A Molecular Toolkit to Visualize Native Protein Assemblies in the Context of Human Disease

Gilmore, Brian L.; Winton, Carly E.; Demmert, Andrew C.; Tanner, Justin R.; Bowman, Sam; Karageorge, Vasilea; Patel, Kaya; Sheng, Zhi; Kelly, Deborah F.

doi:10.1038/srep14440

Cited by 13 publications

(32 citation statements)

References 19 publications

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confidence: 58%

“…K63-linked ubiquitin moieties were also resolved in the structure, indicating a signal for DNA damage repair. Complementary biochemical experiments supported these findings to reveal the first 3D insights of BRCA1 protein assemblies in the context of human disease [4].Additional molecular modeling experiments on the BRCA1 C-terminal domain (BRCT) suggested a mechanism for peptide interactions within the BRCT binding site (Figure 2a, b). Phosphorylated peptide repeats present in the RNAP II core (pSer5) have the proper physical attributes to fit within the BRCT binding site.…”

supporting

confidence: 53%

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Structural Oncology - Determining 3D Structures of Breast Cancer Assemblies

et al. 2016

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Under normal cellular conditions, the breast cancer susceptibility protein (BRCA1) protects the genome by acting as a tumor suppressor. Cells harboring mutations in the BRCA1 gene lose the ability to properly repair DNA damage and transcribe their genome. These effects can contribute to genomic instability and cancer induction [1]. Indeed, mutations in the BRCA1 gene are heavily linked to the development of hereditary breast and ovarian cancers [2]. A major question in the field remains, how do mutations in BRCA1 disrupt molecular processes?Currently, there is little information available for how BRCA1 interacts with other proteins. Conventional biochemical separations do not yield BRCA1 complexes suitable for structural analysis. To address this issue, we have recently developed a tunable microchip system that facilitates the rapid recovery of native protein assemblies from the nuclear material of patient-derived breast cancer cells (Figure 1). We employed the new microchip system to isolate and visualize BRCA1-transcriptional complexes, for the first time. We collected cryo-EM images of BRCA1 assemblies under low-dose conditions and processed the images using the RELION software package [3]. Resulting 3D structures of the assemblies were interpreted by using a combination of antibody-labeling and molecular modeling techniques. This information allowed us to interpret the interactions between the RNAP II core complex and BRCA1 structural elements in the presence of DNA fragments. K63-linked ubiquitin moieties were also resolved in the structure, indicating a signal for DNA damage repair. Complementary biochemical experiments supported these findings to reveal the first 3D insights of BRCA1 protein assemblies in the context of human disease [4].Additional molecular modeling experiments on the BRCA1 C-terminal domain (BRCT) suggested a mechanism for peptide interactions within the BRCT binding site (Figure 2a, b). Phosphorylated peptide repeats present in the RNAP II core (pSer5) have the proper physical attributes to fit within the BRCT binding site. However, models of a prevalent BRCA1 clinical mutation (BRCA1 5382insC ) showed that the mutated BRCT domain does not support these associations (Figure 2c, d). Biochemical analysis of protein interactions with BRCA1 5382insC also indicated decreased affinity for its nuclear partner BARD1 (Figure 2e). Overall, these findings demonstrate new strategies to delineate the multifaceted role of BRCA1 in RNA metabolism while defining opportunities to dissect native BRCA1 protein interactions.

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supporting

confidence: 58%

supporting

confidence: 53%

Structural Oncology - Determining 3D Structures of Breast Cancer Assemblies

et al. 2016

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“…Though the procedure for negative staining is described, tunable microchips also work well in cryo-EM applications [15, 17–19]. Uranyl formate stain should be prepared in advance.…”

Section: Methodsmentioning

confidence: 99%

“…Multi-reference alignment routines are implemented outputting representative 2D class averages (Fig. 3c, right panel) as previously described [19]. …”

Section: Methodsmentioning

confidence: 99%

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Preparation of Tunable Microchips to Visualize Native Protein Complexes for Single-Particle Electron Microscopy

Gilmore

Varano

Dearnaley

et al. 2018

Protein Complex Assembly

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Recent advances in technology have enabled single-particle electron microscopy (EM) to rapidly progress as a preferred tool to study protein assemblies. Newly developed materials and methods present viable alternatives to traditional EM specimen preparation. Improved lipid monolayer purification reagents offer considerable flexibility, while ultrathin silicon nitride films provide superior imaging properties to the structural study of protein complexes. Here, we describe the steps for combining monolayer purification with silicon nitride microchips to create a tunable approach for the EM community.

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Preparation of Disease-Related Protein Assemblies for Single Particle Electron Microscopy

Varano

Harafuji

Dearnaley

et al. 2017

Methods in Molecular Biology

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Electron microscopy (EM) is a rapidly growing area of structural biology that permits us to decode biological assemblies at the nanoscale. To examine biological materials for single particle EM analysis, purified assemblies must be obtained using biochemical separation techniques. Here we describe effective methodologies for isolating histidine (his)-tagged protein assemblies from the nucleus of disease-relevant cell lines. We further demonstrate how Isolated assemblies are visualized using single particle EM techniques and provide representative results for each step in the process.

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A Molecular Toolkit to Visualize Native Protein Assemblies in the Context of Human Disease

Cited by 13 publications

References 19 publications

Structural Oncology - Determining 3D Structures of Breast Cancer Assemblies

Structural Oncology - Determining 3D Structures of Breast Cancer Assemblies

Preparation of Tunable Microchips to Visualize Native Protein Complexes for Single-Particle Electron Microscopy

Preparation of Disease-Related Protein Assemblies for Single Particle Electron Microscopy

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