Gene

Molecular biophysics

Algorithm

Biology

Statistics

Cryo-electron microscopy

Nuclear magnetic resonance

Biochemistry

Computer science

Spliceosome

Physics

Mathematics

RNA splicing

Domain (mathematical analysis)

Mathematical analysis

Artificial intelligence

Chemistry

Subspace topology

Flexibility (engineering)

Biological system

Biophysics

Journal of Structural Biology

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.

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