Cryo-electron Crystallography Research Articles

The SDBC is active in quenching oxidative conditions and bridges the cell envelope layers in Deinococcus radiodurans

Deinococcus radiodurans is known for its remarkable ability to withstand harsh stressful conditions. The outermost layer of its cell envelope is a proteinaceous coat, the S-layer, essential for resistance to and interactions with the environment. The S-layer Deinoxanthin-binding complex (SDBC), one of the main units of the characteristic multilayered cell envelope of this bacterium, protects against environmental stressors and allows exchanges with the environment. So far, specific regions of this complex, the collar and the stalk, remained unassigned. Here, these regions are resolved by cryo-EM and locally refined. The resulting 3D map shows that the collar region of this multiprotein complex is a trimer of the protein DR_0644, a Cu-only superoxide dismutase (SOD) identified here to be efficient in quenching reactive oxygen species. The same data also showed that the stalk region consists of a coiled coil that extends into the cell envelope for ∼280Å, reaching the inner membrane. Finally, the orientation and localization of the complex are defined by in situ cryo-electron crystallography. The structural organization of the SDBC couples fundamental UV antenna properties with the presence of a Cu-only SOD, showing here coexisting photoprotective and chemoprotective functions. These features suggests how the SDBC and similar protein complexes, might have played a primary role as evolutive templates for the origin of photoautotrophic processes by combining primary protective needs with more independent energetic strategies.

Machine learning-based real-time object locator/evaluator for cryo-EM data collection

In cryo-electron microscopy (cryo-EM) data collection, locating a target object is error-prone. Here, we present a machine learning-based approach with a real-time object locator named yoneoLocr using YOLO, a well-known object detection system. Implementation shows its effectiveness in rapidly and precisely locating carbon holes in single particle cryo-EM and in locating crystals and evaluating electron diffraction (ED) patterns in automated cryo-electron crystallography (cryo-EX) data collection. The proposed approach will advance high-throughput and accurate data collection of images and diffraction patterns with minimal human operation.

Structural analysis of the architecture and in situ localization of the main S-layer complex in Deinococcus radiodurans

Bacterial surface layers are paracrystalline assemblies of proteins that provide the first line of defense against environmental shocks. Here, we report the 3D structure, in situ localization, and orientation of the S-layer deinoxanthin-binding complex (SDBC), a hetero-oligomeric assembly of proteins that in Deinococcus radiodurans represents the main S-layer unit. The SDBC is resolved at 11-Å resolution by single-particle analysis, while its in situ localization is determined by cryo-electron crystallography on intact cell-wall fragments leading to a projection map at 4.5-Å resolution. The SDBC exhibits a triangular base with three comma-shaped pores, and a stalk departing orthogonally from the center of the base and oriented toward the intracellular space. Combining state-of-the-art techniques, results show the organization of this S-layer and its connection within the underlying membranes, demonstrating the potential for applications from nanotechnologies to medicine.

Using cryo-electron microscopy maps for X-ray structure determination of homologues.

The combination of cryo-electron microscopy (cryo-EM) and X-ray crystallography reflects an important trend in structural biology. In a previously published study, a hybrid method for the determination of X-ray structures using initial phases provided by the corresponding parts of cryo-EM maps was presented. However, if the target structure of X-ray crystallography is not identical but homologous to the corresponding molecular model of the cryo-EM map, then the decrease in the accuracy of the starting phases makes the whole process more difficult. Here, a modified hybrid method is presented to handle such cases. The whole process includes three steps: cryo-EM map replacement, phase extension by NCS averaging and dual-space iterative model building. When the resolution gap between the cryo-EM and X-ray crystallographic data is large and the sequence identity is low, an intermediate stage of model building is necessary. Six test cases have been studied with sequence identity between the corresponding molecules in the cryo-EM and X-ray structures ranging from 34 to 52% and with sequence similarity ranging from 86 to 91%. This hybrid method consistently produced models with reasonable Rwork and Rfree values which agree well with the previously determined X-ray structures for all test cases, thus indicating the general applicability of the method for X-ray structure determination of homologues using cryo-EM maps as a starting point.

Probing the conformational heterogeneity of complex I by combination of cryo-electron microscopy, crystallography and normal mode analysis

Probing the conformational heterogeneity of complex I by combination of cryo-electron microscopy, crystallography and normal mode analysis

Image processing techniques for high-resolution structure determination from badly ordered 2D crystals.

