Browse
Submit Data
Databases
API
Help

Dataset Information

274 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Pre-40S ribosome biogenesis factor Tsr1 is an inactive structural mimic of translational GTPases

ABSTRACT: Budding yeast Tsr1 is an essential ribosome biogenesis factor that is required for cytoplasmic steps in 40S subunit maturation. S. cerevisiae Tsr1 was expressed as an N-terminal GST fusion protein in E. coli. A mutant Tsr1ΔNΔloop was generated in which residues 410 to 476 of Tsr1 were replaced with a short glycine and serine rich sequence; in addition, the N-ternimal 45 amino acid residues were replaced by five amino acid residues “GPDSD”. Tsr1ΔNΔloop allowed for generating native crystals that diffracted to 3.6 Å. Here we characterize both wild type Tsr1 and Tsr1ΔNΔloop using cross-linking/mass spectrometry.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Zhuo Chen

LAB HEAD: Juri Rappsilber

PROVIDER: PXD004074 | Pride | 2017-02-08

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Publications

Pre-40S ribosome biogenesis factor Tsr1 is an inactive structural mimic of translational GTPases.

McCaughan Urszula M UM Jayachandran Uma U Shchepachev Vadim V Chen Zhuo Angel ZA Rappsilber Juri J Tollervey David D Cook Atlanta G AG

Nature communications 20160602

Budding yeast Tsr1 is a ribosome biogenesis factor with sequence similarity to GTPases, which is essential for cytoplasmic steps in 40S subunit maturation. Here we present the crystal structure of Tsr1 at 3.6 Å. Tsr1 has a similar domain architecture to translational GTPases such as EF-Tu and the selenocysteine incorporation factor SelB. However, active site residues required for GTP binding and hydrolysis are absent, explaining the lack of enzymatic activity in previous analyses. Modelling of T ...[more]

PMID: 27250689

Similar Datasets

A model dataset for quantitative cross-linking/mass spectrometry using isotope-labeled cross-linkers

2016-05-06 | PXD004107 | Pride

Quantitative cross-linking/mass spectrometry reveals subtle protein conformational changes

Project description:We have developed quantitative cross-linking/mass spectrometry (QCLMS) to interrogate conformational rearrangements of proteins in solution. Our workflow was tested using a structurally well-described reference system, the human complement protein C3 and its activated cleavage product C3b. We found that small local conformational changes affect the yields of cross-linking residues that are near in space while larger conformational changes affect the detectability of cross-links. Distinguishing between minor and major changes required robust analysis based on replica analysis and a label-swapping procedure. By providing workflow, code of practice and a framework for semi-automated data processing, we lay the foundation for QCLMS as a tool to monitor the domain choreography that drives binary switching in many protein-protein interaction networks.

2016-06-16 | PXD001675 | Pride

Cross-linking/mass spectrometry analysis of AUGMIN complex

Project description:We applied cross-linking/mass spectrometry to characterize in vivo Augmin from Drosophila in absence of any other structural information. The identified cross-links revealed topology of the Augmin complex and allowed us to predict potential interfaces between Augmin and γ-TuRC.

2017-04-04 | PXD006246 | Pride

Capturing protein communities by structural proteomics in a thermophilic eukaryote

Project description:The arrangement of proteins into complexes is a key organizational principle for many cellular functions. Although the topology of many complexes has been systematically analyzed in isolation, their molecular sociology in situ remains elusive. Here, we show that crude cellular extracts of a eukaryotic thermophile, Chaetomium thermophilum, retain basic principles of cellular organization. Using a structural proteomics approach, we simultaneously characterized the abundance, interactions and structure of a third of the C. thermophilum proteome within these extracts. We identified 27 distinct protein communities that include 108 interconnected complexes, which dynamically associate with each other and functionally benefit from being in close proximity in the cell. Furthermore, we investigated the structure of fatty acid synthase within these extracts by cryoEM and this revealed multiple, flexible states of the enzyme in adaptation to its association with other complexes, thus exemplifying the need for in situ studies. As the components of the captured protein communities are known – at both the protein and complex level – this study constitutes another step forward towards a molecular understanding ofsubcellular organization.

2017-07-26 | PXD006626 | Pride

A folded conformation of MukBEF and Cohesin

Project description:Structural maintenance of chromosomes (SMC)-kleisin complexes organize chromosomal DNAs in all domains of life, where they have key roles in chromosome segregation, DNA repair and regulation of gene expression. They function through topological entrapment and active translocation of DNA, but the underlying conformational changes are largely unclear. Using structural biology, mass spectrometry and cross-linking, we investigated the architecture of two evolutionarily distant SMC-kleisin complexes: proteobacterial MukBEF and eukaryotic cohesin. We show that both contain a dynamic coiled-coil discontinuity, the elbow, near the middle of their arms that permits a folded conformation. Bending at the elbow brings into proximity the hinge dimerization domain and the head/kleisin module, situated at opposite ends of the arms. Our findings favor SMC activity models that include a large conformational change in the arms, such as a relative movement between DNA binding sites during DNA loading and translocation

