A modified McKinnon-Bates (MKB) algorithm for improved 4D cone-beam computed tomography (CBCT) of the lung.

PURPOSE: Four-dimensional (4D) cone-beam computed tomography (CBCT) of the lung is an effective tool for motion management in radiotherapy but presents a challenge because of slow gantry rotation times. Sorting the individual projections by breathing phase and using an established technique such as Feldkamp-Davis-Kress (FDK) to generate corresponding phase-correlated (PC) three-dimensional (3D) images results in reconstructions (FDK-PC) that often contain severe streaking artifacts due to the sparse angular sampling distributions. These can be reduced by further slowing down the gantry at the expense of incurring unwanted increases in scan times and dose. A computationally efficient alternative is the McKinnon-Bates (MKB) reconstruction algorithm that has shown promise in reducing view aliasing-induced streaking but can produce ghosting artifacts that reduce contrast and impede the determination of motion trajectories. The purpose of this work was to identify and correct shortcomings in the MKB algorithm. METHODS: In the general MKB approach, a time-averaged 3D prior image is first reconstructed. The prior is then forward-projected at the same angles as the original projection data creating time-averaged reprojections. These reprojections are subsequently subtracted from the original (unblurred) projections to create motion-encoded difference projections. The difference projections are reconstructed into PC difference images that are added to the well-sampled 3D prior to create the higher quality 4D image. The cause of the ghosting in the traditional 4D MKB images was studied and traced to motion-induced streaking in the prior that, when reprojected, has the undesirable effect of re-encoding for motion in what should be a purely time-averaged reprojection. A new method, designated as the modified McKinnon-Bates (mMKB) algorithm, was developed based on destreaking the prior. This was coupled with a postprocessing 4D bilateral filter for noise suppression and edge preservation (mMKBbf ). The algorithms were tested with the 4D XCAT phantom using four simulated scan times (57, 60, 120, 180 s) and with two in vivo thorax studies (acquisition time of 60 and 90 s). Contrast-to-noise ratios (CNRs) of the target lesions and overall visual quality of the images were assessed. RESULTS: Prior destreaking (mMKB algorithm) reduced ghosting artifacts and increased CNRs for all cases, with the biggest impacts seen in the end inhale (EI) and end exhale (EE) phases of the respiratory cycle. For the XCAT phantom, mMKB lesion CNR was 44% higher than the MKB lesion CNR and was 81% higher than the FDK-PC lesion CNR (EI and EE phases). The bilateral filter provided a further average CNR improvement of 87% with the highest increases associated with longer scan times. Across all phases and scan times, the maximum mMKBbf -to-FDK-PC CNR improvement was over 300%. In vivo results agreed with XCAT results. Significantly less ghosting was observed throughout the mMKB images including near the lesions-of-interest and the diaphragm allowing for, in one case, visualization of a small tumor with nearly 30 mm of motion. The maximum FDK-PC-to-MKBbf CNR improvement for Patient 1's lesion was 261% and for Patient 2's lesion was 318%. CONCLUSIONS: The 4D mMKB algorithm yields good quality coronal and sagittal images in the thorax that may provide sufficient information for patient verification.

2dx_automator: implementation of a semiautomatic high-throughput high-resolution cryo-electron crystallography pipeline.

The introduction of direct electron detectors (DED) to cryo-electron microscopy has tremendously increased the signal-to-noise ratio (SNR) and quality of the recorded images. We discuss the optimal use of DEDs for cryo-electron crystallography, introduce a new automatic image processing pipeline, and demonstrate the vast improvement in the resolution achieved by the use of both together, especially for highly tilted samples. The new processing pipeline (now included in the software package 2dx) exploits the high SNR and frame readout frequency of DEDs to automatically correct for beam-induced sample movement, and reliably processes individual crystal images without human interaction as data are being acquired. A new graphical user interface (GUI) condenses all information required for quality assessment in one window, allowing the imaging conditions to be verified and adjusted during the data collection session. With this new pipeline an automatically generated unit cell projection map of each recorded 2D crystal is available less than 5 min after the image was recorded. The entire processing procedure yielded a three-dimensional reconstruction of the 2D-crystallized ion-channel membrane protein MloK1 with a much-improved resolution of 5Å in-plane and 7Å in the z-direction, within 2 days of data acquisition and simultaneous processing. The results obtained are superior to those delivered by conventional photographic film-based methodology of the same sample, and demonstrate the importance of drift-correction.

Single particle 3D reconstruction for 2D crystal images of membrane proteins.

In cases where ultra-flat cryo-preparations of well-ordered two-dimensional (2D) crystals are available, electron crystallography is a powerful method for the determination of the high-resolution structures of membrane and soluble proteins. However, crystal unbending and Fourier-filtering methods in electron crystallography three-dimensional (3D) image processing are generally limited in their performance for 2D crystals that are badly ordered or non-flat. Here we present a single particle image processing approach, which is implemented as an extension of the 2D crystallographic pipeline realized in the 2dx software package, for the determination of high-resolution 3D structures of membrane proteins. The algorithm presented, addresses the low single-to-noise ratio (SNR) of 2D crystal images by exploiting neighborhood correlation between adjacent proteins in the 2D crystal. Compared with conventional single particle processing for randomly oriented particles, the computational costs are greatly reduced due to the crystal-induced limited search space, which allows a much finer search space compared to classical single particle processing. To reduce the considerable computational costs, our software features a hybrid parallelization scheme for multi-CPU clusters and computer with high-end graphic processing units (GPUs). We successfully apply the new refinement method to the structure of the potassium channel MloK1. The calculated 3D reconstruction shows more structural details and contains less noise than the map obtained by conventional Fourier-filtering based processing of the same 2D crystal images.