2D electron crystallography can be used to study small membrane proteins in their native environment. Obtaining highly ordered 2D crystals is difficult and time-consuming. However, 2D crystals diffracting to only 10-12 Å can be prepared relatively conveniently in most cases. We have developed image-processing algorithms allowing to generate a high resolution 3D structure from cryo-electron crystallography images of badly ordered crystals. These include movie-mode unbending, refinement over sub-tiles of the images in order to locally refine the sample tilt geometry, implementation of different CTF correction schemes, and an iterative method to apply known constraints in the real and reciprocal space to approximate amplitudes and phases in the so-called missing cone regions. These algorithms applied to a dataset of the potassium channel MloK1 show significant resolution improvements to better than 5 Å.

Pulse EPR distance measurements to study multimers and multimerisation

ABSTRACTPulse dipolar electron paramagnetic resonance (PD-EPR) has become a powerful tool for structural biology determining distances on the nanometre scale. Recent advances in hardware, methodology, and data analysis have widened the scope to complex biological systems. PD-EPR can be applied to systems containing lowly populated conformers or displaying large intrinsic flexibility, making them all but intractable for cryo-electron microscopy and crystallography. Membrane protein applications are of particular interest due to the intrinsic difficulties for obtaining high-resolution structures of all relevant conformations. Many drug targets involved in critical cell functions are multimeric channels or transporters. Here, common approaches for introducing spin labels for PD-EPR cause the presence of more than two electron spins per multimeric complex. This requires careful experimental design to overcome detrimental multi-spin effects and to secure sufficient distance resolution in presence of multiple distances. In addition to obtaining mere distances, PD-EPR can also provide information on multimerisation degrees allowing to study binding equilibria and to determine dissociation constants.

Hexadecameric structure of an invertebrate gap junction channel

Innexins are invertebrate-specific gap junction proteins with four transmembrane helices. These proteins oligomerize to constitute intercellular channels that allow for the passage of small signaling molecules associated with neural and muscular electrical activity. In contrast to the large number of structural and functional studies of connexin gap junction channels, few structural studies of recombinant innexin channels are reported. Here we show the three-dimensional structure of two-dimensionally crystallized Caenorhabditis elegans innexin-6 (INX-6) gap junction channels. The N-terminal deleted INX-6 proteins are crystallized in lipid bilayers. The three-dimensional reconstruction determined by cryo-electron crystallography reveals that a single INX-6 gap junction channel comprises 16 subunits, a hexadecamer, in contrast to chordate connexin channels, which comprise 12 subunits. The channel pore diameters at the cytoplasmic entrance and extracellular gap region are larger than those of connexin26. Two bulb densities are observed in each hemichannel, one in the pore and the other at the cytoplasmic side of the hemichannel in the channel pore pathway. These findings imply a structural diversity of gap junction channels among multicellular organisms.

Three-Dimensional Structure of Innexin GAP Junction Channels Studied by Electron Crystallography

Invertebrate-specific gap junction proteins, termed innexins, form a large family of four-transmembrane proteins. These proteins oligomerize to constitute intercellular channels that allow for the passage of small signaling molecules associated with neural and muscular electrical activity. In contrast to the large number of structural and functional studies of vertebrate connexin gap junction channels, few structural studies of recombinant innexin channels have been published. Here we show a three-dimensional structure of two-dimensionally crystallized Caenorhabditis elegans innexin-6 (INX-6) gap junction channels. An N-terminal deletion INX-6 construct in which amino acids 2 through 19 were removed (INX-6-deltaN) was crystallized in lipid bilayers. The three-dimensional reconstruction based on cryo-electron crystallography revealed that the two-dimensional crystals of INX-6-deltaN comprise two lipid bilayers, including fully docked gap junction channels. A single INX-6-deltaN gap junction channel comprises 16 subunits, a hexa-decamer, in contrast to vertebrate connexin channels, which comprise 12 subunits. Two bulb densities were observed in each hemichannel, one in the pore and the other at the cytoplasmic side of the hemichannel in the channel pore pathway. The former is reminiscent of the plug observed in the connexin26 mutant structure we previously reported. A fluorescent dye transfer assay revealed that INX-6-deltaN junction channels were essentially impermeable. These findings imply the structural diversity of gap junction channels among multicellular organisms, and provide insight into the functional properties characteristic of invertebrates.

[Structure of human erythrocyte band 3: two-dimensional crystallographic analysis of the membrane domain].

