Error analysis in the determination of the electron microscopical contrast transfer function parameters from experimental power Spectra

<p>Abstract</p> <p>Background</p> <p>The transmission electron microscope is used to acquire structural information of macromolecular complexes. However, as any other imaging device, it introduces optical aberrations that must be corrected if high-resolution structural information is to be obtained. The set of all aberrations are usually modeled in Fourier space by the so-called Contrast Transfer Function (CTF). Before correcting for the CTF, we must first estimate it from the electron micrographs. This is usually done by estimating a number of parameters specifying a theoretical model of the CTF. This estimation is performed by minimizing some error measure between the theoretical Power Spectrum Density (PSD) and the experimentally observed PSD. The high noise present in the micrographs, the possible local minima of the error function for estimating the CTF parameters, and the cross-talking between CTF parameters may cause errors in the estimated CTF parameters.</p> <p>Results</p> <p>In this paper, we explore the effect of these estimation errors on the theoretical CTF. For the CTF model proposed in <abbrgrp><abbr bid="B1">1</abbr></abbrgrp> we show which are the most sensitive CTF parameters as well as the most sensitive background parameters. Moreover, we provide a methodology to reveal the internal structure of the CTF model (which parameters influence in which parameters) and to estimate the accuracy of each model parameter. Finally, we explore the effect of the variability in the detection of the CTF for CTF phase and amplitude correction.</p> <p>Conclusion</p> <p>We show that the estimation errors for the CTF detection methodology proposed in <abbrgrp><abbr bid="B1">1</abbr></abbrgrp> does not show a significant deterioration of the CTF correction capabilities of subsequent algorithms. All together, the methodology described in this paper constitutes a powerful tool for the quantitative analysis of CTF models that can be applied to other models different from the one analyzed here.</p

Transfer function restoration in 3D electron microscopy via iterative data refinement

Three-dimensional electron microscopy (3D-EM) is a powerful tool for visualizing complex biological systems. As with any other imaging device, the electron microscope introduces a transfer function (called in this field the contrast transfer function, CTF) into the image acquisition process that modulates the various frequencies of the signal. Thus, the 3D reconstructions performed with these CTF-affected projections are also affected by an implicit 3D transfer function. For high-resolution electron microscopy, the effect of the CTF is quite dramatic and limits severely the achievable resolution. In this work we make use of the iterative data refinement (IDR) technique to ameliorate the effect of the CTF. It is demonstrated that the approach can be successfully applied to noisy data.Partial support is acknowledged to the Comisión Interministerial de Ciencia y Tecnología of Spain through projects BIO98-0761 and BIO2001-1237 and to National Institutes of Health through grant HL70472. The work of Y. Censor was done in part at the Center for Computational Mathematics and Scientific Computation (CCMSC) at the University of Haifa and supported by Research Grant 592/00 from the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities

Fast multiscale reconstruction for Cryo-EM

We present a multiscale reconstruction framework for single-particle analysis (SPA). The representation of three-dimensional (3D) objects with scaled basis functions permits the reconstruction of volumes at any desired scale in the real-space. This multiscale approach generates interesting opportunities in SPA for the stabilization of the initial volume problem or the 3D iterative refinement procedure. In particular, we show that reconstructions performed at coarse scale are more robust to angular errors and permit gains in computational speed. A key component of the proposed iterative scheme is its fast implementation. The costly step of reconstruction, which was previously hindering the use of advanced iterative methods in SPA, is formulated as a discrete convolution with a cost that does not depend on the number of projection directions. The inclusion of the contrast transfer function inside the imaging matrix is also done at no extra computational cost. By permitting full 3D regularization, the framework is by itself a robust alternative to direct methods for performing reconstruction in adverse imaging conditions (e.g., heavy noise, large angular misassignments, low number of projections). We present reconstructions obtained at different scales from a dataset of the 2015/2016 EMDataBank Map Challenge. The algorithm has been implemented in the Scipion package

Consistent and elastic registration of histological sections using vector-spline regularization