Ligand-induced structural changes in the cyclic nucleotide-modulated potassium channel MloK1.

Cyclic nucleotide-modulated ion channels are important for signal transduction and pacemaking in eukaryotes. The molecular determinants of ligand gating in these channels are still unknown, mainly because of a lack of direct structural information. Here we report ligand-induced conformational changes in full-length MloK1, a cyclic nucleotide-modulated potassium channel from the bacterium Mesorhizobium loti, analysed by electron crystallography and atomic force microscopy. Upon cAMP binding, the cyclic nucleotide-binding domains move vertically towards the membrane, and directly contact the S1-S4 voltage sensor domains. This is accompanied by a significant shift and tilt of the voltage sensor domain helices. In both states, the inner pore-lining helices are in an 'open' conformation. We propose a mechanism in which ligand binding can favour pore opening via a direct interaction between the cyclic nucleotide-binding domains and voltage sensors. This offers a simple mechanistic hypothesis for the coupling between ligand gating and voltage sensing in eukaryotic HCN channels.

Structure and substrate-induced conformational changes of the secondary citrate/sodium symporter CitS revealed by electron crystallography.

The secondary Na+/citrate symporter CitS of Klebsiella pneumoniae is the best-characterized member of the 2-hydroxycarboxylate transporter family. The recent projection structure gave insight into its overall structural organization. Here, we present the three-dimensional map of dimeric CitS obtained with electron crystallography. Each monomer has 13 a-helical transmembrane segments; six are organized in a distal helix cluster and seven in the central dimer interface domain. Based on structural analyses and comparison to VcINDY, we propose a molecular model for CitS, assign the helices, and demonstrate the internal structural symmetry. We also present projections of CitS in several conformational states induced by the presence and absence of sodium and citrate as substrates. Citrate binding induces a defined movement of a helices within the distal helical cluster. Based on this, we propose a substrate translocation site and conformational changes that are in agreement with the transport model of ''alternating access''.

Image processing of 2D crystal images.

Electron crystallography of membrane proteins uses cryo-transmission electron microscopy to image frozen-hydrated 2D crystals. The processing of recorded images exploits the periodic arrangement of the structures in the images to extract the amplitudes and phases of diffraction spots in Fourier space. However, image imperfections require a crystal unbending procedure to be applied to the image before evaluation in Fourier space. We here describe the process of 2D crystal image unbending, using the 2dx software system.

Merging of image data in electron crystallography.

Electron crystallography of membrane proteins uses cryo-transmission electron microscopy to record images and diffraction patterns of frozen-hydrated 2D crystals. Each two-dimensional (2D) crystal is only imaged once, at one specific tilt angle, and the recorded images can be automatically processed with the 2dx/MRC software package. Processed image data from non-tilted and tilted 2D crystals then need to be merged into a 3D reconstruction of the membrane protein structure. We here describe the process of the 3D merging, using the 2dx software system.

Automation of image processing in electron crystallography.

Electron crystallography of membrane proteins records images and diffraction patterns of frozen-hydrated two-dimensional (2D) crystals. To reconstruct the high-resolution three-dimensional (3D) structure of a membrane protein, a multitude of images of 2D crystals have to be processed. Certain processing steps are thereby similar for batches of images that were recorded under similar conditions. Here we describe how the 2dx software package can be used to automate the processing of 2D crystal images, and how the 2D and 3D merging results can be used to iteratively reprocess the images. While the processing of 2D crystal images has been fully automated, the merging process is still semi-manual.

Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments.

Dynamo is a new software package for subtomogram averaging of cryo Electron Tomography (cryo-ET) data with three main goals: first, Dynamo allows user-transparent adaptation to a variety of high-performance computing platforms such as GPUs or CPU clusters. Second, Dynamo implements user-friendliness through GUI interfaces and scripting resources. Third, Dynamo offers user-flexibility through a plugin API. Besides the alignment and averaging procedures, Dynamo includes native tools for visualization and analysis of results and data, as well as support for third party visualization software, such as Chimera UCSF or EMAN2. As a demonstration of these functionalities, we studied bacterial flagellar motors and showed automatically detected classes with absent and present C-rings. Subtomogram averaging is a common task in current cryo-ET pipelines, which requires extensive computational resources and follows a well-established workflow. However, due to the data diversity, many existing packages offer slight variations of the same algorithm to improve results. One of the main purposes behind Dynamo is to provide explicit tools to allow the user the insertion of custom designed procedures - or plugins - to replace or complement the native algorithms in the different steps of the processing pipeline for subtomogram averaging without the burden of handling parallelization. Custom scripts that implement new approaches devised by the user are integrated into the Dynamo data management system, so that they can be controlled by the GUI or the scripting capacities. Dynamo executables do not require licenses for third party commercial software. Sources, executables and documentation are freely distributed on http://www.dynamo-em.org.

3D reconstruction from 2D crystal image and diffraction data.

Electron crystallography of 2D protein crystals can determine the structure of membrane embedded proteins at high resolution. Images or electron diffraction patterns are recorded with the electron microscope of the frozen hydrated samples, and the 3D structure of the proteins is then determined by computer data processing. Here we introduce the image-processing algorithms for crystallographic Fourier space based methods using the Medical Research Council (MRC) programs, and illustrate the usage of the software packages 2dx, XDP, and IPLT.