Cryo-EM reconstruction of continuous heterogeneity by Laplacian spectral volumes.

Single-particle electron cryomicroscopy is an essential tool for high-resolution 3D reconstruction of proteins and other biological macromolecules. An important challenge in cryo-EM is the reconstruction of non-rigid molecules with parts that move and deform. Traditional reconstruction methods fail in these cases, resulting in smeared reconstructions of the moving parts. This poses a major obstacle for structural biologists, who need high-resolution reconstructions of entire macromolecules, moving parts included. To address this challenge, we present a new method for the reconstruction of macromolecules exhibiting continuous heterogeneity. The proposed method uses projection images from multiple viewing directions to construct a graph Laplacian through which the manifold of three-dimensional conformations is analyzed. The 3D molecular structures are then expanded in a basis of Laplacian eigenvectors, using a novel generalized tomographic reconstruction algorithm to compute the expansion coefficients. These coefficients, which we name spectral volumes, provide a high-resolution visualization of the molecular dynamics. We provide a theoretical analysis and evaluate the method empirically on several simulated data sets.

Photodamage to isolated mononucleotides--photodissociation spectra and fragment channels.

UV excitation of isolated singly-charged deprotonated mononucleotide anions in the gas phase can lead to their dissociation. We present mass spectrometry results, photodepletion and photofragment action spectra on the UV-photodissociation of deprotonated 2'-deoxyribonucleobase-5'-monophosphates with adenine, cytosine, guanine and thymine as nucleobases. We observe the same anionic fragments as in earlier experiments on collision-induced dissociation, although their relative intensities are quite different, especially with respect to the abundance of the deprotonated base anions. The fragment channels correspond to loss of genetic information by cleavage of the CN glycosidic bond and to strand breaking by severing the phosphate-sugar link. We compare the photodissociation spectra with UV absorption spectra of aqueous solutions of the same species and discuss the photodissociation behavior in the context of possible mechanisms and ergodic versus non-ergodic fragmentation.