Dataset Information

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

ABSTRACT: 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 ?L 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.

SUBMITTER: Schmidli C

PROVIDER: S-EPMC6660723 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Schmidli Claudio C Albiez Stefan S Rima Luca L Righetto Ricardo R Mohammed Inayatulla I Oliva Paolo P Kovacik Lubomir L Stahlberg Henning H Braun Thomas T

Proceedings of the National Academy of Sciences of the United States of America 20190710 30

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 tar ...[more]

PMID: 31292253

Similar Datasets

Project description:Cryo-electron microscopy (cryo-EM) has become a leading technology for determining protein structures. Recent advances in this field have allowed for atomic resolution. However, predicting the backbone trace of a protein has remained a challenge on all but the most pristine density maps (<2.5?Å resolution). Here we introduce a deep learning model that uses a set of cascaded convolutional neural networks (CNNs) to predict C? atoms along a protein's backbone structure. The cascaded-CNN (C-CNN) is a novel deep learning architecture comprised of multiple CNNs, each predicting a specific aspect of a protein's structure. This model predicts secondary structure elements (SSEs), backbone structure, and C? atoms, combining the results of each to produce a complete prediction map. The cascaded-CNN is a semantic segmentation image classifier and was trained using thousands of simulated density maps. This method is largely automatic and only requires a recommended threshold value for each protein density map. A specialized tabu-search path walking algorithm was used to produce an initial backbone trace with C? placements. A helix-refinement algorithm made further improvements to the ?-helix SSEs of the backbone trace. Finally, a novel quality assessment-based combinatorial algorithm was used to effectively map protein sequences onto C? traces to obtain full-atom protein structures. This method was tested on 50 experimental maps between 2.6?Å and 4.4?Å resolution. It outperformed several state-of-the-art prediction methods including Rosetta de-novo, MAINMAST, and a Phenix based method by producing the most complete predicted protein structures, as measured by percentage of found C? atoms. This method accurately predicted 88.9% (mean) of the C? atoms within 3?Å of a protein's backbone structure surpassing the 66.8% mark achieved by the leading alternate method (Phenix based fully automatic method) on the same set of density maps. The C-CNN also achieved an average root-mean-square deviation (RMSD) of 1.24?Å on a set of 50 experimental density maps which was tested by the Phenix based fully automatic method. The source code and demo of this research has been published at https://github.com/DrDongSi/Ca-Backbone-Prediction.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets