Biology subjects

Karius, K.

Publications and source records attributed to Karius, K..

3 recordsLinked to original sources

bioRxiv2021

Integrative structural modelling of macromolecular complexes using Assembline

Integrative modelling enables structure determination of macromolecular complexes by combining data from multiple experimental sources such as X-ray crystallography, electron microscopy (EM), or crosslinking mass spectrometry (XL-MS). It is particularly useful for complexes not amenable to high-resolution EM--complexes that are flexible, heterogenous, or imaged in cells with cryo-electron tomography. We have recently developed an integrative modelling protocol that allowed us to model multi-megadalton complexes as large as the nuclear pore complex. Here, we describe the Assembline software package, which combines multiple programs and libraries with our own algorithms in a streamlined modelling pipeline. Assembline builds ensembles of models satisfying data from atomic structures or homology models, EM maps and other experimental data, and provides tools for their analysis. Comparing to other methods, Assembline enables efficient sampling of conformational space through a multi-step procedure, provides new modeling restraints, and includes a unique configuration system for setting up the modelling project. Our protocol achieves exhaustive sampling in less than 100 - 1,000 CPU-hours even for complexes in the megadalton range. For larger complexes, resources available in institutional or public computer clusters are needed and sufficient to run the protocol. We also provide step-by-step instructions for preparing the input, running the core modelling steps, and assessing modelling performance at any stage.

bioinformatics↗

bioRxiv2020

Structure of the TFIIIC subcomplex {tau} A provides insights into RNA polymerase III pre-initiation complex formation

Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex {tau}A, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that {tau}A binding to its promoter is auto-inhibited by a disordered acidic tail of subunit {tau}95. We further provide a negative stain reconstruction of {tau}A bound to the TFIIIB subunits Brf1 and TBP with an unexpected location of Brf1 and TBP. This shows that a ruler element in {tau}A achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.

biochemistry↗

bioRxiv2020

In cell architecture of the nuclear pore complex and snapshots of its turnover

Nuclear pore complexes (NPCs) mediate exchange across the nuclear envelope. They consist of hundreds of proteins called nucleoporins (Nups) that assemble in multiple copies to fuse the inner and outer nuclear membranes. Elucidating the molecular function and architecture of NPCs imposes a formidable challenge and requires the convergence of in vitro and in situ approaches. How exactly NPC architecture accommodates processes such as mRNA export or NPC assembly and turnover inside of cells remains poorly understood. Here we combine integrated in situ structural biology, correlative light and electron microscopy with yeast genetics to structurally analyze NPCs within the native context of Saccharomyces cerevisiae cells under conditions of starvation and exponential growth. We find an unanticipated in situ layout of nucleoporins with respect to overall dimensions and conformation of the NPC scaffold that could not have been predicted from previous in vitro analysis. Particularly striking is the configuration of the Nup159 complex, which appears critical to spatially accommodate not only mRNA export but also NPC turnover by selective autophagy. We capture structural snapshots of NPC turnover, revealing that it occurs through nuclear envelope herniae and NPC-containing nuclear vesicles. Our study provides the basis for understanding the various membrane remodeling events that happen at the interface of the nuclear envelope with the autophagy apparatus and emphasizes the need of investigating macromolecular complexes in their cellular context.

biophysics↗