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Electron Crystallography of Membrane Proteins [chapter]

Hui-Ting Chou, James E. Evans, Henning Stahlberg
2007 Msphere  
Electron crystallography studies the structure of two-dimensional crystals of membrane proteins or other crystalline arrays. This method has been used to determine the atomic structures of six membrane proteins and tubulin, as well as several other structures at a slightly lower resolution, where secondary structure motifs could be identified. To preserve the high-resolution structure of 2D crystals, the meticulous sample preparation for electron crystallography is of outmost importance.
more » ... induced specimen drift and lack of specimen flatness can severely affect the resolution of images for tilted samples. However, sample preparations that sandwich the two-dimensional crystals between symmetrical carbon films reduce the charge-induced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol. Data collection in the cryoelectron microscope using either the imaging or the electron diffraction mode has to be performed after low-dose procedures. Spot scanning further reduces the charge-induced specimen drift.
doi:10.1007/978-1-59745-294-6_16 pmid:17656758 fatcat:apcxz272ifechi37vqv4sjcyku

Cryo-electron Microscopy of Membrane Proteins [chapter]

Kenneth N. Goldie, Priyanka Abeyrathne, Fabian Kebbel, Mohamed Chami, Philippe Ringler, Henning Stahlberg
2013 Msphere  
Electron crystallography is used to study membrane proteins in the form of planar, twodimensional (2D) crystals, or other crystalline arrays such as tubular crystals. This method has been used to determine the atomic resolution structures of bacteriorhodopsin, tubulin, aquaporins, and several other membrane proteins. In addition, a large number of membrane protein structures were studied at a slightly lower resolution, whereby at least secondary structure motifs could be identified.In order to
more » ... onserve the structural details of delicate crystalline arrays, cryo-electron microscopy (cryo-EM) allows imaging and/or electron diffraction of membrane proteins in their close-to-native state within a lipid bilayer membrane.To achieve ultimate high-resolution structural information of 2D crystals, meticulous sample preparation for electron crystallography is of outmost importance. Beam-induced specimen drift and lack of specimen flatness can severely affect the attainable resolution of images for tilted samples. Sample preparations that sandwich the 2D crystals between symmetrical carbon films reduce the beaminduced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol.Data collection in the cryo-electron microscope using either the imaging or the electron diffraction mode has to be performed applying low-dose procedures. Spot-scanning further reduces the effects of beam-induced drift. Data collection using automated acquisition schemes, along with improved and user-friendlier data processing software, is increasingly being used and is likely to bring the technique to a wider user base.
doi:10.1007/978-1-62703-776-1_15 pmid:24357370 fatcat:nwp6mqj7uzbgxfiwkktydqu7rq

Electron crystallography of membrane proteins

Ben Hankamer, Robert Glaeser, Henning Stahlberg
2007 Journal of Structural Biology  
Electron crystallography studies the structure of two-dimensional crystals of membrane proteins or other crystalline arrays. This method has been used to determine the atomic structures of six membrane proteins and tubulin, as well as several other structures at a slightly lower resolution, where secondary structure motifs could be identified. To preserve the high-resolution structure of 2D crystals, the meticulous sample preparation for electron crystallography is of outmost importance.
more » ... induced specimen drift and lack of specimen flatness can severely affect the resolution of images for tilted samples. However, sample preparations that sandwich the two-dimensional crystals between symmetrical carbon films reduce the charge-induced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol. Data collection in the cryoelectron microscope using either the imaging or the electron diffraction mode has to be performed after low-dose procedures. Spot scanning further reduces the charge-induced specimen drift.
doi:10.1016/j.jsb.2007.11.001 pmid:18022538 pmcid:PMC2170452 fatcat:gfgdsjt2jjhevmoj7ka5xa37nm

Cryo-EM structure of native human thyroglobulin [article]