The final publication is available at Springer via http://dx.doi.org/10.1007/11889762_8Revised Papers on Second International ECCV Workshop, CVAMIA 2006 Graz, Austria, May 12, 2006Here we present a new image registration algorithm for the alignment of histological sections that combines the ideas of B-spline based elastic registration and consistent image registration, to allow simultaneous registration of images in two directions (direct and inverse). In principle, deformations based on B-splines are not invertible. The consistency term overcomes this limitation and allows registration of two images in a completely symmetric way. This extension of the elastic registration method simplifies the search for the optimum deformation and allows registering with no information about landmarks or deformation regularization. This approach can also be used as the first step to solve the problem of group-wise registration.Ignacio Arganda-Carreras is being supported by a predoctoral FPI-CAM fellow- ship since October 2003. Carlos Ortiz-de-Solorzano is supported by a Ramon y Cajal (Spanish Ministry of Education and Science ryc-2004-002353) and a Marie Curie International Reintegration Grant (FP6-518688). Jan Kybic was sponsored by the Czech Ministery of Education under project number MSM210000012. Par- tial support is acknowledged to Comunidad de Madrid through grant GR/SAL/0234, to Instituto de Salud Carlos III-Fondo de Investigaciones Sanitarias (FIS) through the IM3 Network and grant 040683 and to the Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+I)

Optimization problems in electron microscopy of single particles

The final publication is available at Springer via http://dx.doi.org/10.1007/s10479-006-0078-8Electron Microscopy is a valuable tool for the elucidation of the three-dimensional structure of macromolecular complexes. Knowledge about the macromolecular structure provides important information about its function and how it is carried out. This work addresses the issue of three-dimensional reconstruction of biological macromolecules from electron microscopy images. In particular, it focuses on a methodology known as “single-particles” and makes a thorough review of all those steps that can be expressed as an optimization problem. In spite of important advances in recent years, there are still unresolved challenges in the field that offer an excellent testbed for new and more powerful optimization techniques.We acknowledge partial support from the “Comunidad Autónoma de Madrid” through grants CAM-07B-0032-2002, GR/SAL/0653/2004 and GR/SAL/0342/2004, the “Comisión Interministerial de Ciencia yTecnologia” of Spain through grants BIO2001-1237, BIO2001-4253-E, BIO2001-4339-E, BIO2002- 10855-E, BFU2004-00217/BMC, the Spanish FIS grant (G03/185), the European Union through grants QLK2- 2000-00634, QLRI-2000-31237, QLRT-2000-0136, QLRI-2001-00015, FP6-502828 and the NIH through grant HL70472. Alberto Pascual and Roberto Marabini acknowledge support by the Spanish Ramon y Cajal Program

On the development of three new tools for organizing and sharing information in three-dimensional electron microscopy

This work was funded by the Spanish Ministerio de Economía y Competividad through grants BFU2009-09331, BIO2010-16566, ACI2009-1022, ACI2010-1088 and AIC-A- 2011-0638, by the Comunidad Autonoma de Madrid through grant S2010/BMD-2305, by NFS grant No. 1114901 and by the Spanish National Institute of Bioinformatics (a project funded by the Instituto de Salud Carlos III). This work was conducted using the Protégé resource, which is supported by grant LM007885 from the United States National Library of Medicine. COSS is a Ramón y Cajal researcher financed by the European Social Fund and the Ministerio de Economía y Competitividad. JV is a Juan de la Cierva Postdoctoral Fellow (JCI-2011-10185). This work was funded by Instruct, which is part of the European Strategy Forum on Research Infrastructures (ESFRI) and is supported by national member subscriptions

FSC-Q: a CryoEM map-to-atomic model quality validation based on the local Fourier shell correlation

