Grigore Pintilie

Quantitative analysis of cryo-EM density map segmentation by watershed and scale-space filtering, and fitting of structures by alignment to regions.

Cryo-electron microscopy produces 3D density maps of molecular machines, which consist of various molecular components such as proteins and RNA. Segmentation of individual components in such maps is a challenging task, and is mostly accomplished interactively. We present an approach based on the immersive watershed method and grouping of the resulting regions using progressively smoothed maps. The method requires only three parameters: the segmentation threshold, a smoothing step size, and the number of smoothing steps. We first apply the method to maps generated from molecular structures and use a quantitative metric to measure the segmentation accuracy. The method does not attain perfect accuracy, however it produces single or small groups of regions that roughly match individual proteins or subunits. We also present two methods for fitting of structures into density maps, based on aligning the structures with single regions or small groups of regions. The first method aligns centers and principal axes, whereas the second aligns centers and then rotates the structure to find the best fit. We describe both interactive and automated ways of using these two methods. Finally, we show segmentation and fitting results for several experimentally-obtained density maps.

Structure of a Biologically Active Estrogen Receptor- Coactivator Complex on DNA

Estrogen receptor (ER/ESR1) is a transcription factor critical for development, reproduction, metabolism, and cancer. ER function hinges on its ability to recruit primary and secondary coactivators, yet structural information on the full-length receptor-coactivator complex to complement preexisting and sometimes controversial biochemical information is lacking. Here, we use cryoelectron microscopy (cryo-EM) to determine the quaternary structure of an active complex of DNA-bound ERα, steroid receptor coactivator 3 (SRC-3/NCOA3), and a secondary coactivator (p300/EP300). Our structural model suggests the following assembly mechanism for the complex: each of the two ligand-bound ERα monomers independently recruits one SRC-3 protein via the transactivation domain of ERα; the two SRC-3s in turn bind to different regions of one p300 protein through multiple contacts. We also present structural evidence for the location of activation function 1 (AF-1) in a full-length nuclear receptor, which supports a role for AF-1 in SRC-3 recruitment.

EMDataBank unified data resource for 3DEM.

Three-dimensional Electron Microscopy (3DEM) has become a key experimental method in structural biology for a broad spectrum of biological specimens from molecules to cells. The EMDataBank project provides a unified portal for deposition, retrieval and analysis of 3DEM density maps, atomic models and associated metadata (emdatabank.org). We provide here an overview of the rapidly growing 3DEM structural data archives, which include maps in EM Data Bank and map-derived models in the Protein Data Bank. In addition, we describe progress and approaches toward development of validation protocols and methods, working with the scientific community, in order to create a validation pipeline for 3DEM data.

Comparison of Segger and other methods for segmentation and rigid-body docking of molecular components in cryo-EM density maps.

Segmentation and docking are useful methods for the discovery of molecular components in electron cryo-microscopy (cryo-EM) density maps of macromolecular complexes. In this article, we describe the segmentation and docking methods implemented in Segger. For 11 targets posted in the 2010 cryo-EM challenge, we segmented the regions corresponding to individual molecular components using Segger. We then used the segmented regions to guide rigid-body docking of individual components. Docking results were evaluated by comparing the docked components with published structures, and by calculation of several scores, such as atom inclusion, density occupancy, and geometry clash. The accuracy of the component segmentation using Segger and other methods was assessed by comparing segmented regions with docked components.

