In situ cryo-FIB/SEM Specimen Preparation Using the Waffle Method.

Cryo-focused ion beam (FIB) milling of vitrified specimens is emerging as a powerful method for in situ specimen preparation. It allows for the preservation of native and near-native conditions in cells, and can reveal the molecular structure of protein complexes when combined with cryo-electron tomography (cryo-ET) and sub-tomogram averaging. Cryo-FIB milling is often performed on plunge-frozen specimens of limited thickness.

Waffle Method: A general and flexible approach for improving throughput in FIB-milling.

Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens with vitrification issues, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and specimens that distribute poorly across grid squares. Here we present a general approach called the 'Waffle Method' which leverages high-pressure freezing to address these challenges.

Label-free visual proteomics: Coupling MS- and EM-based approaches in structural biology.

Combining diverse experimental structural and interactomic methods allows for the construction of comprehensible molecular encyclopedias of biological systems. Typically, this involves merging several independent approaches that provide complementary structural and functional information from multiple perspectives and at different resolution ranges. A particularly potent combination lies in coupling structural information from cryoelectron microscopy or tomography (cryo-EM or cryo-ET) with interactomic and structural information from mass spectrometry (MS)-based structural proteomics.

Structure and desensitization of AMPA receptor complexes with type II TARP γ5 and GSG1L.

AMPA receptors (AMPARs) mediate the majority of excitatory neurotransmission. Their surface expression, trafficking, gating, and pharmacology are regulated by auxiliary subunits. Of the two types of TARP auxiliary subunits, type I TARPs assume activating roles, while type II TARPs serve suppressive functions.

Notch-Jagged signaling complex defined by an interaction mosaic.

The Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear how the 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand, Jagged1, to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical, and structural techniques on the full extracellular complex and targeted sites, we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network.

Structure of the Arabidopsis thaliana glutamate receptor-like channel GLR3.4.

Glutamate receptor-like channels (GLRs) play vital roles in various physiological processes in plants, such as wound response, stomatal aperture control, seed germination, root development, innate immune response, pollen tube growth, and morphogenesis. Despite the importance of GLRs, knowledge about their molecular organization is limited. Here we use X-ray crystallography and single-particle cryo-EM to solve structures of the Arabidopsis thaliana GLR3.

Missing regions within the molecular architecture of human fibrin clots structurally resolved by XL-MS and integrative structural modeling.

Upon activation, fibrinogen forms large fibrin biopolymers that coalesce into clots which assist in wound healing. Limited insights into their molecular architecture, due to the sheer size and the insoluble character of fibrin clots, have restricted our ability to develop novel treatments for clotting diseases. The, so far resolved, disparate structural details have provided insights into linear elongation; however, molecular details like the C-terminal domain of the α-chain, the heparin-binding domain on the β-chain, and other functional domains remain elusive.

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results.

Cross-ID: Analysis and Visualization of Complex XL-MS-Driven Protein Interaction Networks.

Protein interactions enable much more complex behavior than the sum of the individual protein parts would suggest and represents a level of biological complexity requiring full understanding when unravelling cellular processes. Cross-linking mass spectrometry has emerged as an attractive approach to study these interactions, and recent advances in mass spectrometry and data analysis software have enabled the identification of thousands of cross-links from a single experiment. The resulting data complexity is, however, difficult to understand and requires interactive software tools.

Efficient and robust proteome-wide approaches for cross-linking mass spectrometry.

Cross-linking mass spectrometry (XL-MS) has received considerable interest, owing to its potential to investigate protein-protein interactions (PPIs) in an unbiased fashion in complex protein mixtures. Recent developments have enabled the detection of thousands of PPIs from a single experiment. A unique strength of XL-MS, in comparison with other methods for determining PPIs, is that it provides direct spatial information for the detected interactions.

Regulation of KIF1A-Driven Dense Core Vesicle Transport: Ca/CaM Controls DCV Binding and Liprin-α/TANC2 Recruits DCVs to Postsynaptic Sites.

Tight regulation of neuronal transport allows for cargo binding and release at specific cellular locations. The mechanisms by which motor proteins are loaded on vesicles and how cargoes are captured at appropriate sites remain unclear. To better understand how KIF1A-driven dense core vesicle (DCV) transport is regulated, we identified the KIF1A interactome and focused on three binding partners, the calcium binding protein calmodulin (CaM) and two synaptic scaffolding proteins: liprin-α and TANC2.

Spacer capture and integration by a type I-F Cas1-Cas2-3 CRISPR adaptation complex.

CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1-Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3.

LC-MS/MS with 2D mass mapping of skin secretions' peptides as a reliable tool for interspecies identification inside Rana esculenta complex.

Identification of species constituting Rana esculenta complex represents a certain problem as two parental species Rana ridibunda and Rana lessonae form their hybrid R. esculenta, while external signs and sizes of the members of this complex are intersected. However the composition of skin secretion consisting mainly of peptides is different for the species of the complex.