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. 2020 Sep 18;11(1):4734.
doi: 10.1038/s41467-020-18403-x.

Retrieving functional pathways of biomolecules from single-particle snapshots

Ali Dashti #  1 , Ghoncheh Mashayekhi #  1 , Mrinal Shekhar  2   3 , Danya Ben Hail  4 , Salah Salah  4   5   6 , Peter Schwander  1 , Amedee des Georges  7   8   9 , Abhishek Singharoy  10 , Joachim Frank  11   12 , Abbas Ourmazd  13
Affiliations

Retrieving functional pathways of biomolecules from single-particle snapshots

Ali Dashti et al. Nat Commun. .

Abstract

A primary reason for the intense interest in structural biology is the fact that knowledge of structure can elucidate macromolecular functions in living organisms. Sustained effort has resulted in an impressive arsenal of tools for determining the static structures. But under physiological conditions, macromolecules undergo continuous conformational changes, a subset of which are functionally important. Techniques for capturing the continuous conformational changes underlying function are essential for further progress. Here, we present chemically-detailed conformational movies of biological function, extracted data-analytically from experimental single-particle cryo-electron microscopy (cryo-EM) snapshots of ryanodine receptor type 1 (RyR1), a calcium-activated calcium channel engaged in the binding of ligands. The functional motions differ substantially from those inferred from static structures in the nature of conformationally active structural domains, the sequence and extent of conformational motions, and the way allosteric signals are transduced within and between domains. Our approach highlights the importance of combining experiment, advanced data analysis, and molecular simulations.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Energy landscapes with and without ligands, and transition probability map.
a Energy landscapes without ligands (upper surface), and with ligands (Ca2+, ATP, and caffeine) (lower surface). The landscapes are described in terms of the most important two of a common set of orthogonal conformational coordinates. The curved path represents a high-probability route to the binding of ligands. This path starts at the minimum-energy conformation of RyR1 without ligands (“START”), follows the conduit of lowest energy to a point with a high probability of transition to the with-ligands energy landscape (“HOT”), and terminates at the minimum-energy conformation with ligands (“FINISH”). Six representative conformational states along the path, in white, are selected for validation with molecular dynamics simulations (S1–S6). b Probability map for transitions from the energy landscape without ligands to the energy landscape with ligands. The axes are the same as in Fig. 1a, with magenta discs indicating the positions of the minima of the energy landscapes. The magenta arrow indicates the position of the transition point.
Fig. 2
Fig. 2. Conformational changes at calcium and ATP-binding sites along the functional path.
Ryanodine receptor 1 (RyR1) conformational changes at calcium and ATP-binding sites along the functional trajectory of Fig. 1a, augmented with excursions from the minimum-energy points along CC1. These excursions are in the increasing CC1 direction on the no-ligand landscape, and in the decreasing CC1 direction on the with-ligand landscape. Top: The general region and the specific sites examined in detail. Distance variations between two amino acid backbones on the opposite side of the ATP and calcium are shown. The amino acids used for measurement are represented as sticks, the rest of the molecule in blue ribbon for the model corresponding to “START” in Fig. 1a, and in green ribbon for model corresponding to “FINISH”. Bottom: Distance variations between carbon-alpha backbone atoms of two opposing residues at each of the calcium and ATP binding sites for 50 structures along the functional and interpolated trajectories. For the latter, 50 structures were extracted from a morph between a model of RyR1 without ligands (PDB: 5TB4) and a model of RyR1 with calcium, ATP, and caffeine (PDB: 5T9V). To analyze the functional route, six published models (PDB: 5TB4, 5T9R, 5TAP, 5T9V, 5TAL, 5TAQ) were each fitted and refined into the maps along the trajectory. Error bars represent the full scatter (not standard deviation) of the results obtained with different starting models.
Fig. 3
Fig. 3. Conformational changes at the caffeine binding site along the functional path.
Ryanodine receptor 1 (RyR1) conformational changes at the caffeine binding site along the functional trajectory of Fig. 1a, augmented with excursions from the minimum-energy points along CC1. These excursions are in the increasing CC1 direction on the no-ligand landscape, and in the decreasing CC1 direction on the with-ligand landscape. Top: The general region and the specific site (inset) are examined in detail. The molecule is shown in blue ribbon for the model corresponding to “START”, and in green ribbon for model corresponding to “FINISH” in Fig. 1a. Bottom: Distance variations between two pairs of amino acid backbones on the opposite side of caffeine. The amino acids used for measurement are represented as sticks. Error bars represent the full scatter (not standard deviation) of the results obtained with six different starting models.
Fig. 4
Fig. 4. Conformational changes in the pore and EF-hand domains along the augmented functional path.
The general region and the specific sites (insets) are examined in detail. Distance variations between two amino acid backbones: in the pore measured at Ile4937 (left); and between EF hand (EF) at Glu4075 and S2S3 domain at Phe4732 (right). The amino acids used for measurement are represented as sticks, the rest of the molecule in blue ribbon for the model corresponding to “START” in Fig. 1a, and in green ribbon for model corresponding to “FINISH”. Error bars represent the full scatter (not standard deviation) of the results obtained with six different starting models.
Fig. 5
Fig. 5. Conformational changes at interprotomer contact sites along the augmented functional path.
The monomers are shown in different colors. Red markers highlight known point-mutations involved in malignant hyperthermia-1 and central core disease of muscle. Distance variations are between two amino acid backbones at contact sites between the monomers. The amino acids used for measurement are shown as sticks. Distance variations between carbon-alpha backbone atoms of two opposing residues along the augmented functional path. Error bars represent the full scatter (not standard deviation) of the results obtained with six different starting models.
Fig. 6
Fig. 6. Functional path vs. interpolation between discrete clusters.
a Distribution on the energy landscape of Fig. 1a of snapshots assigned by maximum likelihood to two discrete conformational clusters. Each white curve encloses a region of high snapshot density. These two classes were previously identified as representing the extremes of the conformational range, and used to infer function by discrete cluster analysis. b Overall conformational changes along the functional path vs. linear interpolation between the two maximum-likelihood classes previously used for functional inference (see panel a). The ryanodine receptor 1 (RyR1) atomic model is shown in sphere representation, with color indicating the displacement of each atom from its “START” position. c Conformational changes in the activation core domain (residues: 3614-5037) along the functional path of Fig. 1a. The RyR1 backbone structure is shown as sticks, with the color of each atom indicating its atomic displacement (see color bar). The largest atomic displacements along the functional trajectory are confined to a narrow allosteric conduit connecting the ligand-binding sites to the EF hand.
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