Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals

Area of Science:

• Structural biology
• Biophysics
• Crystallography

Background:

• Crystallization of recombinant proteins within living cells offers a novel structural biology approach.
• Efficient diffraction data collection is crucial for analyzing these intracellular crystals.
• Serial femtosecond crystallography (SFX) at X-ray Free Electron Lasers (XFELs) can detect diffraction from small crystals but requires rapid sample exchange.

Purpose of the Study:

• To present an efficient method for high-resolution structure elucidation using serial femtosecond *in cellulo* diffraction.
• To demonstrate the utility of a fixed-target approach with micro-patterned silicon chips for *in cellulo* crystallography.
• To determine the structure of the HEX-1 protein from *Neurospora crassa* using this novel technique.

Main Methods:

• Utilized serial femtosecond *in cellulo* diffraction on micrometer-sized HEX-1 protein crystals within living insect cells.
• Employed a fixed-target system with micro-patterned, single-crystalline silicon chips and a high-speed translation stage.
• Collected diffraction data at the Linac Coherent Light Source (LCLS).

Main Results:

• Achieved high hit rates (up to 30%) and reduced background scattering compared to liquid-jet methods.
• Determined the 1.8 Å resolution structure of HEX-1 using data from a single chip collected in 12 minutes.
• Demonstrated significantly reduced sample consumption compared to previous SFX experiments.
• Validated the approach by obtaining a structure nearly identical to one determined by traditional methods.

Conclusions:

• Fixed-target SFX with micro-patterned silicon chips is highly effective for *in cellulo* diffraction data collection from living cells.
• This method offers significant potential for straightforward structure elucidation of proteins forming intracellular crystals.
• The approach is suitable for both XFELs and synchrotron sources, advancing structural biology research.