New Frontiers in Gold Labeling

Hainfeld, James F.; Powell, Richard D.

doi:10.1177/002215540004800404

Cited by 239 publications

(175 citation statements)

References 53 publications

Supporting

Mentioning

174

Contrasting

Unclassified

Order By: Relevance

“…This was expected even in negatively stained samples because of the use of methylamine vanadate (25).…”

Section: Resultsmentioning

confidence: 92%

“…Specifically, the unlabeled particle mass is 1.2 MDa (1). 4 To account for the gold particle (25) and tRNA Leu , the mass value used for the threshold of the reconstruction from the sample labeled with goldtRNA Leu was 1.24 MDa. For that labeled with total yeast tRNA, a value of 1.3 MDa was used, which assumes an average of four bound tRNAs of 25 kDa each.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

Wolfe

Warrington

Treadwell

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

It has become evident that the process of protein synthesis is performed by many cellular polypeptides acting in concert within the structural confines of protein complexes. In multicellular eukaryotes, one of these assemblies is a multienzyme complex composed of eight proteins that have aminoacyl-tRNA synthetase activities as well as three non-synthetase proteins (p43, p38, and p18) with diverse functions. This study uses electron microscopy and three-dimensional reconstruction to explore the arrangement of proteins and tRNA substrates within this "core" multisynthetase complex. Binding of unfractionated tRNA establishes that these molecules are widely distributed on the exterior of the structure. Binding of gold-labeled tRNALeu places leucyl-tRNA synthetase and the bifunctional glutamyl-/prolyl-tRNA synthetase at the base of this asymmetric "V"-shaped particle. A stable cell line has been produced that incorporates hexahistidine-labeled p43 into the multisynthetase complex. Using a gold-labeled nickel-nitrilotriacetic acid probe, the polypeptides of the p43 dimer have been located along one face of the particle. The results of this and previous studies are combined into an initial three-dimensional working model of the multisynthetase complex. This is the first conceptualization of how the protein constituents and tRNA substrates are arrayed within the structural confines of this multiprotein assembly.It has been known for many years that a "core" complex of aminoacyltRNA synthetases can be isolated from all multicellular eukaryotes (1). Its characteristic composition is three non-synthetase proteins and eight polypeptides having nine aminoacyl-tRNA synthetase activities. With the enzymes abbreviated as the three-letter amino acid name plus RS for the enzyme, the components are ArgRS, AspRS, GluProRS, GlnRS, IleRS, LeuRS, LysRS, MetRS, p43, p38, and p18. Several of these are known dimers, so the total polypeptide count in the multisynthetase complex is at least fifteen. This is a particularly intriguing protein assembly as all of the enzymes catalyze the same reaction, albeit with individual and highly specific substrates. The reaction catalyzed is the covalent attachment of an amino acid to either the 2Ј-or 3Ј-hydroxyl of the 3Ј-terminal adenosine of tRNA. Recent increased interest in these enzymes has been generated by studies indicating that they have roles in a variety of other cellular processes (2-4). Such activities range from cytokine-mediated chemoattraction to priming of reverse transcription of the human immunodeficiency virus, type 1 genome. These enzymes are also viewed as targets in the development of new antimicrobial agents (5). The multisynthetase complex also contains three non-synthetase proteins. The first of these, p18, has been shown to interact with EF-1␥, which may facilitate transfer of aminoacyl-tRNAs to the ribosome (6). Data from yeast two-hybrid screens (7), in vitro binding assays (8), and deletion analysis (9) indicate that p38 functions as a scaffolding protein and appears to be...

show abstract

“…This was expected even in negatively stained samples because of the use of methylamine vanadate (25).…”

Section: Resultsmentioning

confidence: 92%

Section: Methodsmentioning

confidence: 99%

A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

Wolfe

Warrington

Treadwell

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…S4B displays a representative field view of negatively stained LRRK2 showing abundant elongated particles with a few protein aggregates. To ascertain the identity of the recorded particles and the oligomeric state they represent, we used gold markers in combination with antibody labeling (31). LRRK2 was first labeled with either monoclonal anti-FLAG or polyclonal anti-LRR antibodies, followed by the addition of 5 nm of gold-conjugated anti-IgG secondary antibody, and revealed particles marked with two gold moieties when imaged (Fig.…”

Section: Architecture Of Dimeric Lrrk2 Revealed By Negative-stain Elementioning

confidence: 99%

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

Guaitoli

Raimondi

Gilsbach

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

139

163

View full text Add to dashboard Cite

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson's disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity-together with the LRR domain-to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD.LRRK2 | Parkinson's disease | structural modeling | EM | CL-MS

show abstract

“…Here we use a specimen of Nanogold particles deposited onto a 20 nm-thick carbon film to test whether the proposed method gives a reasonable value for the gold content of a Nanogold particle, which is expected to be ∼ 67 atoms (Hainfeld and Powell, 2000). To calculate the number of gold atoms, N Au , we applied Eq.…”

Section: Quantification Of Nanogold Onto a 20 Nm-thick Carbon Filmmentioning

confidence: 99%

Determination of quantitative distributions of heavy-metal stain in biological specimens by annular dark-field STEM

Sousa

Hohmann‐Marriott

Aronova

et al. 2008

Journal of Structural Biology

View full text Add to dashboard Cite

It is shown that dark-field images collected in the scanning transmission electron microscope (STEM) at two different camera lengths yield quantitative distributions of both the heavy and light atoms in a stained biological specimen. Quantitative analysis of the paired STEM images requires knowledge of the elastic scattering cross sections, which are calculated from the NIST Elastic-Scattering CrossSection Database. The results reveal quantitative information about the distribution of fixative and stain within the biological matrix, and provide a basis for assessing detection limits for heavy metal clusters used to label intracellular proteins. In sectioned cells that have been stained only with osmium tetroxide, we find an average of 1.2 ± 0.1 Os atom per nm 3 , corresponding to an atomic ratio of Os:C atoms of approximately 0.02, which indicates that small heavy atom clusters of Undecagold and Nanogold can be detected in lightly stained specimens.

show abstract

New Frontiers in Gold Labeling

Cited by 239 publications

References 53 publications

A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

Determination of quantitative distributions of heavy-metal stain in biological specimens by annular dark-field STEM

Contact Info

Product

Resources

About