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 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 Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex

2018 ◽  
Author(s):  
Diego Gauto ◽  
Leandro Estrozi ◽  
Charles Schwieters ◽  
Gregory Effantin ◽  
Pavel Macek ◽  
...  
Keyword(s):  
Structure Determination ◽  
Protein Function ◽  
Structural Information ◽  
Magic Angle Spinning ◽  
Atomic Resolution ◽  
Magic Angle ◽  
Nmr Structure Determination ◽  
Structure Information ◽  
Resolution Structure

Atomic-resolution structure determination is the key requirement for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enabled determination of the high-resolution structure of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Angstrom by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and specifically labelled methyl groups, and a 4.1 Angstrom resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution (up to 8 Angstrom) cryo-EM data are available, thus paving avenues for the structure determination of challenging biological assemblies.

scholarly journals Von der „Blobology“ zur atomaren Auflösung der Kryo-Elektronenmikroskopie

BIOspektrum ◽  
2020 ◽  
Vol 26 (7) ◽  
pp. 710-713
Author(s):  
Holger Stark
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structure Determination ◽  
Protein Complexes ◽  
Pharmaceutical Research ◽  
New Drugs ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
First Time

AbstractIt took almost a century to develop electron microscopy into a powerful method for high-resolution structure determination of proteins. Technical improvements in microscopy, detector technology, and image processing software contributed to the exponential growth of high-resolution structures of protein complexes determined by cryo-electron microscopy in recent years. We now succeeded in breaking another resolution barrier in cryo-electron microscopy and for the first time in achieving true atomic resolution, where single atoms in the protein can indeed be visualized individually. These improvements in cryo-EM indicate that the method will continue to gain importance, not only as a method for structure determination but also in the development of new drugs in pharmaceutical research.

scholarly journals Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins

FEBS Letters ◽  
2010 ◽  
Vol 584 (12) ◽  
pp. 2539-2547 ◽  
Author(s):  
Yo Sonoda ◽  
Alex Cameron ◽  
Simon Newstead ◽  
Hiroshi Omote ◽  
Yoshinori Moriyama ◽  
...  
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Structure Determination ◽  
High Resolution Structure ◽  
Resolution Structure

scholarly journals 3P-094 High resolution and precise X-ray crystal structure determination of Cyctochrome c Oxidase(The 46th Annual Meeting of the Biophysical Society of Japan)

2008 ◽  
Vol 48 (supplement) ◽  
pp. S142
Author(s):  
Michihiro Suga ◽  
Kyoko Ito-Shinzawa ◽  
Hiroshi Aoyama ◽  
Kazumasa Muramoto ◽  
Eiki Yamashita ◽  
...  
Keyword(s):  
Crystal Structure ◽  
High Resolution ◽  
Annual Meeting ◽  
Structure Determination ◽  
Crystal Structure Determination ◽  
X Ray ◽  
Biophysical Society

scholarly journals Multidimensional high-resolution solid-state NMR for the structure determination of ^C/^N-labeled biomolecules

2000 ◽  
Vol 40 (supplement) ◽  
pp. S174
Author(s):  
Y. Todokoro ◽  
H. Yanagishita ◽  
T. Fujiwara ◽  
H. Akutsu
Keyword(s):  
Solid State ◽  
High Resolution ◽  
Structure Determination ◽  
Solid State Nmr
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