Band 3 (also known as anion exchanger 1, AE1) is one of the most abundant membrane proteins in human erythrocytes. Band 3 has 911 amino acids and consists of two structurally and functionally distinct domains. One is a 40-kDa N-terminal cytoplasmic domain and the other is a 55-kDa C-terminal membrane domain. The cytoplasmic domain maintains red cell shape through interactions with cytoskeletal proteins, such as protein 4.1, protein 4.2, ankyrin, and spectrin. On the other hand, the membrane domain mediates electroneutral exchange of anions, such as bicarbonate and chloride across the erythrocyte membrane. We reported the three-dimensional structure of the outward-open membrane domain of band 3, which was cross-linked between K539 and K851 with H2DIDS, at 7.5 Å resolution using cryo-electron crystallography. Although the results showed significantly improved resolution as compared with previous structural analyses, we could not assign all α-helices because of low resolution and uncertainty persists regarding the fold of band 3. However, we recognized that band 3 has internal repeats, because the structure exhibited distinctive anti-parallel V-shaped motifs, which protrude from the membrane bilayer on both sides. One of the helices in the motif is very long and highly tilted with respect to the normal structure of the bilayer.

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.

Data‐driven homology modelling of P‐glycoprotein in the ATP‐bound state indicates flexibility of the transmembrane domains

Human P-glycoprotein is an ATP-binding cassette transporter that plays an important role in the defence against potentially harmful molecules from the environment. It is involved in conferring resistance against cancer therapeutics and plays an important role for the pharmacokinetics of drugs. The lack of a high resolution structure of P-glycoprotein has hindered its functional understanding and represents an obstacle for structure based drug development. The homologous bacterial exporter Sav1866 has been shown to share a common architecture and overlapping substrate specificity with P-glycoprotein. The structure of Sav1866 suggests that helices in the transmembrane domains diverge at the extracytoplasmic face, whereas cross-link information and a combination of small angle X-ray scattering and cryo-electron crystallography data indicate that helices 6 and 12 of P-glycoprotein are closer in P-glycoprotein than in the crystal structure of Sav1866. Using homology modelling, we present evidence that the protein possesses intrinsic structural flexibility to allow cross-links to occur between helices 6 and 12 of P-glycoprotein, thereby reconciling crystallographic models with available experimental data from cross-linking.

Structural insights into P-glycoprotein (ABCB1) by small angle X-ray scattering and electron crystallography

P-glycoprotein (ABCB1) is an ATP-binding cassette protein that is associated with the acquisition of multi-drug resistance in cancer and the failure of chemotherapy in humans. Structural insights into this protein are described using a combination of small angle X-ray scattering data and cryo-electron crystallography data. We have compared the structures with bacterial homologues, and discuss the development of homology models for P-glycoprotein based on the bacterial Sav1866 structure.

