scholarly journals Sparsely-sampled, high-resolution 4-D omit spectra for detection and assignment of intermolecular NOEs of protein complexes

2014 ◽  
Vol 59 (2) ◽  
pp. 51-56 ◽  
Author(s):  
Su Wang ◽  
Pei Zhou
Keyword(s):  
High Resolution ◽  
Protein Complexes

Three-dimensional crystallization of membrane proteins

1988 ◽  
Vol 21 (4) ◽  
pp. 429-477 ◽  
Author(s):  
W. Kühlbrandt
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Membrane Protein Complexes ◽  
Essential Prerequisite

As recently as 10 years ago, the prospect of solving the structure of any membrane protein by X-ray crystallography seemed remote. Since then, the threedimensional (3-D) structures of two membrane protein complexes, the bacterial photosynthetic reaction centres of Rhodopseudomonas viridis (Deisenhofer et al. 1984, 1985) and of Rhodobacter sphaeroides (Allen et al. 1986, 1987 a, 6; Chang et al. 1986) have been determined at high resolution. This astonishing progress would not have been possible without the pioneering work of Michel and Garavito who first succeeded in growing 3-D crystals of the membrane proteins bacteriorhodopsin (Michel & Oesterhelt, 1980) and matrix porin (Garavito & Rosenbusch, 1980). X-ray crystallography is still the only routine method for determining the 3-D structures of biological macromolecules at high resolution and well-ordered 3-D crystals of sufficient size are the essential prerequisite.

LOPIT-DC: A simpler approach to high-resolution spatial proteomics

2018 ◽  
Author(s):  
Aikaterini Geladaki ◽  
Nina Kočevar Britovšek ◽  
Lisa M. Breckels ◽  
Tom S. Smith ◽  
Claire M. Mulvey ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
High Resolution Mass Spectrometry ◽  
Protein Complexes ◽  
Subcellular Localisation ◽  
Proteomics Data ◽  
Large Protein ◽  
Subcellular Resolution ◽  
First Time ◽  
Human Dataset

AbstractHyperplexed Localisation of Organelle Proteins by Isotope Tagging (hyperLOPIT) is a well-established method for studying protein subcellular localisation in complex biological samples. As a simpler alternative we developed a second workflow named Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC) which is faster and less resource-intensive. We present the most comprehensive high-resolution mass spectrometry-based human dataset to date and deliver a flexible set of subcellular proteomics protocols for sample preparation and data analysis. For the first time, we methodically compare these two different mass spectrometry-based spatial proteomics methods within the same study and also apply QSep, the first tool that objectively and robustly quantifies subcellular resolution in spatial proteomics data. Using both approaches we highlight suborganellar resolution and isoform-specific subcellular niches as well as the locations of large protein complexes and proteins involved in signalling pathways which play important roles in cancer and metabolism. Finally, we showcase an extensive analysis of the multilocalising proteome identified via both methods.

scholarly journals The molecular appearance of native TRPM7 channel complexes identified by high-resolution proteomics

2021 ◽  
Author(s):  
Astrid Kollewe ◽  
Vladimir Chubanov ◽  
Fong Tsuen Tseung ◽  
Alexander Haupt ◽  
Catrin Swantje M&uumlller ◽  
...  
Keyword(s):  
High Resolution ◽  
Transient Receptor Potential ◽  
Protein Complexes ◽  
Divalent Cations ◽  
Affinity Purification ◽  
Receptor Potential ◽  
Metal Transporter ◽  
Transient Receptor Potential Melastatin ◽  
Transient Receptor ◽  
And Function

The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed membrane protein consisting of ion channel and protein kinase domains. TRPM7 plays a fundamental role in the cellular uptake of divalent cations such as Zn2+, Mg2+ and Ca2+, and thus shapes cellular excitability, plasticity and metabolic activity. The molecular appearance and operation of TRPM7 channel complexes in native tissues have remained unresolved. Here, we investigated the subunit composition of endogenous TRPM7 channels in rodent brain by multi-epitope affinity purification and high-resolution quantitative MS analysis. We found that native TRPM7 channels are high molecular-weight multi-protein complexes that contain the putative metal transporter proteins CNNM1-4 and a small G-protein ARL15. Heterologous reconstitution experiments confirmed the formation of TRPM7/CNNM/ARL15 ternary complexes and indicated that ARL15 effectively and specifically impacts TRPM7 channel activity. These results open up new avenues towards a mechanistic understanding of the cellular regulation and function of TRPM7 channels.

scholarly journals Cryo-EM structure of haemoglobin at 3.2 Å determined with the Volta phase plate

2016 ◽  
Author(s):  
Maryam Khoshouei ◽  
Mazdak Radjainia ◽  
Wolfgang Baumeister ◽  
Radostin Danev
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Protein Complexes ◽  
Protein Structure Determination ◽  
Cost Effective ◽  
Particle Analysis ◽  
Phase Plate ◽  
Human Haemoglobin ◽  
Low Contrast ◽  
Macromolecular Assemblies

