Molecular dynamics-based model refinement and validation for sub-5 Å cryo-electron microscopy maps
- University of Illinois at Urbana Champaign, United States
- University of Illinois at Urbana-Champaign, United States
- University of California, San Francisco, United States
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
-
NIZN[FE4S4] AND NINI[FE4S4] CLUSTERS IN CLOSED AND OPEN ALPHA SUBUNITS OF ACETYL-COA SYNTHASE/CARBON MONOXIDE DEHYDROGENASEPublicly available at the Protien Data Bank (accession no. 1OAO).
-
Structure of TRPV1 ion channel determined by single particle electron cryo-microscopyPublicly available at the Protien Data Bank (accession no. 3J5P).
-
Structure of the capsaicin receptor, TRPV1, determined by single particle electron cryo-microscopyPublicly available at the EMDataBank (accesion no. EMD-5778).
-
2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitorPublicly available at the Protien Data Bank (accession no. 5A1A).
-
2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitorPublicly available at the EMDataBank (accesion no. EMD-2984).
-
Cryo-EM structure of the human gamma-secretase complex at 3.4 angstrom resolution.Publicly available at the Protien Data Bank (accession no. 5A63).
-
Cryo-EM structure of the human gamma-secretase complex at 3.4 angstrom resolutionPublicly available at the EMDataBank (accesion no. EMD-3061).
-
Structure of a extracellular domainPublicly available at the Protien Data Bank (accession no. 4UPC).
-
Three-dimensional structure of human gamma-secretase at 4.5 angstrom resolutionPublicly available at the EMDataBank (accesion no. EMD-2677).
-
Thermoplasma acidophilum 20S proteasomePublicly available at the Protien Data Bank (accession no. 3J9I).
-
3D reconstruction of archaeal 20S proteasomePublicly available at the EMDataBank (accesion no. EMD-5623).
-
Structure of beta-galactosidase at 3.2-A resolution obtained by cryo-electron microscopyPublicly available at the Protien Data Bank (accession no. 3J7H).
-
Structure of beta-galactosidase at 3.2-A resolution obtained by cryo-electron microscopyPublicly available at the EMDataBank (accesion no. EMD-5995).
Article and author information
Author details
Reviewing Editor
- Axel T Brunger, Howard Hughes Medical Institute, Stanford University, United States
Publication history
- Received: March 16, 2016
- Accepted: July 6, 2016
- Accepted Manuscript published: July 7, 2016 (version 1)
- Accepted Manuscript updated: July 8, 2016 (version 2)
- Version of Record published: August 18, 2016 (version 3)
- 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
-
- 5,416
- Page views
-
- 1,064
- Downloads
-
- 102
- 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)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Molecular dynamics-based model refinement and validation for sub-5 Å cryo-electron microscopy maps
eLife 5:e16105.
https://doi.org/10.7554/eLife.16105
Further reading
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Aurora B, together with IN-box, the C-terminal part of INCENP, forms an enzymatic complex that ensures faithful cell division. The [Aurora B/IN-box] complex is activated by autophosphorylation in the Aurora B activation loop and in IN-box, but it is not clear how these phosphorylations activate the enzyme. We used a combination of experimental and computational studies to investigate the effects of phosphorylation on the molecular dynamics and structure of [Aurora B/IN-box]. In addition, we generated partially phosphorylated intermediates to analyze the contribution of each phosphorylation independently. We found that the dynamics of Aurora and IN-box are interconnected, and IN-box plays both positive and negative regulatory roles depending on the phosphorylation status of the enzyme complex. Phosphorylation in the activation loop of Aurora B occurs intramolecularly and prepares the enzyme complex for activation, but two phosphorylated sites are synergistically responsible for full enzyme activity.
-
- Neuroscience
- Structural Biology and Molecular Biophysics
The cation-permeable TRPV2 channel is important for cardiac and immune cell function. Cannabidiol (CBD), a non-psychoactive cannabinoid of clinical relevance, is one of the few molecules known to activate TRPV2. Using the patch-clamp technique, we discover that CBD can sensitize current responses of the rat TRPV2 channel to the synthetic agonist 2-aminoethoxydiphenyl borate (2-APB) by over two orders of magnitude, without sensitizing channels to activation by moderate (40°C) heat. Using cryo-EM, we uncover a new small-molecule binding site in the pore domain of rTRPV2 in addition to a nearby CBD site that had already been reported. The TRPV1 and TRPV3 channels are also activated by 2-APB and CBD and share multiple conserved features with TRPV2, but we find that strong sensitization by CBD is only observed in TRPV3, while sensitization for TRPV1 is much weaker. Mutations at non-conserved positions between rTRPV2 and rTRPV1 in either the pore domain or the CBD sites failed to confer strong sensitization by CBD in mutant rTRPV1 channels. Together, our results indicate that CBD-dependent sensitization of rTRPV2 channels engages multiple channel regions, and that the difference in sensitization strength between rTRPV2 and rTRPV1 channels does not originate from amino acid sequence differences at the CBD binding site or the pore domain. The remarkably robust effect of CBD on TRPV2 and TRPV3 channels offers a promising new tool to both understand and overcome one of the major roadblocks in the study of these channels – their resilience to activation.