Ricardo Adaixo, Eva M. Steiner, Ricardo D. Righetto, Alexander Schmidt, Henning Stahlberg, Nicholas M. I. Taylor
2021 bioRxiv   pre-print
The thyroglobulin (Tg) protein is essential to thyroid hormone synthesis, playing a vital role in the regulation of metabolism, development and growth. Its structure is conserved among vertebrates. Tg is delivered through the secretory pathway of the thyroid follicular unit to the central colloid depository, where it is iodinated at specific tyrosine sites to form mono- or diiodotyrosine, which combine to produce triiodothyronine (T3) and thyroxine (T4), respectively. Synthesis of these
more » ... depends on the precise 3D structure of Tg, which has remained unknown despite decades of research. Here, we present the cryo-electron microscopy structure of human thyroglobulin (hTg) to a global resolution of 3.2 Å. The structure provides detailed information on the location of the hTg hormonogenic sites and reveals the position as well as the role of many of its glycosylation sites. Our results offer structural insight into thyroid hormonogenesis and provide a fundamental understanding of clinically relevant hTg mutations, which can improve treatment of thyroid diseases.
doi:10.1101/2021.06.06.447243 fatcat:p4ik66uzcnei3c3z6dtc7p5owa

Structural basis of drug recognition by the multidrug transporter ABCG2 [article]

Julia Kowal, Dongchun Ni, Scott M Jackson, Ioannis Manolaridis, Henning Stahlberg, Kaspar P Locher
2021 bioRxiv   pre-print
Manolaridis I, Jackson SM, Taylor NMI, Kowal J, Stahlberg H, Locher KP.  ...  ACS Chem Neurosci. 2011;2:82-9. 540 [26] Taylor NMI, Manolaridis I, Jackson SM, Kowal J, Stahlberg H, Locher KP. Structure of the 541 human multidrug transporter ABCG2.  ... 
doi:10.1101/2021.02.18.431786 fatcat:kgwenpn3pbhkxhddvdqruh4ahu

Molecular Electron Microscopy: State of the Art and Current Challenges

Henning Stahlberg, Thomas Walz
2008 ACS Chemical Biology  
The objective of molecular electron microscopy (EM) is to use electron microscopes to visualize the structure of biological molecules. This Review provides a brief overview of the methods used in molecular EM, their respective strengths and successes, and current developments that promise an even more exciting future for molecular EM in the structural investigation of proteins and macromolecular complexes, studied in isolation or in the context of cells and tissues. E lectron microscopy (EM)
more » ... been a longstanding tool in the ultrastructural analysis of cells and tissues. Over the last 3 decades, it has also evolved into a powerful technique for the structural study of biological macromolecules. The main difference between this molecular EM and the more conventional EM of fixed tissue sections is its ability to deliver three-dimensional (3D) structures of the studied complexes at the higher resolution necessary to visualize structural details of molecules (on the scale of nanometers) rather than of the gross architecture of cells (on the scale of micrometers). Whereas modern electron microscopes can routinely deliver images of inorganic material at atomic resolution, biological specimens pose great difficulties for EM imaging, significantly reducing the attainable resolution. Biological specimens consist of up to 80% water, requiring the samples to be prepared in a way that prevents structural collapse upon dehydration in the vacuum of the electron microscope. Biological specimens also consist mainly of light atoms, and the density of proteins is very close to that of vitrified ice (see below), making them low-contrast objects. For thin biological samples, different materials influence mainly the phases of the passing electron beam, not its intensity. To increase image contrast, data are collected out of focus, with the amount of contrast increasing with increasing underfocus. The general effect of defocusing is described, in the weak-phase approximation (1), by the socalled contrast transfer function (CTF), a semiperiodic function in reciprocal space (2). The main consequences of defocusing are the lack of frequency information around the zero transitions of the CTF in the imaged object, the inversion of phases in some regions of the reciprocal space, and a rapid decrease of the Fourier amplitudes in the high spatial frequency region. Therefore,
doi:10.1021/cb800037d pmid:18484707 pmcid:PMC2660199 fatcat:ljpfnv2rnzfetgktjm26cxgufy

Cryo-EM structure of alpha-synuclein fibrils [article]

Ricardo Guerrero-Ferreira, Nicholas M. I. Taylor, Daniel Mona, Philippe Ringler, Matthias E. Lauer, Roland Riek, Markus Britschgi, Henning Stahlberg
2018 biorxiv/medrxiv   pre-print
Data collection Magnification https://orcid.org/0000-0003-3252-8718 Markus Britschgi http://orcid.org/0000-0001-6151-4257 Henning Stahlberg http://orcid.org/0000-0002-1185-4592 Decision letter and  ...  Author response Decision letter https://doi.org/10.7554/eLife.36402.021 Author response https://doi.org/10.7554/eLife.36402.022 Guerrero-Ferreira R, Taylor N M I, Mona D, Riek R, Britschgi M, Stahlberg  ... 
doi:10.1101/276436 fatcat:vvht2hccuzfq7fhiw2lc376ram

