Structural biology performed inside cells can capture molecular machines in action within their native context. Here we developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combined whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpointed NusA...

Structural biology performed inside cells can capture molecular machines in action within their native context. Here we developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combined whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpointed NusA...

Transferrin receptor protein 1 (TfR1) is an important molecule in anti-cancer therapy. Targeted delivery of such therapeutic compounds improves their cellular uptake and circulation time, thereby enhancing therapeutic efficacy.Drug designing is therefore used to engineer molecules with structures that facilitate specific interactions. However, this process requires a thorough knowledge of all the interactions, including the three-dimensional (3D) and quaternary structures (QS) of the interacting molecules. Since structural information is available for only a part of...

The activation of heterotrimeric G proteins is typically achieved by guanine nucleotide exchange factor (GEF) proteins that facilitate the exchange of GDP for GTP on G-alpha subunits. While this exchange activity is typically performed by G protein-coupled receptors (GPCRs), cytosolic non-receptor type GEFs have been more recently described as G-alpha activators. A subset of the described non-receptor GEFs contain a Galpha-Binding and Activating (GBA) motif. This short, evolutionarily conserved motif is known to confer nucleotide...

The YolD family (that includes both Bacillus subtilis YolD (this model) and YqjX) is part of the WYL-like superfamily, predicted to have an SH3 β-barrel fold related to Sm domains. In our analysis, among the closest detected structural matches were E. coli YaeO, a Rho-specific inhibitor of transcription termination, and a cyanobacterial Hfq homolog. Model assessment showed that YqjX and YolD sequences are indeed compatible with the SH3 β-barrel structure. Interestingly, UmuD' has an all...

The DXO family of proteins participates in eukaryotic mRNA 5′-end quality control, removal of non-canonical NAD+ cap and maturation of fungal rRNA precursors. In this work, we characterize the Arabidopsis thaliana DXO homolog, DXO1. We demonstrate that the plant-specific modification within the active site negatively affects 5′-end capping surveillance properties of DXO1, but has only a minor impact on its strong deNADding activity. Unexpectedly, catalytic activity does not contribute to striking morphological and molecular aberrations...

The structure of 'SARS-CoV-2 NON-STRUCTURAL PROTEIN 2 (nsp2)” is a part of the project “In silico Proteome analysis of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)” (Baruah et al., 2020; In communication) based on the sequence analysis and complete coordinate structure prediction of full SARS-CoV-2 proteome based on the NCBI reference sequence NC_045512 (29903 bp ss-RNA) which is identical to GenBank entry MN908947 and MT415321.

The structure of 'SARS-CoV-2 ORF3a protein' is a part of the project “In silico Proteome analysis of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)” (Baruah et al., 2020; In communication) based on the sequence analysis and complete coordinate structure prediction of full SARS-CoV-2 proteome based on the NCBI reference sequence NC_045512 (29903 bp ss-RNA) which is identical to GenBank entry MN908947 and MT415321. (Citation: bioRxiv preprint doi: https://doi.org/10.1101/2020.05.23.104919)

The aim of this study was to use molecular modelling and simulation to investigate how cytochrome P450 1A1 and its redox partner, cytochrome P450 reductase, form an electron transfer competent complex in a phospholipid bilayer. We used a multiresolution computational approach, combining Brownian dynamics, coarse-grained and all-atom molecular dynamics simulations to model the complex. The structural parameters and electron transfer rates agree well with experimental data.

We identified a novel class of proteins of the cryptochrome/photolyase family (CPF), which are shorter than most CPF proteins. We named this class of proteins Short Photolyase-Like (SPL). A member of SPL from Rhodococcus fascians (RfSPL) was selected, and its homology structural model was built with SWISS-MODEL using the known crystal structure of Synechococcus elongatus class I CPD photolyase (SeCPDI, PDB ID: 1TEZ) as the templates. Among the total of 406 residues of RfSPL, a...

We have taken advantage of cross-linking mass spectrometry (XL-MS) to interrogate the quaternary structure of the human MSC. Using the MS-cleavable cross-linker, DSSO, 19 unique inter-protein cross-links spanning all MSC constituents, and 118 unique intra-protein cross-links, were observed in MSC isolated from human HEK293T cells. The cross-links established spatial relationships between constituent pairs, and between domains in individual constituents. The protein-protein interactions detected by XL-MS were further improved by docking, revealing for the first time...

The essential feature of replication initiation proteins is their ability to bind to DNA. In this work, we describe a new domain that contributes to a replication initiator sequence-specific interaction with DNA. Applying biochemical assays and structure prediction methods coupled with DNA-protein cross-linking, mass spectrometry, and construction and analysis of mutant proteins, we identified that the replication initiator of the broad-host-range plasmid RK2, in addition to two winged helix domains, contains a third DNA binding...