Identifying components in 3D density maps of protein nanomachines by multi-scale segmentation

Segmentation of density maps obtained using cryo-electron microscopy (cryo-EM) is a challenging task, and is typically accomplished by time-intensive interactive methods. The goal of segmentation is to identify the regions inside the density map that correspond to individual components. We present a multi-scale segmentation method for accomplishing this task that requires very little user interaction. The method uses the concept of scale space, which is created by convolution of the input density map with a Gaussian filter. The latter process smoothes the density map. The standard deviation of the Gaussian filter is varied, with smaller values corresponding to finer scales and larger values to coarser scales. Each of the maps at different scales is segmented using the watershed method, which is very efficient, completely automatic, and does not require the specification of seed points. Some detail is lost in the smoothing process. A sharpening process reintroduces detail into the segmentation at the coarsest scale by using the segmentations at the finer scales. We apply the method to simulated density maps, where the exact segmentation (or ground truth) is known, and rigorously evaluate the accuracy of the resulting segmentations.

The 3.5-Å CryoEM Structure of Nanodisc-Reconstituted Yeast Vacuolar ATPase Vo Proton Channel

The molecular mechanism of transmembrane proton translocation in rotary motor ATPases is not fully understood. Here, we report the 3.5-Å resolution cryoEM structure of the lipid nanodisc-reconstituted Vo proton channel of the yeast vacuolar H+-ATPase, captured in a physiologically relevant, autoinhibited state. The resulting atomic model provides structural detail for the amino acids that constitute the proton pathway at the interface of the proteolipid ring and subunit a. Based on the structure and previous mutagenesis studies, we propose the chemical basis of transmembrane proton transport. Moreover, we discovered that the C terminus of the assembly factor Voa1 is an integral component of mature Vo. Voa1s C-terminal transmembrane α helix is bound inside the proteolipid ring, where it contributes to the stability of the complex. Our structure rationalizes possible mechanisms by which mutations in human Vo can result in disease phenotypes and may thus provide new avenues for therapeutic interventions.

The first single particle analysis Map Challenge: A summary of the assessments

The recent successes of cryo-electron microscopy fostered great expectation of solving many new and previously recalcitrant biomolecular structures. However, it also brings with it the danger of compromising the validity of the outcomes if not done properly. The Map Challenge is a first step in assessing the state of the art and to shape future developments in data processing. The organizers presented seven cases for single particle reconstruction, and 27 members of the community responded with 66 submissions. Seven groups analyzed these submissions, resulting in several assessment reports, summarized here. We devised a range of analyses to evaluate the submitted maps, including visual impressions, Fourier shell correlation, pairwise similarity and interpretation through modeling. Unfortunately, we did not find strong trends. We ascribe this to the complexity of the challenge, dealing with multiple cases, software packages and processing approaches. This puts the user in the spotlight, where his/her choices becomes the determinant of map quality. The future focus should therefore be on promulgating best practices and encapsulating these in the software. Such practices include adherence to validation principles, most notably the processing of independent sets, proper resolution-limited alignment, appropriate masking and map sharpening. We consider the Map Challenge to be a highly valuable exercise that should be repeated frequently or on an ongoing basis.

Interactive design and simulation of net sculptures

We present a graphical user interface that allows an artist to virtually design and visualize net sculptures. Net sculptures consist of net pieces that are seamlessly connected to each other and to fixed rails. They are flexible and hence dynamic under external forces such as gravity and wind. The interface that we describe allows an artist to create net sculpturesmade up of multiple net pieces. Simple operations such as clicking on points and click-and-drag gestures are used to create and modify individual net pieces, and drag-and-drop gestures are used to connect net pieces to multiple rails. The effect of gravity on the net sculpture is simulated, allowing the artist to simultaneously design and visualize net sculptures as they would appear once installed in a real setting.

GLUBs: games for learning and understanding biology

This paper introduces GLUBs, which are games based on molecular mechanisms that take place in biological systems. Such games make use of interactive 3D graphics, and allow the player to interact with molecular components using simple 2D mouse gestures. They are similar to puzzle games, since the goal is to discover the correct interactions between molecular components. When the correct interactions are found, the player observes a molecular mechanism taking place. GLUBs thus have the potential to communicate much of the growing structural and biochemical knowledge in a fun and interesting way. In the game presented in this paper, the molecular mechanism involves the assisted folding of a protein by a chaperone.