3D variability analysis: Resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM

• Published in: Journal of structural biology Vol. 213; no. 2; p. 107702
• Main Authors: Punjani, Ali, Fleet, David J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2021
• Subjects: Algorithms; Archaeal Proteins - chemistry; Continuous heterogeneity; Cryoelectron Microscopy - methods; Databases, Protein; Electron microscopy; Endopeptidases - chemistry; Expectation maximization algorithm; Image processing; Imaging, Three-Dimensional - methods; NAV1.7 Voltage-Gated Sodium Channel - chemistry; NAV1.7 Voltage-Gated Sodium Channel - metabolism; Plasmodium falciparum - chemistry; Principle component analysis; Receptors, Cannabinoid - chemistry; Ribosome Subunits, Large, Bacterial - chemistry; Ribosomes - chemistry; Signal-To-Noise Ratio; Single particle analysis; Spliceosomes - chemistry; Single particle analysis; Continuous heterogeneity; Image processing; Expectation maximization algorithm; Electron microscopy; Principle component analysis
• ISSN: 1047-8477; 1095-8657
• DOI: 10.1016/j.jsb.2021.107702

[Display omitted] •Continuous structural heterogeneity of proteins is often functionally relevant.•Existing single particle cryo-EM reconstruction algorithms yield static structures.•Algorithm that fits linear subspace model at high resolution is introduced.•New method resolves detailed molecular flexibility from single particle data. Single particle cryo-EM excels in determining static structures of protein molecules, but existing 3D reconstruction methods have been ineffective in modelling flexible proteins. We introduce 3D variability analysis (3DVA), an algorithm that fits a linear subspace model of conformational change to cryo-EM data at high resolution. 3DVA enables the resolution and visualization of detailed molecular motions of both large and small proteins, revealing new biological insight from single particle cryo-EM data. Experimental results demonstrate the ability of 3DVA to resolve multiple flexible motions of α-helices in the sub-50 kDa transmembrane domain of a GPCR complex, bending modes of a sodium ion channel, five types of symmetric and symmetry-breaking flexibility in a proteasome, large motions in a spliceosome complex, and discrete conformational states of a ribosome assembly. 3DVA is implemented in the cryoSPARC software package.