NIH Grant Watch

BioTechniquesVol. 45, No. 1 NIH Grant WatchOpen AccessNIH Grant WatchD. McCormick† & B. Perry†D. McCormick††D. McCormick is editorial director of BioTechniques. B. Perry is president of NIH Sales, Rockville, MD.Search for more papers by this author & B. Perry††D. McCormick is editorial director of BioTechniques. B. Perry is president of NIH Sales, Rockville, MD.Search for more papers by this authorPublished Online:16 May 2018https://doi.org/10.2144/000112878AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Protein Isolation and PurificationBioTechniques and NIH Sales sought out National Institutes of Health research grants awarded to proposals that featured protein purification and isolation. The search returned 499 grants awarded between Nov. 12, 2007, and June 1, 2008. The National Institute of General Medical Sciences contributed 21.6% of the aggregate $165.4 million distributed, followed by the National Institute of Allergy and Infectious Diseases (19.9%) and the National Cancer Institute (16.0%). Grants ranged from $2.5 million for prion research at the University of California at San Francisco (see below) to $3500 to support research on structure-function relationships in kinesins at the Institute for Molecular Cell Biology in Dresden, Germany.Degenerative and Dementing Diseases of Aging $2,545,642(5P01AG002132-28, National Institute on Aging, 2/11/2008)Stanley B. Prusiner (University Of California, San Francisco, CA)Goal: To understand the replication of prions and the pathogenesis of prion diseases, and to identify therapeutic targets. The research program will investigate 1) the structure of the pathogenic isoform of the prion protein (PrPSc) using cryo-electron crystallography, computational modeling, and small-angle X-ray fiber; 2) suitability of well-established prion strains for structural analysis by electron crystallography and fiber; 3) PrP structural transitions; 4) the possibility that a mixture of PrP conformers can be manipulated by previously unappreciated features of PrP's topogenic sequences; and 5) the molecular basis of the species barrier. The proposed studies use Random Homozygous Knockout (RHKO), a novel mutational technology that enables the random homozygous inactivation of chromosomal genes in initially susceptible cell populations and the identification and isolation of cells that consequently survive prion-induced lethality.Medical Structural Genomics of Pathogenic Protozoa (MSGPP) $2,265,689(5P01AI067921-03, National Institute of Allergy and Infectious Diseases, 4/14/2008)Wilhelmus G. J. Hol (University Of Washington, Seattle, WA)Goal: To accelerate development of drugs to combat the 10 major pathogenic protozoa that infect humans by 1) analyzing the genome sequences from the targeted protozoa for suitable MSGPP protein targets from a functional and ligand perspective; 2) expressing, crystallizing, and determining three-dimensional structures of the selected crucial proteins, focusing on complexes with small-molecule ligands; 3) applying computational processes to these structures to predict new ligands targeting key sites of the selected proteins; 4) discovering in solution small-molecule ligands binding to target proteins; and 5) using such ligands for the development of new leads and drug candidates, in collaboration with interested institutions. Protein targets will be selected from the following pathogenic protozoa: Plasmodium falciparum, Plasmodium vivax, Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, Leishmania infantum, Entamoeba histolytica, Giardia lamblia, Cryptosporidium parvum, and Toxoplasma gondii.Structure and Function of Beta3 Integrins on Blood Cells $1,888,035(5P01HL073311-05, National Heart, Lung, and Blood Institute, 3/28/2008)Edward Plow (Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland, OH)Goal: To study the structure and function of the beta3 integrins, alpha-IIb-beta3 and alpha-v-beta3, targeting platelets, and endothelial cells. The program seeks to understand the molecular mechanisms that regulate the contribution of alpha-IIb-beta3 and alpha-v-beta3 to the adhesive and migratory responses of these cells. These data will, in turn, illuminate complex biological responses, including platelet aggregation and the angiogenic response of endothelial cells.Combinatorial Chemistry and Cancer Target $1,341,169(5P01CA078045-10, National Cancer Institute, 2/8/2008)Peter K Vogt (Scripps Research Institute, La Jolla, CA)Goal: To meld combinatorial chemistry and modern cancer biology and genetics, and to identify novel anticancer compounds. The program includes 1) production and testing of libraries targeted to protein-protein and protein-DNA interactions; 2) isolation of angiogenesis inhibitors (specifically of matrix metalloproteinase 2 and integrin alpha3beta1); and 3) isolation of small-molecule regulators of the Myc network and of beta-catenin-LEF interaction.FiguresReferencesRelatedDetails Vol. 45, No. 1 Follow us on social media for the latest updates Metrics Downloaded 163 times History Published online 16 May 2018 Published in print July 2008 Information© 2008 Author(s)PDF download

Cryo-Electron Microscopy of Biological Samples

A brief summary of current cryo–electron microscopy methods for processing and imaging biological tissues is provided. The main emphasis is given to two preparation procedures: frozen-hydrated samples because of the remarkable success of cryo-electron crystallography in obtaining near atomic resolution of integral membrane proteins, and high-pressure freezing because of the wide applicability for vitrification of large samples of normal and diseased tissues for ultrastructural and immunolabeling analysis. Methods for examining certain samples with a TEM cryo-stage are summarized. This includes an introduction to the relatively new area of cryo-electron tomography, which offers the possibility to observe the three-dimensional structure of subcellular components using only their natural variations in composition to generate contrast.

Trimeric structure of OprN and OprM efflux proteins from Pseudomonas aeruginosa, by 2D electron crystallography

OprM and OprN belong to the outer membrane factor family of multidrug efflux proteins from Pseudomonas aeruginosa, a bacterium responsible of nosocomial infections. We report here the two-dimensional (2D) crystallization of OprN and OprM into lipid bilayers and the determination of their 2D projected structure by cryo-electron crystallography, at 1 and 1.4 nm, respectively. Both proteins present a dense ring of protein density, of ∼7 nm diameter. An additional thin peripheral ring is resolved in OprN structure. Both proteins are assembled as trimers. The results presented here indicate a high structural homology between OprN (and OprM) and TolC, a multidrug efflux protein from Escherichia coli.