In recent years, advances in cryoEM have dramatically increased the resolution of reconstructions and, with it, the number of solved atomic models. It is widely accepted that the quality of cryoEM maps varies locally; therefore, the evaluation of the maps-derived structural models must be done locally as well. In this article, a method for the local analysis of the map-to-model fit is presented. The algorithm uses a comparison of two local resolution maps. The first is the local FSC (Fourier shell correlation) between the full map and the model, while the second is calculated between the half maps normally used in typical single particle analysis workflows. We call the quality measure “FSC-Q”, and it is a quantitative estimation of how much of the model is supported by the signal content of the map. Furthermore, we show that FSC-Q may be helpful to detect overfitting. It can be used to complement other methods, such as the Q-score method that estimates the resolvability of atomsWe thank Prof. David Veesler for providing us the half maps of the spike glycoprotein of SARS-CoV-2. The authors would like to acknowledge financial support from: the Comunidad de Madrid through grant CAM (S2017/BMD-3817), the Spanish National Research Council (PIE/COVID-19 number 202020E079), the Spanish Ministry of Economy and Competitiveness through grants SEV 2017-0712, PID2019-104757RB-I00/AEI/10.13039/501100011033, the Instituto de Salud Carlos III through grant PT17/0009/0010 (ISCIII-GEFI/ERDF-). Instruct-ULTRA (Grant 731005), an EU H2020 project to further develop the services of Instruct-ERIC. UE H2020 grant HighResCells (ERC-2018-SyG, Proposal: 810057). This work was supported by the Intramural Research Program of the National Institute for Arthritis, musculoskeletal, and Skin Diseases, NIH. The authors acknowledge the support and the use of resources of Instruct, a Landmark ESFRI projec

Miro-1 links mitochondria and microtubule dynein motors to control lymphocyte migration and polarity

The recruitment of leukocytes to sites of inflammation is crucial for a functional immune response. In the present work, we explored the role of mitochondria in lymphocyte adhesion, polarity, and migration. We show that during adhesion to the activated endothelium under physiological flow conditions, lymphocyte mitochondria redistribute to the adhesion zone together with the microtubule-organizing center (MTOC) in an integrin-dependent manner. Mitochondrial redistribution and efficient lymphocyte adhesion to the endothelium require the function of Miro-1, an adaptor molecule that couples mitochondria to microtubules. Our data demonstrate that Miro-1 associates with the dynein complex. Moreover, mitochondria accumulate around the MTOC in response to the chemokine CXCL12/SDF-1α this redistribution is regulated by Miro-1. CXCL12-dependent cell polarization and migration are reduced in Miro-1-silenced cells, due to impaired myosin II activation at the cell uropod and diminished actin polymerization. These data point to a key role of Miro-1 in the control of lymphocyte adhesion and migration through the regulation of mitochondrial redistribution.This study was supported by SAF2011-25834 from the Spanish Ministry of Science and Innovation, INDISNET-S2011/BMD-2332 from the Comunidad de Madrid, Red Cardiovascular RD 12-0042-0056 from Instituto Salud Carlos III (ISCIII), and ERC-2011-AdG 294340- GENTRIS. J.M.G.-G. received salary support from the Miguel Servet (CP11/00145) ISCIII program. R.V.-B. was supported by a Juan de la Cierva postdoctoral contract from the Spanish Ministry of Economy and Competiveness (JCI-2011-09663

Marker-free image registration of electron tomography tilt-series

<p>Abstract</p> <p>Background</p> <p>Tilt series are commonly used in electron tomography as a means of collecting three-dimensional information from two-dimensional projections. A common problem encountered is the projection alignment prior to 3D reconstruction. Current alignment techniques usually employ gold particles or image derived markers to correctly align the images. When these markers are not present, correlation between adjacent views is used to align them. However, sequential pairwise correlation is prone to bias and the resulting alignment is not always optimal.</p> <p>Results</p> <p>In this paper we introduce an algorithm to find regions of the tilt series which can be tracked within a subseries of the tilt series. These regions act as landmarks allowing the determination of the alignment parameters. We show our results with synthetic data as well as experimental cryo electron tomography.</p> <p>Conclusion</p> <p>Our algorithm is able to correctly align a single-tilt tomographic series without the help of fiducial markers thanks to the detection of thousands of small image patches that can be tracked over a short number of images in the series.</p