Cryo-EM map interpretation and protein model-building using iterative map segmentation.

A procedure for building protein chains into maps produced by single-particle electron cryo-microscopy (cryo-EM) is described. The procedure is similar to the way an experienced structural biologist might analyze a map, focusing first on secondary structure elements such as helices and sheets, then varying the contour level to identify connections between these elements. Since the high density in a map typically follows the main-chain of the protein, the main-chain connection between secondary structure elements can often be identified as the unbranched path between them with the highest minimum value along the path. This chain-tracing procedure is then combined with finding side-chain positions based on the presence of density extending away from the main path of the chain, allowing generation of a Cα model. The Cα model is converted to an all-atom model and is refined against the map. We show that this procedure is as effective as other existing methods for interpretation of cryo-EM maps and that it is considerably faster and produces models with fewer chain breaks than our previous methods that were based on approaches developed for crystallographic maps

Peptide model helices in lipid membranes: insertion, positioning, and lipid response on aggregation studied by X-ray scattering

Studying membrane active peptides or protein fragments within the lipid bilayer environment is particularly challenging in the case of synthetically modified, labeled, artificial, or recently discovered native structures. For such samples the localization and orientation of the molecular species or probe within the lipid bilayer environment is the focus of research prior to an evaluation of their dynamic or mechanistic behavior. X-ray scattering is a powerful method to study peptide/lipid interactions in the fluid, fully hydrated state of a lipid bilayer. For one, the lipid response can be revealed by observing membrane thickening and thinning as well as packing in the membrane plane; at the same time, the distinct positions of peptide moieties within lipid membranes can be elucidated at resolutions of up to several angstroms by applying heavy-atom labeling techniques. In this study, we describe a generally applicable X-ray scattering approach that provides robust and quantitative information about peptide insertion and localization as well as peptide/lipid interaction within highly oriented, hydrated multilamellar membrane stacks. To this end, we have studied an artificial, designed β-helical peptide motif in its homodimeric and hairpin variants adopting different states of oligomerization. These peptide lipid complexes were analyzed by grazing incidence diffraction (GID) to monitor changes in the lateral lipid packing and ordering. In addition, we have applied anomalous reflectivity using synchrotron radiation as well as in-house X-ray reflectivity in combination with iodine-labeling in order to determine the electron density distribution ρ(z) along the membrane normal (z axis), and thereby reveal the hydrophobic mismatch situation as well as the position of certain amino acid side chains within the lipid bilayer. In the case of multiple labeling, the latter technique is not only applicable to demonstrate the peptide’s reconstitution but also to generate evidence about the relative peptide orientation with respect to the lipid bilayer