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
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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).
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Structure of TRPV1 ion channel determined by single particle electron cryo-microscopyPublicly available at the Protien Data Bank (accession no. 3J5P).
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Structure of the capsaicin receptor, TRPV1, determined by single particle electron cryo-microscopyPublicly available at the EMDataBank (accesion no. EMD-5778).
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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).
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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).
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Cryo-EM structure of the human gamma-secretase complex at 3.4 angstrom resolution.Publicly available at the Protien Data Bank (accession no. 5A63).
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Cryo-EM structure of the human gamma-secretase complex at 3.4 angstrom resolutionPublicly available at the EMDataBank (accesion no. EMD-3061).
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Structure of a extracellular domainPublicly available at the Protien Data Bank (accession no. 4UPC).
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Three-dimensional structure of human gamma-secretase at 4.5 angstrom resolutionPublicly available at the EMDataBank (accesion no. EMD-2677).
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Thermoplasma acidophilum 20S proteasomePublicly available at the Protien Data Bank (accession no. 3J9I).
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3D reconstruction of archaeal 20S proteasomePublicly available at the EMDataBank (accesion no. EMD-5623).
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Structure of beta-galactosidase at 3.2-A resolution obtained by cryo-electron microscopyPublicly available at the Protien Data Bank (accession no. 3J7H).
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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.
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Further reading
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- Structural Biology and Molecular Biophysics
Lipid droplets (LDs) are organelles formed in the endoplasmic reticulum (ER) to store triacylglycerol (TG) and sterol esters. The ER protein seipin is key for LD biogenesis. Seipin forms a cage-like structure, with each seipin monomer containing a conserved hydrophobic helix and two transmembrane (TM) segments. How the different parts of seipin function in TG nucleation and LD budding is poorly understood. Here, we utilized molecular dynamics simulations of human seipin, along with cell-based experiments, to study seipin’s functions in protein–lipid interactions, lipid diffusion, and LD maturation. An all-atom simulation indicates that seipin TM segment residues and hydrophobic helices residues located in the phospholipid tail region of the bilayer attract TG. Simulating larger, growing LDs with coarse-grained models, we find that the seipin TM segments form a constricted neck structure to facilitate conversion of a flat oil lens into a budding LD. Using cell experiments and simulations, we also show that conserved, positively charged residues at the end of seipin’s TM segments affect LD maturation. We propose a model in which seipin TM segments critically function in TG nucleation and LD growth.
