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Latest News from the NCDIR

1302, 2021

Research Spotlight—Mass spectrometry-based diagnostic and screening platform for optimizing sample preparation in single particle cryo-EM

February 13th, 2021|

A native mass spectrometry (nMS)-based platform developed at the NCDIR for evaluating and optimizing sample quality for single particle cryogenic microscopy (cryo-EM) is featured in the current issue of the journal Structure (https://www.cell.com/structure/home).

The exponential increase in high-resolution biomolecular structures determined by cryo-EM is yielding unprecedented structural insights into cellular processes. Despite its extraordinary capabilities, one major bottleneck in single-particle cryo-EM involves sample preparation and the need to have an efficient means for assessing the quality of samples prior to committing the substantial resources, time and effort needed for a typical cryo-EM analysis. To address this need, Dom Olinares, a Research Associate in Brian Chait’s lab at the NCDIR worked with structural biologists to develop an nMS platform that readily integrates into the cryo-EM sample preparation workflow providing rapid feedback on sample integrity/purity and optimal preparation conditions.

Figure. nMS-based screening to assemble the intact SARS-CoV-2 helicase-RTC. (A) Screening initial conditions for RTC with varying RNA scaffolds and ammonium acetate (AmOAc) concentrations did not form any assembled complex. Detection of unassembled subunits and a truncated nsp12 indicated issues with nsp12 expression. (B) nMS results after optimizing expression of the full-length nsp12 and subsequent reconstitution of RTC. Incubation of the assembled RTC with the nsp13 helicase yielded a single peak corresponding to nsp13-RTC at 1:1 stoichiometry. (C) cryo-EM structure of the nsp13-bound RTC (PDB ID: 6xez) (Olinares et. al., 2020).

Because nMS enables accurate mass analysis of intact protein complexes, it is well-suited for routine evaluation of the composition, integrity, and homogeneity of samples prior to their plunge-freezing on EM grids. In addition, once the EM data is collected, the nMS spectra acquired from the screening provide critical information on subunit composition, stoichiometry, protein modifications, and bound cofactors/ligands that serve as key inputs for EM structure reconstruction and validation.

The utility of the nMS-based diagnostic and screening platform was demonstrated through examples that characterized several bacterial transcription complexes as well as the replication-transcription complex (RTC) coupled to nsp13 helicase from SARS-CoV-2. Most of these structural projects started out with reconstitution conditions that did not yield the desired target assemblies for cryo-EM. In each case, with the aid of iterative nMS-based screening, it proved possible to rapidly establish optimized sample conditions that yielded cryo-EM structures at high resolution.

Publication:

Olinares PDB, Kang JY, Llewellyn E, Chiu C, Chen J, Malone B, Saecker RM, Campbell EA, Darst SA, Chait BT. “Native Mass Spectrometry-Based Screening for Optimal Sample Preparation in Single-Particle Cryo-EM.” Structure. 2020 Nov 17:S0969-2126(20)30413-5. PMID: 33217329.

The full text of the paper is freely available here:

https://www.cell.com/structure/fulltext/S0969-2126(20)30413-5

910, 2020

NCDIR alumni in the news!

October 9th, 2020|

Our NCDIR 2019 fellow Maria Benitez Guijarro was recently featured as Cold Spring Harbor laboratory (CSHL)  “Visitor of the week”.

Click on the link below to read more of Maria’s time at CSHL!

 

3009, 2020

Research Spotlight – “Defining the proteolytic landscape during enterovirus infection”

September 30th, 2020|

In collaboration with Charles Rice’s lab at Rockefeller University, NCDIR researchers have contributed to a new study that illuminates how a class of viruses called enteroviruses disrupt the machinery of normal human host cells during infection. 

Enteroviruses cause a number of human diseases including gastroenteritis, the common cold, hand-foot-and-mouth disease, acute hemorrhagic conjunctivitis, and skin rash. In this study, researchers identified which host proteins are broken apart, or “cleaved”, by proteins produced by enteroviruses. By disrupting proteins this way, these viruses remodel infected cells to avoid detection by the host’s immune system and create a favorable environment for replication. The researchers identified proteins commonly targeted across five different enteroviruses and were able to identify specific cell functions in which these proteins participate, providing valuable insight into which cellular processes are subverted or disrupted during infection. As the first global assessment of enterovirus protein cleavage in human cells, the study is the first of its kind and provides a valuable community resource for understanding enterovirus biology and for generating new approaches to combat enterovirus-related diseases.

Figure: Analysis of proteins broadly targeted by enteroviruses.

(A) Venn diagram showing the numbers of cleaved proteins common or unique to different viruses. (B) STRING interaction network of proteins targeted by at least two of the five enteroviruses as rendered in Cytoscape (disconnected nodes are not shown). Coloring indicates groups of five or more proteins identified as highly interconnected by the MCODE clustering algorithm. Most significantly enriched biological processes are summarized below the network. Cleaved proteins validated by western blot are shown in diamond shaped nodes. (C) Top 15 significantly enriched biological processes targeted by at least two of the five viruses.

Full citation and article access:

Saeed M, Kapell S, Hertz NT, Wu X, Bell K, et al. (2020) Defining the proteolytic landscape during enterovirus infection. PLOS Pathogens 16(9): e1008927. https://doi.org/10.1371/journal.ppat.1008927

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