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

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.

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

10.1101/087841 ◽

2016 ◽

Cited By ~ 11

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.

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Faculty Opinions recommendation of An efficient protocol for NMR-spectroscopy-based structure determination of protein complexes in solution.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.3548964.4042059 ◽

2010 ◽

Author(s):

Linda Ball ◽

Victoria Ann Higman

Keyword(s):

Nmr Spectroscopy ◽

Structure Determination ◽

Protein Complexes

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Faculty Opinions recommendation of A method for integrative structure determination of protein-protein complexes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717961400.793466183 ◽

2012 ◽

Author(s):

Anastassis Perrakis

Keyword(s):

Structure Determination ◽

Protein Complexes

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An Efficient Protocol for NMR-Spectroscopy-Based Structure Determination of Protein Complexes in Solution

Angewandte Chemie ◽

10.1002/ange.200906147 ◽

2010 ◽

Vol 122 (11) ◽

pp. 2011-2014 ◽

Cited By ~ 5

Author(s):

Bernd Simon ◽

Tobias Madl ◽

Cameron D. Mackereth ◽

Michael Nilges ◽

Michael Sattler

Keyword(s):

Nmr Spectroscopy ◽

Structure Determination ◽

Protein Complexes

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A method for integrative structure determination of protein-protein complexes

Bioinformatics ◽

10.1093/bioinformatics/bts628 ◽

2012 ◽

Vol 28 (24) ◽

pp. 3282-3289 ◽

Cited By ~ 61

Author(s):

Dina Schneidman-Duhovny ◽

Andrea Rossi ◽

Agustin Avila-Sakar ◽

Seung Joong Kim ◽

Javier Velázquez-Muriel ◽

...

Keyword(s):

Structure Determination ◽

Protein Complexes

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

10.1101/556068 ◽

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.

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