A maximum likelihood approach to two-dimensional crystals

Xiangyan Zeng, Henning Stahlberg, Nikolaus Grigorieff
2007 Journal of Structural Biology  
Stahlberg et al. (1998) used single particle methods to detect and correct for the non-crystallographic orientation of the photosynthetic reaction center within well-ordered 2D crystal images of the surrounding  ... 
doi:10.1016/j.jsb.2007.09.013 pmid:17964808 pmcid:PMC2147720 fatcat:plfkh4b6gzctjlvzld5n5aosha

MRCZ - A proposed fast compressed MRC file format and direct detector normalization strategies [article]

Robert A McLeod, Ricardo Diogo Righetto, Andy Stewart, Henning Stahlberg
2017 bioRxiv   pre-print
The introduction of high-speed CMOS detectors is fast marching the field of transmission electron microscopy into an intersection with the computer science field of big data. Automated data pipelines to control the instrument and the initial processing steps are imposing more and more onerous requirements on data transfer and archiving. We present a proposal for expansion of the venerable MRC file format to combine integer decimation and lossless compression to reduce storage requirements and
more » ... prove file read/write times by >1000 % compared to uncompressed floating-point data. The integer decimation of data necessitates application of the gain normalization and outlier pixel removal at the data destination, rather than the source. With direct electron detectors, the normalization step is typically provided by the vendor and is not open-source. We provide robustly tested normalization algorithms that perform at-least as well as vendor software. We show that the generation of hot pixels is a highly dynamic process in direct electron detectors, and that outlier pixels must be detected on a stack-by-stack basis. In comparison, the low-frequency bias features of the detectors induced by the electronics on-top of the active layer, are extremely stable with time. Therefore we introduce a stochastic-based approach to identify outlier pixels and smoothly filter them, such that the degree of correlated noise in micrograph stacks is reduced. Both a priori and a posteriori gain normalization approaches that are compatible with pipeline image processing are discussed. The a priori approach adds a gamma-correction to the gain reference, and the a posteriori approach normalized by a moving average of time-adjacent stacks, with the current stack being knocked-out, known as the KOMA (knock-out moving average) filter. The combination of outlier filter and KOMA normalization over ~25 frames can reduce the correlated noise in movies to nearly zero. Sample libraries and a command-line utility are hosted at github.com/em-MRCZ and released under the BSD license.
doi:10.1101/116533 fatcat:i2f6khuldjeo7fya2w55akcgpu

Retrieving High-Resolution Information from Disordered 2D Crystals by Single Particle Cryo-EM [article]

Ricardo Righetto, Nikhil Biyani, Julia Kowal, Mohamed Chami, Henning Stahlberg
2018 bioRxiv   pre-print
McLeod RA, Diogo Righetto R, Stewart A, Stahlberg H. MRCZ -A file format for cryo-TEM data with fast compression. J. Struct. Biol. 201, 252-257 (2018).  ... 
doi:10.1101/488403 fatcat:hrtkvumobbhuxdahwsw2fpzbvm

2dx_merge: Data management and merging for 2D crystal images

Bryant Gipson, Xiangyan Zeng, Henning Stahlberg
2007 Journal of Structural Biology  
Electron crystallography of membrane proteins determines the structure of membrane-reconstituted and two-dimensionally (2D) crystallized membrane proteins by low-dose imaging with the transmission electron microscope, and computer image processing. We have previously presented the software system 2dx, for user-friendly image processing of 2D crystal images. Its central component 2dx_image is based on the MRC program suite, and allows the optionally fully automatic processing of one 2D crystal
more » ... age. We present here the program 2dx_merge, which assists the user in the management of a 2D crystal image-processing project, and facilitates the merging of the data from multiple images. The merged dataset can be used as a reference to re-process all images, which usually improves the resolution of the final reconstruction. Image processing and merging can be applied iteratively, until convergence is reached. 2dx is available under the GNU General Public License at http://2dx.org.
doi:10.1016/j.jsb.2007.09.011 pmid:17967545 pmcid:PMC2157552 fatcat:7m4i4kkujjf67didbl6zxsqnly

