Wilmanns, M. | bioRxiv Science

bioRxiv2018

Structural variability of EspG chaperones from mycobacterial ESX-1, ESX-3 and ESX-5 type VII secretion systems

Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central {beta}-sheet and two -helical bundles. Additionally, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3 and EspG5 chaperones using small-angle X-ray scattering (SAXS) reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3-PE5-PPE4 complex based on the SAXS analysis.\n\nHighlightsO_LIThe crystal structure of EspG1 reveals the common architecture of the type VII secretion system chaperones\nC_LIO_LIStructures of EspG3 chaperones display a number of conformations that could reflect alternative substrate binding modes\nC_LIO_LIEspG3 chaperones dimerize in solution\nC_LIO_LIA model of EspG3 in complex with its substrate PE-PPE dimer is proposed based on SAXS data\nC_LI

biochemistry↗

bioRxiv2017

Iterative model-based density improvement yields better atomic structures from cryo-EM maps

Atomic models based on high-resolution density maps are the ultimate result of the cryo-EM structure determination process. Current cryo-EM model refinement procedures work with the experimental density map that remains constant throughout the process. Here, we introduce a general procedure that iteratively improves cryo-EM density maps based on prior knowledge of an atomic reference structure. The procedure optimizes contrast of cryo-EM densities by local amplitude scaling (LocScale) based on an atomic model without introducing model bias. We alternate the procedure with consecutive rounds of model refinement and tested it on four cryo-EM structures of TRPV1, {beta}-galactosidase, {gamma}-secretase and RNA polymerase III. We demonstrate that LocScale density improvement reveals previously undiscovered map features and improves the quality of atomic models. The presented approach enhances the interpretability of cryo-EM density maps and provides an implementation reminiscent of iterative density improvement as it is routinely employed in the refinement of X-ray crystallographic models.

biophysics↗