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 Microfluidic protein isolation and sample preparation for high-resolution cryo-EM

2019 ◽  
Author(s):  
Claudio Schmidli ◽  
Stefan Albiez ◽  
Luca Rima ◽  
Ricardo Righetto ◽  
Inayatulla Mohammed ◽  
...  
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Protein Function ◽  
Structural Information ◽  
Protein Complexes ◽  
Protein Structure Determination ◽  
Cell Lysate ◽  
Endogenous Protein ◽  
Direct Preparation

AbstractHigh-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousands to a few million protein particles required for structure-determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 μL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens new opportunities for the isolation of sensitive, endogenous protein complexes.

scholarly journals Microfluidic protein isolation and sample preparation for high-resolution cryo-EM

2019 ◽  
Vol 116 (30) ◽  
pp. 15007-15012 ◽  
Author(s):  
Claudio Schmidli ◽  
Stefan Albiez ◽  
Luca Rima ◽  
Ricardo Righetto ◽  
Inayatulla Mohammed ◽  
...  
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Protein Function ◽  
Structural Information ◽  
Protein Complexes ◽  
Protein Structure Determination ◽  
Cell Lysate ◽  
Endogenous Protein ◽  
Direct Preparation

High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 μL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes.

scholarly journals Zernike Phase Plate Cryoelectron Microscopy Facilitates Single Particle Analysis of Unstained Asymmetric Protein Complexes

Structure ◽  
2010 ◽  
Vol 18 (1) ◽  
pp. 17-27 ◽  
Author(s):  
Wei-Hau Chang ◽  
Michael T.-K. Chiu ◽  
Chin-Yu Chen ◽  
Chi-Fu Yen ◽  
Yen-Cheng Lin ◽  
...  
Keyword(s):  
Single Particle ◽  
Protein Complexes ◽  
Cryoelectron Microscopy ◽  
Particle Analysis ◽  
Phase Plate ◽  
Single Particle Analysis

scholarly journals De Novo High-Resolution Protein Structure Determination from Sparse Spin-Labeling EPR Data

Structure ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 181-195 ◽  
Author(s):  
Nathan Alexander ◽  
Ahmad Al-Mestarihi ◽  
Marco Bortolus ◽  
Hassane Mchaourab ◽  
Jens Meiler
Keyword(s):  
Protein Structure ◽  
High Resolution ◽  
Structure Determination ◽  
De Novo ◽  
Protein Structure Determination ◽  
Spin Labeling

scholarly journals High-resolution structure determination of sub-100 kilodalton complexes using conventional cryo-EM

2018 ◽  
Author(s):  
Mark A. Herzik ◽  
Mengyu Wu ◽  
Gabriel C. Lander
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Drug Target ◽  
Phase Plate ◽  
Biological Macromolecules ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
High Resolution Structure ◽  
Resolution Structure

Determining high-resolution structures of biological macromolecules with masses of less than 100 kilodaltons (kDa) has long been a goal of the cryo-electron microscopy (cryo-EM) community. While the Volta Phase Plate has enabled cryo-EM structure determination of biological specimens of this size range, use of this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine the high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.9 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity, indicating that these methodologies can be used to investigate the molecular details of drug-target interactions. Our ~2.8 Å and ~3.2 Å resolution structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the first sub-nanometer cryo-EM structure of a protein smaller than 50 kDa.

scholarly journals CryoEM structure determination of small protein complexes with the Volta phase plate

2017 ◽  
Vol 73 (a1) ◽  
pp. a137-a137
Author(s):  
Maryam Khoshouei ◽  
Radostin Danev ◽  
Mazdak Radjainia ◽  
Wolfgang Baumeister
Keyword(s):  
Structure Determination ◽  
Protein Complexes ◽  
Phase Plate ◽  
Small Protein

scholarly journals Comparative structural analysis of 20S proteasome ortholog protein complexes by native mass spectrometry

2020 ◽  
Author(s):  
Shay Vimer ◽  
Gili Ben Nissan ◽  
David Morgenstern ◽  
Fanindra Kumar-Deshmukh ◽  
Caley Polkinghorn ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Structural Diversity ◽  
Native Mass Spectrometry ◽  
Hek293 Cells ◽  
Particle Analysis ◽  
20S Proteasome ◽  
Related Protein

<p>Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multi-level experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the <i>T. acidophilum</i> archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (<i>S. cerevisiae</i>) and mammals (rat - <i>R.</i> <i>norvegicus</i>, rabbit - <i>O. cuniculus</i> and human - HEK293 cells), the proteasome increased both in size and stability. Native Ms structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution three-dimensional structures, highly resembled that of the human complex. Using cryo-electron microscopy single-particle analysis we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size, and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the <a></a><a>PSMA7 </a>subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies. </p>

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