Goc, G. | Search bioRxiv

bioRxiv2023

Combined workflow for XL-MS data processing: Application to the FtsH-HflK-HflC membrane protein complex

Recent advances in mass spectrometry (MS) yielding sensitive and accurate measurements along with developments in software tools have enabled the characterization of complex systems routinely. Thus, structural proteomics and cross-linking mass spectrometry (XL-MS) have become a useful method for structural modeling of protein complexes. Here we utilized commonly used XL-MS software tools to elucidate the protein interactions within a membrane protein complex containing FtsH, HflK, and HflC, over-expressed in E.coli. The MS data were processed using MaxLynx, MeroX, MS Annika, xiSEARCH, and XlinkX software tools. The number of identified inter- and intra-protein cross-links varied among software. Each interaction was manually checked using the raw MS and MS/MS data and distance restraints to verify inter- and intra-protein cross-links. A total of 37 inter-protein and 148 intra-protein cross-links were determined in the FtsH-HflK-HflC complex. The 59 of them were new interactions on the lacking region of recently published structures. These newly identified interactions, when combined with molecular docking and structural modeling, present opportunities for further investigation. The results provide valuable information regarding the complex structure and function to decipher the intricate molecular mechanisms underlying the mega FtsH-HflK-HflC complex. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/544024v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@8e10f5org.highwire.dtl.DTLVardef@fb809borg.highwire.dtl.DTLVardef@1b84df3org.highwire.dtl.DTLVardef@105c8e6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO The overall workflow of XL-MS analysis: the steps of XL-MS studies applied to the two samples taken from different stages of purification process of mega membrane protein complex FtsH-HflC-HflK. C_FIG

biochemistry↗

bioRxiv2022

Cryogenic X-ray crystallographic studies of biomacromolecules at Turkish Light Source: Turkish DeLight

X-ray crystallography is a robust and powerful structural biology technique that provides high-resolution atomic structures of biomacromolecules. Scientists use this technique to unravel mechanistic and structural details of biological macromolecules (e.g. proteins, nucleic acids, protein complexes, protein-nucleic acid complexes, or large biological compartments). Since its inception, single-crystal cryo-crystallography has never been performed in Turkiye due to the lack of a single-crystal X-ray diffractometer. The X-ray diffraction facility recently established at the University of Health Sciences, Istanbul, Turkiye will enable Turkish and international researchers to easily perform high-resolution structural analysis of biomacromolecules from single crystals. Here, we describe the technical and practical outlook of a state-of-the-art home-source X-ray, using lysozyme as a model protein. The methods and practice described in this article can be applied to any biological sample for structural studies. Therefore, this article will be a valuable practical guide from sample preparation to data analysis.

biophysics↗