffraction.
• The sample delivery process can impact the quality and concentration of NCs, necessitating careful optimization.
• This evaluation method ensures efficient use of XFEL beamtime and improves the success rate of structure determination from small protein crystals.