1. Structural Biology and Molecular Biophysics
  2. Computational and Systems Biology
Download icon

Molecular dynamics-based model refinement and validation for sub-5 Å cryo-electron microscopy maps

  1. Abhishek Singharoy  Is a corresponding author
  2. Ivan Teo
  3. Ryan McGreevy
  4. John E Stone
  5. Jianhua Zhao
  6. Klaus Schulten  Is a corresponding author
  1. University of Illinois at Urbana Champaign, United States
  2. University of Illinois at Urbana-Champaign, United States
  3. University of California, San Francisco, United States
Tools and Resources
  • Cited 77
  • Views 4,887
  • Annotations
Cite this article as: eLife 2016;5:e16105 doi: 10.7554/eLife.16105

Abstract

Two structure determination methods, based on the molecular dynamics flexible fitting (MDFF) paradigm, are presented that resolve sub-5-Å cryo-electron microscopy (EM) maps with either single structures or ensembles of such structures. The methods, denoted cascade MDFF and resolution exchange MDFF, sequentially re-refine a search model against a series of maps of progressively higher resolutions, which ends with the original experimental resolution. Application of sequential re-refinement enables MDFF to achieve a convergence radius of ~25Å demonstrated with the accurate modeling of β-galactosidase and TRPV1 proteins at 3.2Å and 3.4Å resolution. The MDFF refinements uniquely offer map-model validation and B-factor determination criteria based on the inherent dynamics of the respective macromolecules studied, captured employing local root mean square fluctuations. The MDFF tools are made available to researchers through an easy-to-use and cost-effective cloud computing resource on Amazon Web Services.

Data availability

The following previously published data sets were used

Article and author information

Author details

  1. Abhishek Singharoy

    Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana Champaign, Urbana, United States
    For correspondence
    singharo@illinois.edu
    Competing interests
    The authors declare that no competing interests exist.

  2. Ivan Teo

    Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Ryan McGreevy

    Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. John E Stone

    Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Jianhua Zhao

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Klaus Schulten

    Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States
    For correspondence
    kschulte@ks.uiuc.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7192-9632

Reviewing Editor

  1. Axel T Brunger, Howard Hughes Medical Institute, Stanford University, United States

Publication history

  1. Received: March 16, 2016
  2. Accepted: July 6, 2016
  3. Accepted Manuscript published: July 7, 2016 (version 1)
  4. Accepted Manuscript updated: July 8, 2016 (version 2)
  5. Version of Record published: August 18, 2016 (version 3)
  6. Version of Record updated: October 7, 2016 (version 4)

Copyright

© 2016, singharoy et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 4,887
    Page views
  • 1,015
    Downloads
  • 77
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Categories and tags

Further reading

    1. Immunology and Inflammation
    2. Structural Biology and Molecular Biophysics

    Neutrophil-mediated oxidative stress and albumin structural damage predict COVID-19-associated mortality

    Mohamed A Badawy et al.
    Research Article

    Human serum albumin (HSA) is the frontline antioxidant protein in blood with established anti-inflammatory and anticoagulation functions. Here we report that COVID-19-induced oxidative stress inflicts structural damages to HSA and is linked with mortality outcome in critically ill patients. We recruited 39 patients who were followed up for a median of 12.5 days (1-35 days), among them 23 had died. Analyzing blood samples from patients and healthy individuals (n=11), we provide evidence that neutrophils are major sources of oxidative stress in blood and that hydrogen peroxide is highly accumulated in plasmas of non-survivors. We then analyzed electron paramagnetic resonance (EPR) spectra of spin labelled fatty acids (SLFA) bound with HSA in whole blood of control, survivor, and non-survivor subjects (n=10-11). Non-survivor' HSA showed dramatically reduced protein packing order parameter, faster SLFA correlational rotational time, and smaller S/W ratio (strong-binding/weak-binding sites within HSA), all reflecting remarkably fluid protein microenvironments. Following loading/unloading of 16-DSA we show that transport function of HSA maybe impaired in severe patients. Stratified at the means, Kaplan–Meier survival analysis indicated that lower values of S/W ratio and accumulated H2O2 in plasma significantly predicted in-hospital mortality (S/W≤0.15, 81.8% (18/22) vs. S/W>0.15, 18.2% (4/22), p=0.023; plasma [H2O2]>8.6 mM, 65.2% (15/23) vs. 34.8% (8/23), p=0.043). When we combined these two parameters as the ratio ((S/W)/[H2O2]) to derive a risk score, the resultant risk score lower than the mean (< 0.019) predicted mortality with high fidelity (95.5% (21/22) vs. 4.5% (1/22), logrank c2 = 12.1, p=4.9x10-4). The derived parameters may provide a surrogate marker to assess new candidates for COVID-19 treatments targeting HSA replacements and/or oxidative stress.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics

    EGFR transactivates RON to drive oncogenic crosstalk

    Carolina Franco Nitta et al.
    Research Article

    Crosstalk between different receptor tyrosine kinases (RTKs) is thought to drive oncogenic signaling and allow therapeutic escape. EGFR and RON are two such RTKs from different subfamilies, which engage in crosstalk through unknown mechanisms. We combined high-resolution imaging with biochemical and mutational studies to ask how EGFR and RON communicate. EGF stimulation promotes EGFR-dependent phosphorylation of RON, but ligand stimulation of RON does not trigger EGFR phosphorylation – arguing that crosstalk is unidirectional. Nanoscale imaging reveals association of EGFR and RON in common plasma membrane microdomains. Two-color single particle tracking captured formation of complexes between RON and EGF-bound EGFR. Our results further show that RON is a substrate for EGFR kinase, and that transactivation of RON requires formation of a signaling competent EGFR dimer. These results support a role for direct EGFR/RON interactions in propagating crosstalk, such that EGF-stimulated EGFR phosphorylates RON to activate RON-directed signaling.

    1. Structural Biology and Molecular Biophysics

    Structure of Mycobacterium tuberculosis cytochrome bcc in complex with Q203 and TB47, two anti-TB drug candidates

    Shan Zhou et al.
    Research Article

    Pathogenic mycobacteria pose a sustained threat to global human health. Recently, cytochrome bcc complexes have gained interest as targets for antibiotic drug development. However, there is currently no structural information for the cytochrome bcc complex from these pathogenic mycobacteria. Here, we report the structures of Mycobacterium tuberculosis cytochrome bcc alone (2.68 Å resolution) and in complex with clinical drug candidates Q203 (2.67 Å resolution) and TB47 (2.93 Å resolution) determined by single-particle cryo-electron microscopy. M. tuberculosis cytochrome bcc forms a dimeric assembly with endogenous menaquinone/menaquinol bound at the quinone/quinol-binding pockets. We observe Q203 and TB47 bound at the quinol-binding site and stabilized by hydrogen bonds with the side chains of QcrBThr313 and QcrBGlu314, residues that are conserved across pathogenic mycobacteria. These high-resolution images provide a basis for the design of new mycobacterial cytochrome bcc inhibitors that could be developed into broad-spectrum drugs to treat mycobacterial infections.