Conformational space exploration of cryo-EM structures by variability refinement

Afonine, Pavel V.; Gobet, Alexia; Moissonnier, Loïck; Martin, Juliette; Poon, Billy K.; Chaptal, Vincent

doi:10.1016/j.bbamem.2023.184133

Cited by 7 publications

(12 citation statements)

References 38 publications

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“…Model series were generated using the 3DVA display job using 10 frames. To better analyze motion associated with the first two variability components, a series of models were generated corresponding to each volume series using phenix.varref (39). Supplemental movies were generated with Chimera and ChimeraX.…”

Section: D Variability Analysismentioning

confidence: 99%

Conformational landscape of soluble α-klotho revealed by cryogenic electron microscopy

Schnicker,

Xu,

Amir

et al. 2024

Preprint

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α-Klotho (sKLA) is a type-1 membranous protein that can associate with fibroblast growth factor receptor (FGFR) to form co-receptor for FGF23. The ectodomain of unassociated KLA is shed as soluble KLA (sKLA) to exert FGFR/FGF23-independent pleiotropic functions. The previously determined X-ray crystal structure of the extracellular region of sKLA in complex with FGF23 and FGFR1c suggests that sKLA functions solely as an on-demand coreceptor for FGF23. To understand the FGFR/FGF23-independent pleiotropic functions of sKLA, we investigated biophysical properties and structure of apo-sKLA. Mass photometry revealed that sKLA can form a stable structure with FGFR and/or FGF23 as well as sKLA dimer in solution. Single particle cryogenic electron microscopy (cryo-EM) supported the dimeric structure of sKLA. Cryo-EM further revealed a 3.3A resolution structure of apo-sKLA that overlays well with its counterpart in the ternary complex with several distinct features. Compared to the ternary complex, the KL2 domain of apo-sKLA is more flexible. 3D variability analysis revealed that apo-sKLA adopts conformations with different KL1-KL2 interdomain bending and rotational angles. The potential multiple forms and shapes of sKLA support its role as FGFR-independent hormone with pleiotropic functions. A comprehensive understanding of the sKLA conformational landscape will provide the foundation for developing klotho-related therapies for diseases.

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Section: D Variability Analysismentioning

confidence: 99%

Conformational landscape of soluble α-klotho revealed by cryogenic electron microscopy

Schnicker,

Xu,

Amir

et al. 2024

Preprint

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“…5 components were asked as output but the main movements are observed in the 3 first components, which were subjected to variability refinement. Each 20 maps originating from 3DVA were input to variability refinement (phenix.varref [18]) along with the final refined model. 50 models were generated for each map, with restraints adapted to the resolution of 3DVA calculations, and only the best model was kept for each map.…”

Section: Variability Analysis and Variability Refinementmentioning

confidence: 99%

R6G narrows BmrA conformational spectrum for a more efficient use of ATP

Gobet,

Moissonnier,

Zarkadas

et al. 2024

Preprint

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Multidrug ABC transporters harness the energy of ATP binding and hydrolysis to change conformation and thereby translocate substrates out of the cell to detoxify them. While this general access mechanism scheme is well accepted, molecular details of this interplay is still elusive. Rhodamine6G binding on a catalytic mutant of the homodimeric multidrug ABC transporter BmrA triggers a cooperative binding of ATP on the two identical nucleotide-binding-sites, otherwise Michaelian. We investigated this asymmetric behavior via a structural-enzymology approach, solving cryoEM structure of BmrA at defined ATP ratio along the enzymatic transition, highlighting the plasticity of BmrA as it undergoes the transition from inward to outward facing conformations. Analysis of continuous heterogeneity within cryoEM data and structural dynamics, revealed that Rhodamine6G narrows the conformational spectrum explored by the nucleotide-binding-domains, describing the allosteric effect of drug binding that optimizes the ATP-dependent conversion of the transporter to the outward-facing state. Following on these findings, the effect of drug-binding showed an ATPase stimulation and a maximal transport activity of the wild-type protein at the concentration-range where the allosteric transition occurs. Drug diffusion rate is the likely rate-limiting step of the reaction, while drug transport and ATPase activities are in effect uncoupled

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“…114 For high-resolution maps (<4 Å), it is often possible to use standard interactive refinement tools adapted from x-ray crystallography such as REFMAC5 and Phenix, 115,116 which have been updated to account for the differences in how atoms scatter x-rays and electrons, 117,118 the differences in the data processing approaches 119 and the handling of initial models from machine learning methods 120,121 such as AlphaFold 31,32 and Modelangelo 122 and more recently the large numbers of structures coming from continuous heterogeneity analyses. 123 For lower resolution maps, the common approaches are rigid and flexible fitting using pieces from existing or modeled structures (as discussed below) and more CG modeling approaches (see Section 7).…”

Section: Flexible Fitting: Structural Refinement and Characterization...mentioning

confidence: 99%

Computational biophysics meets cryo‐EM revolution in the search for the functional dynamics of biomolecular systems

Costa,

Gur,

Krieger

et al. 2023

WIREs Comput Mol Sci

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There is a variety of experimental and computational techniques available to explore protein dynamics, each presenting advantages and limitations. One promising experimental technique that is driving the development of computational methods is cryo‐electron microscopy (cryo‐EM). Cryo‐EM provides molecular‐level structural data and first estimates of conformational landscape from single particle analysis but cannot track real‐time protein dynamics and may contain uncertainties in atomic positions especially at highly dynamic regions. Molecular simulations offer atomic‐level insights into protein dynamics; however, their computing time requirements limit the conformational sampling accuracy, and it is often hard, to assess by full‐atomic simulations the cooperative movements of biological interest for large assemblies such as those resolved by cryo‐EM. Coarse‐grained (CG) simulations permit us to explore such systems, but at the costs of lower resolution and potentially incomplete sampling of conformational space. On the other hand, analytical methods may circumvent sampling limitations. In particular, elastic network models‐based normal mode analyses (ENM‐NMA) provide unique solutions for the complete mode spectra near equilibrium states, even for systems of megadaltons, and may thus deliver information on mechanisms of motions relevant to biological function. Yet, they lack atomic resolution as well as temporal information for non‐equilibrium systems. Given the complementary nature of these methods, the integration of molecular simulations and ENM‐NMA into hybrid methodologies has gained traction. This review presents the current state‐of‐the‐art in structure‐based computations and how they are helping us gain a deeper understanding of biological mechanisms, with emphasis on the development of hybrid methods accompanying the advances in cryo‐EM.This article is categorized under: Structure and Mechanism > Computational Biochemistry and Biophysics

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Conformational space exploration of cryo-EM structures by variability refinement

Cited by 7 publications

References 38 publications

Conformational landscape of soluble α-klotho revealed by cryogenic electron microscopy

Conformational landscape of soluble α-klotho revealed by cryogenic electron microscopy

R6G narrows BmrA conformational spectrum for a more efficient use of ATP

Computational biophysics meets cryo‐EM revolution in the search for the functional dynamics of biomolecular systems

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