Structural Characterization of HC-Pro, a Plant Virus Multifunctional Protein

The helper component proteinase (HC-Pro) is a key protein encoded by plant viruses of the genus Potyvirus. HC-Pro is involved in different steps of the viral cycle, aphid transmission, replication, and virus cell-to-cell and systemic movement and is a suppressor of post-transcriptional gene silencing. Structural knowledge of HC-Pro is required to better understand its multiple functions. To this aim, we purified His-tagged wild-type HC-Pro and a N-terminal deletion mutant (DeltaHC-Pro) from plants infected with recombinant potyviruses. Biochemical analysis of the recombinant proteins confirmed that HC-Pro is a dimer in solution, that the N terminus is not essential for self-interaction, and that a large C-terminal domain is highly resistant to proteolysis. Two-dimensional crystals of the recombinant proteins were successfully grown on Ni2+-chelating lipid monolayers. Comparison of projection maps of negatively stained crystals revealed that HC-Pro is composed of two domains separated by a flexible constriction. Cryo-electron crystallography of DeltaHC-Pro allowed us to calculate a projection map at 9-A resolution. Our data from electron microscopy, biochemical analysis, and secondary structure predictions lead us to suggest a model for structure/function relationships in the HC-Pro protein.

Structure of the major membrane protein complex from urinary bladder epithelial cells by cryo-electron crystallography

Numerous protein plaques cover the apical surface of mammalian urinary bladder epithelial cells. These plaques contain four integral membrane proteins, called uroplakins, which form a well-ordered array of hexameric complexes. The 3D structure of these naturally occurring 2D crystals was studied by cryo-electron-crystallographic methods using a slow-scan charged-coupled device (CCD) camera to record the electron micrographs. A 1.2 nm projection map calculated from untilted crystals shows that each hexamer comprises a ring of six inner and six outer domains at a radius of 5.7 nm and 9.2 nm respectively. The 3D structure shows that the mass is distributed strongly asymmetrically with respect to the membrane, with most of the mass protruding from the luminal face. Both domains in the asymmetric unit traverse the membrane and protrude from the membrane on the cytoplasmic side. On the luminal side, the two domains are bridged forming a stretched arc. The total thickness of the complex is about 13.2 nm. A model of the urothelial plaque reveals that contacts between the hexamers are much less extended than within the hexamers.

New Concept in Helium Cooled Transmission Microscopes Philips Tecnai F30 Helium: Design and First Results

To date it has been possible to solve the structure of several membrane proteins to atomic resolution with cryo-electron crystallography (1-4). in all studies two-dimensional crystals were required as well as special provisions on the transmission microscope. in all cases a field emission gun provided the necessary coherence and liquid-Helium cooling the specimen protection (4,5).Although repeatedly studies have been done on the cryo-protection factor of beam sensitive specimen at liquid-Nitrogen and liquid-Helium temperatures (6-10), the data available are still too limited for a clear demonstration of the superiority of liquid-Helium. Especially investigations are needed with individual particles embedded in amorphous ice (11).To meet the increasing demand for liquid-Helium cooling, FEI developed the Tecnai F30 Helium. The challenge was to provide an instrument with a very stable cryo-stage, which can work at liquid- Nitrogen as well as liquid-Helium temperature.

Structural studies of cyanobacterial photosystem II

The determination of the structure of photosystem II (PSII) at high resolution is required in order to understand its reaction mechanisms. Two-dimensional crystals of purified highly active Synechococcus elongatus PSII dimers were obtained by in vitro reconstitution. Images of these crystals were recorded by cryo-electron microscopy and their analysis revealed they belong to the space group p22121, with unit cell parameters a=121A, b=333A and a =90o. From these crystals a projection map was calculated to a resolution of ~16A. The reliability of this projection map is confirmed by its very good agreement with the three-dimensional model of the same complex, obtained by X-ray crystallography, which has been recently published (1). Comparison of the cyanobacterial PSII structure with a 10A three-dimensional map recently obtained by cryo-electron crystallography of spinach PSII core dimers indicates a similar organisation of the transmembrane domain of the major subunits. However, differences are found between the cyanobacterial and the higher plant PSII complexes, related to the content and organisation of the small subunits and extrinsic proteins of the PSII complex in each organism. These data on the structure of cyanobacterial PSII core can also be used to understand its interaction with the external antennae complex, the hydrophilic phycobilisomes. Although some models have been suggested, the molecular interactions and the mechanisms of energy transfer between PSII and the phycobilisome core are far from fully understood. These interactions will be studied by combining the electron crystallographic and X-ray data with information obtained by single particle analysis of purified PSII-phycobilisome complexes. (1) Zouni et al., Nature (2001) 409: 739.