Rapid screening of in cellulo grown protein crystals via a small-angle X-ray scattering/X-ray powder diffraction synergistic approach

Abstract

Crystallization of recombinant proteins in living cells is an exciting new approach for structural biology that provides an alternative to the time-consuming optimization of protein purification and extensive crystal screening steps. Exploiting the potential of this approach requires a more detailed understanding of the cellular processes involved and versatile screening strategies for crystals in a cell culture. Particularly if the target protein forms crystalline structures of unknown morphology only in a small fraction of cells, their detection by applying standard visualization techniques can be time consuming and difficult owing to the environmental challenges imposed by the living cells. In this study, a high-brilliance and low-background bioSAXS beamline is employed for rapid and sensitive detection of protein microcrystals grown within insect cells. On the basis of the presence of Bragg peaks in the recorded small-angle X-ray scattering profiles, it is possible to assess within seconds whether a cell culture contains microcrystals, even in a small percentage of cells. Since such information cannot be obtained by other established detection methods in this time frame, this screening approach has the potential to overcome one of the bottlenecks of intracellular crystal detection. Moreover, the association of the Bragg peak positions in the scattering curves with the unit-cell composition of the protein crystals raises the possibility of investigating the impact of environmental conditions on the crystal structure of the intracellular protein crystals. This information provides valuable insights helping to further understand the in cellulo crystallization process.

A simple vapor-diffusion method enables protein crystallization inside the HARE serial crystallography chip

“…Applying the parameters of a primitive hexagonal unit cell, 5520 of the recorded detector images were successfully indexed (a 27% indexing rate). The refined unit-cell parameters are comparable to those extracted from previous X-ray diffraction experiments (Yuan et al, 2003;Lahey-Rudolph et al, 2020, confirming the indicated similar composition of the in vitro-grown and in cellulo-grown HEX-1 crystals. The structural model of HEX-1 refined at 2.30 Å resolution is superimposable on that obtained from FT-SFX of in cellulo-grown HEX-1 (Lahey-Rudolph et al, 2021), with an overall r.m.s.d.…”

“…Its spontaneous self-assembly into intracellular crystals also occurs in insect cells after infection by a recombinant baculovirus (rBV) encoding the HEX-1 gene. Regular, micrometresized hexagonal crystals grow reproducibly and with high efficiency in living insect cells (Lahey-Rudolph et al, 2020). In the High Five insect cells used here, crystal growth is observed 48 h after rBV infection at the earliest, with a maximum of crystal-containing cells 96 h after infection.…”

“…Cloning of the HEX-1 gene (GenBank accession No. XM_958614) from the filamentous fungus Neurospora crassa has been described previously (Lahey-Rudolph et al, 2020). In brief, the HEX-1 coding sequence was amplified using the primers 5 0 -TACTACGACGACGACGXT CACG-3 0 (sense) and 5 0 -GAGGCGGGAACCGTGGACG-3 0 (antisense) and blunt end-ligated into the EheI site of a pFastBac1 vector containing the sequence 5 0 -ATGGGCG CCTAA-3 0 between the BamHI and HindIII restriction sites.…”

“…For baculovirus production, recombinant bacmid DNA was generated in E. coli DH10EmBacY cells (Geneva Biotech) and used for lipofection of Spodoptera frugiperda Sf9 insect cells. The virus titer was calculated using the TCID 50 (tissueculture infectious dose) in a serial dilution assay as described previously (Lahey-Rudolph et al, 2020).…”

Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals

“…We recently reported that spontaneous self-assembly of HEX-1 into intracellular crystals is not restricted to the native environment of the fungal cells. On infection with a recombinant baculovirus encoding the HEX-1 gene, living insect cells also form regular, micrometre-sized hexagonal crystals (Lahey-Rudolph et al, 2020), supporting a strong intrinsic crystallization tendency of HEX-1 that might have evolved along with its physiological function.…”