LIMITING FACTORS IN SINGLE PARTICLE CRYO ELECTRON TOMOGRAPHY

Mikhail Kudryashev, Daniel Castaño-Díez, Henning Stahlberg
2012 Computational and Structural Biotechnology Journal  
Introduction The combination of vitreous preservation of biological samples [1] with 3D electron tomographic imaging [2] has opened a unique opportunity to study living matter at nanometer scale [3] . Importantly, in addition to observing the ultrastructure of cells, it is also possible to perform structural analysis of large macromolecules and their complexes in situ. Since the inherent contrast of cryopreserved material is low, a large number of electron microscopy datasets of identical
more » ... ens is needed to allow statistical image processing with specialized algorithms and methods. This approach is fundamentally similar to single particle cryo-electron microscopy (cryo-EM) [4] , except that each tomographic sub-volume is a 3dimensional dataset with anisotropic resolution, and that the particles are imaged in a crowded cellular context rather than isolated in solution. The process of producing an average structure is commonly referred to as "cryo electron tomography sub-volume averaging", or CET SVA. The typical resolution that may be achieved by CET SVA in situ is 2-6 nm, dependent upon on a number of factors. We here discuss four major sources of resolution degradation: 1) sample thickness, 2) cryo-EM instrumentation, 3) sample heterogeneity, and the 4) effect of the contrast transfer function of the instrument. First, the quality of cryo-EM images worsens with increasing electron density thickness of the sample. The fraction of inelastically scattered electrons increases with growing sample thickness, degrading the resolution of structures derived from SVA. For the commonly used intermediate voltage transmission electron microscopes, operating at electron acceleration voltages of 200 or 300 kV, the sample thickness limit for tomographic imaging of biological specimens is around 0.5 -1 µm. Second, the choice of cryo-EM instrumentation is important: electrons accelerated with higher voltages are capable of penetrating thicker samples; electron energy loss imaging filters eliminate inelastically scattered electrons of lower energy, thereby improving the signal to noise ratio of the resulting images. The point resolution of modern electron microscopes is in the angstrom range, while the resolution of CET SVA is so far typically in nanometer range so this aspect currently does not limit the resolution. Thirdly, large protein complexes in the native context are subject to flexibility originating from external forces and from their own structural heterogeneity. Sub-volume averaging requires all volumes to contain structures in exactly the same conformation. Structural variations among sub-volumes would otherwise blur the calculated average, thereby reducing the resolution. Fourth, the low contrast in cryo-EM images is usually compensated for by the instrument's operator by defocusing the microscope's objective lens, which introduces strong oscillations to the instrument's contrast transfer function (CTF) that have to be accounted for during image processing (see below). Typical objects approached by CET SVA are large protein complexes inside intact bacterial [5] [6] [7] [8] or eukaryotic cells [9] [10] [11] ; large protein or protein-DNA complexes in cryo-sectioned cells or tissues [12] [13] [14] ; viral cores [15] [16] [17] [18] and outer surface proteins [19] [20] [21] ; bacteriophages [22, 23] ; microtubule bound proteins in situ [24] [25] [26] or in vitro [27] ; membrane protein complexes in simplified systems such as lipid vesicles [28] or isolated membrane fractions [29] and proteins and their complexes that have a preferential orientation on the EM support [30, 31] . The popularity of CET SVA is growing fast, and the number of laboratories in the world with access to the expensive instrumentation and image processing expertise is rising rapidly. Although progress in single particle electron tomography has recently been reviewed [32], here we use the rapidly increasing number of publications to present a comprehensive analysis of recently published structures and employed imaging parameters from cryo electron tomography and sub-volume averaging. Table S1 summarizes the acquisition parameters and resulting resolutions of 123 structures solved by CET SVA that were found by CSBJ Abstract: Modern methods of cryo electron microscopy and tomography allow visualization of protein nanomachines in their native state at the nanometer scale. Image processing methods including sub-volume averaging applied to repeating macromolecular elements within tomograms allow exploring their structures within the native context of the cell, avoiding the need for protein isolation and purification. Today, many different data acquisition protocols and software solutions are available to researchers to determine average structures of macromolecular complexes and potentially to classify structural intermediates. Here, we list the density maps reported in the literature, and analyze each structure for the chosen instrumental settings, sample conditions, main processing steps, and obtained resolution. We present conclusions that identify factors currently limiting the resolution gained by this approach. Overview
doi:10.5936/csbj.201207002 pmid:24688638 pmcid:PMC3962116 fatcat:qetrvvslu5glzi6tllcbva6mb4