With the advent of direct electron detectors, the perspectives of cryo-electron microscopy (cryo-EM) have changed in a profound way1. These cameras are superior to previous detectors in coping with the intrinsically low contrast of radiation-sensitive organic materials embedded in amorphous ice, and so they have enabled the structure determination of several macromolecular assemblies to atomic or near-atomic resolution. According to one theoretical estimation, a few thousand images should suffice for calculating the structure of proteins as small as 17 kDa at 3 Å resolution2. In practice, however, we are still far away from this theoretical ideal. Thus far, protein complexes that have been successfully reconstructed to high-resolution by single particle analysis (SPA) have molecular weights of ~100 kDa or larger3. Here, we report the use of Volta phase plate in determining the structure of human haemoglobin (64 kDa) at 3.2 Å. Our results demonstrate that this method can be applied to complexes that are significantly smaller than those previously studied by conventional defocus-based approaches. Cryo-EM is now close to becoming a fast and cost-effective alternative to crystallography for high-resolution protein structure determination.

scholarly journals Identification of Protein Complexes Associated with the Usher Syndrome 2C and Epilepsy-Associated Protein VLGR1 Applying Affinity Proteomics

2017 ◽  
Vol 4 (1) ◽  
pp. 100051 ◽  
Author(s):  
Barbara Knapp ◽  
Deva Krupakar Kusuluri ◽  
Nicola Horn ◽  
Karsten Boldt ◽  
Marius Ueffing ◽  
...  
Keyword(s):  
High Resolution ◽  
Signaling Pathways ◽  
Protein Complexes ◽  
Usher Syndrome ◽  
Protein Networks ◽  
Cellular Compartments ◽  
Affinity Proteomics ◽  
Shed Light

Authors aimed to identify novel VLGR1-associated protein networks to shed light on its integration into signaling pathways and the cellular compartments in which VLGR1 functions using high-resolution affinity proteomics based on tandem affinity purifications (TAPs).

scholarly journals Bidirectional intraflagellar transport is restricted to two sets of microtubule doublets in the trypanosome flagellum

2018 ◽  
Author(s):  
Eloïse Bertiaux ◽  
Adeline Mallet ◽  
Cécile Fort ◽  
Thierry Blisnick ◽  
Serge Bonnefoy ◽  
...  
Keyword(s):  
High Resolution ◽  
Focused Ion Beam ◽  
Protein Complexes ◽  
Ion Beam ◽  
Intraflagellar Transport ◽  
Sem Analysis ◽  
Large Individual ◽  
Resolution Electron ◽  
Cilia And Flagella ◽  
Bidirectional Movement

SummaryIntraflagellar transport (IFT) is the rapid bidirectional movement of large protein complexes driven by kinesin and dynein motors along microtubule doublets of cilia and flagella. Here we used a combination of high-resolution electron and light microscopy to investigate how and where these IFT trains move within the flagellum of the protist Trypanosoma brucei. Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) analysis of trypanosomes showed that trains are found almost exclusively along two sets of doublets (3-4 and 7-8) and distribute in two categories according to their length. High-resolution live imaging of cells expressing mNeonGreen::IFT81 or GFP::IFT52 revealed for the first time IFT trafficking on two parallel lines within the flagellum. Anterograde and retrograde IFT occur on each of these lines. At the distal end, a large individual anterograde IFT train is converted in several smaller retrograde trains in the space of 3-4 seconds while remaining on the same side of the axoneme.

Stoichiometry determination of macromolecular membrane protein complexes

2017 ◽  
Vol 398 (2) ◽  
pp. 155-164 ◽  
Author(s):  
Susann Zilkenat ◽  
Iwan Grin ◽  
Samuel Wagner
Keyword(s):  
High Resolution ◽  
Membrane Protein ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Transmembrane Protein ◽  
Protein Structures ◽  
Membrane Protein Complexes ◽  
Improved Methods ◽  
Structure Calculations

Abstract Gaining knowledge of the structural makeup of protein complexes is critical to advance our understanding of their formation and functions. This task is particularly challenging for transmembrane protein complexes, and grows ever more imposing with increasing size of these large macromolecular structures. The last 10 years have seen a steep increase in solved high-resolution membrane protein structures due to both new and improved methods in the field, but still most structures of large transmembrane complexes remain elusive. An important first step towards the structure elucidation of these difficult complexes is the determination of their stoichiometry, which we discuss in this review. Knowing the stoichiometry of complex components not only answers unresolved structural questions and is relevant for understanding the molecular mechanisms of macromolecular machines but also supports further attempts to obtain high-resolution structures by providing constraints for structure calculations.

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