2019-03-01 | PXD012377 | Pride

Quantitative cross-linking/mass spectrometry

Project description:Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue- resolution data on static proteinaceous structures. Here we investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation. We cross-linked human serum albumin (HSA) with the readily available cross-linker BS3-d0/4 in different heavy/light ratios. Wefound two limitations. First, isotope labelling reduced the number of identified cross-links. This is in line with similar findings when identifying proteins. Second, standard quantitative proteomics software was not suitable for work with cross-linking. To ameliorate this we wrote a basic open source application, XiQ. Using XiQ we could establish that quantitative CLMS was technically feasible. Biological significance Cross-linking/mass spectrometry (CLMS) has become a powerful tool for providing residue- resolution data on static proteinaceous structures. Adding quantitation to CLMS will extend its ability of recording dynamic processes. Here we introduce a cross-linking specific quantitation strategy by using isotope labelled cross-linkers. Using a model system, we demonstrate the principle and feasibility of quantifying cross-linking data and discuss challenges one may encounter while doing so.We then provide a basic open source application, XiQ, to carry out automated quantitation of CLMS data. Ourwork lays the foundations of studying themolecular details of biological processes at greater ease than this could be done so far.

2015-06-08 | PXD002142 | Pride

Cross-linking/mass spectrometry analysis of the AOC complex

Project description:As a part of a study on how kinetochors are assembled at the centromeres of the chromosomes, cross-linking/mass spectrometry has been applied to investigate interactions between Okp1/ Ame1 heterodimer, which is part of the COMA complex, and CENP-A from Saccharomyces cerevisiae.

2018-10-30 | PXD009648 | Pride

On the reproducibility of label-free quantitative cross-linking/mass spectrometry

Project description:Quantitative cross-linking/mass spectrometry (QCLMS) is an emerging approach to study conformational changes of proteins and multi-subunit complexes. Distinguishing protein conformations requires reproducibly identifying and quantifying cross-linked peptides. Here we analyzed the variation between multiple cross-linking reactions using bis[sulfosuccinimidyl] suberate (BS3)-cross-linked human serum albumin (HSA) and evaluated how reproducible cross-linked peptides can be identified and quantified by LC-MS analysis. To make QCLMS accessible to a broader research community we developed a workflow that integrates the established software tools MaxQuant for spectra pre-processing, Xi for cross-linked peptide identification and finally Skyline for quantification (MS1 filtering). Out of the 221 unique residue pairs identified in our sample, 146 (66% of those identified) could be imported into Skyline and 124 (84% of those imported) were subsequently quantified with coefficient of variation (CV) values of 14% (injection replica) and 32% (reaction replica). Thus our results demonstrate that the reproducibility of QCLMS is in line with the reproducibility of general quantitative proteomics and we establish a robust workflow for MS1-based quantitation of cross-linked peptides.

2018-01-02 | PXD007250 | Pride

A folded conformation of MukBEF and Cohesin

Project description:PleasStructural maintenance of chromosomes (SMC)-kleisin complexes organize chromosomal DNAs in all domains of life, where they have key roles in chromosome segregation, DNA repair and regulation of gene expression. They function through topological entrapment and active translocation of DNA, but the underlying conformational changes are largely unclear. Using structural biology, mass spectrometry and cross-linking, we investigated the architecture of two evolutionarily distant SMC-kleisin complexes: proteobacterial MukBEF and eukaryotic cohesin. We show that both contain a dynamic coiled-coil discontinuity, the elbow, near the middle of their arms that permits a folded conformation. Bending at the elbow brings into proximity the hinge dimerization domain and the head/kleisin module, situated at opposite ends of the arms. Our findings favor SMC activity models that include a large conformational change in the arms, such as a relative movement between DNA binding sites during DNA loading and translocatione provide an overall description of your study, think something similar in scope to the manuscript abstract

2019-03-01 | PXD012370 | Pride

Quantitative cross-linking/mass spectrometry analysis revealed unknown structure of complement iC3

Project description:We applied quantitative cross-linking/mass spectrometry (QCLMS) to interrogate the structure of iC3 (or C3(H2O)), the activated hydrolytic product of the abundant human complement protein C3. The slow but spontaneous and ubiquitous formation of iC3 from C3 initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. QCLMS revealed structural differences and similarities between iC3 and C3, as well as between iC3 and C3b that is a pivotal proteolytic cleavage product of C3 and is functionally similar to iC3. Considered in combination with the crystal structures of C3 and C3b, our data support a model wherein the thioester-containing domain of C3 swings to the other end of the molecule creating, in iC3, a stable C3b-like platform for binding the zymogen, factor B, or the regulator, factor H. The integration of available crystallographic and QCLMS data allowed the determination of a 3D model for iC3. The unique arrangement of domains in iC3, which retains the anaphylatoxin (ANA) domain (while ANA is excised when C3 is enzymatically activated to C3b), is consistent with observed differences in activation and regulation between iC3 and C3b.

2016-06-17 | PXD003486 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data