The aquaporin sidedness revisited

Simon Scheuring, Peter Tittmann, Henning Stahlberg, Philippe Ringler, Mario Borgnia, Peter Agre, Heinz Gross, Andreas Engel
2000 Journal of Molecular Biology  
Aquaporins are transmembrane water channel proteins, which play important functions in the osmoregulation and water balance of microorganisms, plants, and animal tissues. All aquaporins studied to date are thought to be tetrameric assemblies of four subunits each containing its own aqueous pore. Moreover, the subunits contain an internal sequence repeat forming two obversely symmetric hemichannels predicted to resemble an hour-glass. This unique arrangement of two highly related protein domains
more » ... oriented at 180 to each other poses a signi®cant challenge in the determination of sidedness. Aquaporin Z (AqpZ) from Escherichia coli was reconstituted into highly ordered two-dimensional crystals. They were freeze-dried and metal-shadowed to establish the relationship between surface structure and underlying protein density by electron microscopy. The shadowing of some surfaces was prevented by protruding aggregates. Thus, images collected from freeze-dried crystals that exhibited both metal-coated and uncoated regions allowed surface relief reconstructions and projection maps to be obtained from the same crystal. Cross-correlation peak searches along lattices crossing metalcoated and uncoated regions allowed an unambiguous alignment of the surface reliefs to the underlying density maps. AqpZ topographs previously determined by AFM could then be aligned with projection maps of AqpZ, and ®nally with human erythrocyte aquaporin-1 (AQP1). Thereby features of the AqpZ topography could be interpreted by direct comparison to the 6 A Ê three-dimensional structure of AQP1. We conclude that the sidedness we originally proposed for aquaporin density maps was inverted. Abbreviations used: AQP1, aquaporin-1; AqpZ, aquaporin Z; AFM, atomic force microscopy.
doi:10.1006/jmbi.2000.3811 pmid:10873451 fatcat:wslidfzhvnclzdz7kp7lj36zvm

Cryo-EM structure of alpha-synuclein fibrils

Ricardo Guerrero-Ferreira, Nicholas MI Taylor, Daniel Mona, Philippe Ringler, Matthias E Lauer, Roland Riek, Markus Britschgi, Henning Stahlberg
2018 eLife  
Parkinson's disease is a progressive neuropathological disorder that belongs to the class of synucleinopathies, in which the protein alpha-synuclein is found at abnormally high concentrations in affected neurons. Its hallmark are intracellular inclusions called Lewy bodies and Lewy neurites. We here report the structure of cytotoxic alpha-synuclein fibrils (residues 1–121), determined by cryo-electron microscopy at a resolution of 3.4 Å. Two protofilaments form a polar fibril composed of
more » ... ed β-strands. The backbone of residues 38 to 95, including the fibril core and the non-amyloid component region, are well resolved in the EM map. Residues 50–57, containing three of the mutation sites associated with familial synucleinopathies, form the interface between the two protofilaments and contribute to fibril stability. A hydrophobic cleft at one end of the fibril may have implications for fibril elongation, and invites for the design of molecules for diagnosis and treatment of synucleinopathies.
doi:10.7554/elife.36402 pmid:29969391 pmcid:PMC6092118 fatcat:ykzkcdn26zabza6b2fcgrmjck4

2dx—User-friendly image processing for 2D crystals

Bryant Gipson, Xiangyan Zeng, Zi Yan Zhang, Henning Stahlberg
2007 Journal of Structural Biology  
Electron crystallography determines the structure of two-dimensional (2D) membrane protein crystals and other 2D crystal systems. Cryotransmission electron microscopy records high-resolution electron micrographs, which require computer processing for three-dimensional structure reconstruction. We present a new software system 2dx, which is designed as a user-friendly, platform-independent software package for electron crystallography. 2dx assists in the management of an image-processing
more » ... guides the user through the processing of 2D crystal images, and provides transparence for processing tasks and results. Algorithms are implemented in the form of script templates reminiscent of c-shell scripts. These templates can be easily modified or replaced by the user and can also execute modular stand-alone programs from the MRC software or from other image processing software packages. 2dx is available under the GNU General Public License at 2dx.org.
doi:10.1016/j.jsb.2006.07.020 pmid:17055742 fatcat:76xzmzz4hnd2pahg3nnowxl7v4
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