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Supercell refinement: a cautionary tale.

ABSTRACT: Theoretically, crystals with supercells exist at a unique crossroads where they can be considered as either a large unit cell with closely spaced reflections in reciprocal space or a higher dimensional superspace with a modulation that is commensurate with the supercell. In the latter case, the structure would be defined as an average structure with functions representing a modulation to determine the atomic location in 3D space. Here, a model protein structure and simulated diffraction data were used to investigate the possibility of solving a real incommensurately modulated protein crystal using a supercell approximation. In this way, the answer was known and the refinement method could be tested. Firstly, an average structure was solved by using the `main' reflections, which represent the subset of the reflections that belong to the subcell and in general are more intense than the `satellite' reflections. The average structure was then expanded to create a supercell and refined using all of the reflections. Surprisingly, the refined solution did not match the expected solution, even though the statistics were excellent. Interestingly, the corresponding superspace group had multiple 3D daughter supercell space groups as possibilities, and it was one of the alternate daughter space groups that the refinement locked in on. The lessons learned here will be applied to a real incommensurately modulated profilin-actin crystal that has the same superspace group.

SUBMITTER: Lovelace J

PROVIDER: S-EPMC6719663 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Supercell refinement: a cautionary tale.

Lovelace Jeffrey J Petrícek Václav V Murshudov Garib G Borgstahl Gloria E O GEO

Acta crystallographica. Section D, Structural biology 20190828 Pt 9

Theoretically, crystals with supercells exist at a unique crossroads where they can be considered as either a large unit cell with closely spaced reflections in reciprocal space or a higher dimensional superspace with a modulation that is commensurate with the supercell. In the latter case, the structure would be defined as an average structure with functions representing a modulation to determine the atomic location in 3D space. Here, a model protein structure and simulated diffraction data wer ...[more]

PMID: 31478908

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Project description:FURIN is a pro-protein convertase previously shown to be important for placental syncytialisation (Zhou et al. [1]), a process of cell fusion whereby placental cytotrophoblast cells fuse to form a multinucleated syncytium. This finding has been broadly accepted however, we have evidence suggesting the contrary. Spontaneously syncytialising term primary human trophoblast cells and BeWo choriocarcinoma cells were treated with either FURIN siRNA or negative control siRNA or the protease inhibitor, DEC-RVKR-CMK, or vehicle. Cells were then left to either spontaneously syncytialise (primary trophoblasts) or were induced to syncytialise with forskolin (BeWo). Effects on syncytialisation were measured by determining human chorionic gonadotrophin secretion and E-cadherin protein levels. We showed that FURIN is not important for syncytialisation in either cell type. However, in primary trophoblasts another protease also inhibited by DEC-RVKR-CMK, may be involved. Our results directly contrast with those published by Zhou et al. Zhou et al. however, used first trimester villous explants to study syncytialisation, and we used term primary trophoblasts. Therefore, we suggest that FURIN may be involved in syncytialisation of first trimester trophoblasts, but not term trophoblasts. What is more concerning is that our results using BeWo cells do not agree with their results, even though for the most part, we used the same experimental design. It is unclear why these experiments yielded different results, however we wanted to draw attention to simple differences in measuring syncytialisation or flaws in method reporting (including omission of cell line source and passage numbers, siRNA concentration and protein molecular weights) and choice of immunoblot loading controls, that could impact on experimental outcomes. Our study shows that careful reporting of methods by authors and thorough scrutiny by referees are vital. Furthermore, a universal benchmark for measuring syncytialisation is required so that various studies of syncytialisation can be validated.

Dataset Information

Supercell refinement: a cautionary tale.

Publications

Supercell refinement: a cautionary tale.

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