scholarly journals Routine sub-2.5 Å cryo-EM structure determination of GPCRs

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Radostin Danev ◽  
Matthew Belousoff ◽  
Yi-Lynn Liang ◽  
Xin Zhang ◽  
Fabian Eisenstein ◽  
...  
Keyword(s):  
Objective Lens ◽  
Phase Plate ◽  
Structure Based Drug Design ◽  
Energy Filtering ◽  
Methods Development ◽  
Small Proteins ◽  
Cryo Electron Microscopy ◽  
Depth Analysis ◽  
G Protein Coupled

AbstractCryo-electron microscopy (cryo-EM) of small membrane proteins, such as G protein-coupled receptors (GPCRs), remains challenging. Pushing the performance boundaries of the technique requires quantitative knowledge about the contribution of multiple factors. Here, we present an in-depth analysis and optimization of the main experimental parameters in cryo-EM. We combined actual structural studies with methods development to quantify the effects of the Volta phase plate, zero-loss energy filtering, objective lens aperture, defocus magnitude, total exposure, and grid type. By using this information to carefully maximize the experimental performance, it is now possible to routinely determine GPCR structures at resolutions better than 2.5 Å. The improved fidelity of such maps enables the building of better atomic models and will be crucial for the future expansion of cryo-EM into the structure-based drug design domain. The optimization guidelines given here are not limited to GPCRs and can be applied directly to other small proteins.

scholarly journals Routine sub-2.5 Å cryo-EM structure determination of B-family G protein-coupled receptors

2020 ◽  
Author(s):  
Radostin Danev ◽  
Matthew Belousoff ◽  
Yi-Lynn Liang ◽  
Xin Zhang ◽  
Denise Wootten ◽  
...  
Keyword(s):  
G Protein ◽  
Structure Determination ◽  
G Protein Coupled Receptors ◽  
Objective Lens ◽  
Phase Plate ◽  
Performance Limits ◽  
Experimental Conditions ◽  
Structure Based Drug Design ◽  
Depth Analysis ◽  
G Protein Coupled

AbstractCryo-electron microscopy (cryo-EM) experienced game-changing hardware and software advances about a decade ago. Since then, there have been gradual and steady improvements in experimental and data analysis methods. Nonetheless, structural analysis of nonsymmetric membrane proteins, such as G protein-coupled receptors (GPCRs), remains challenging. Their relatively low molecular weight and obstruction by the micelle/nanodisc result in marginal signal levels, which combined with the intrinsic flexibility of such complexes creates difficult structural study scenarios. Pushing the performance limits of cryo-EM requires careful optimization of all experimental aspects. To this end, it is necessary to build quantitative knowledge of the effect each parameter has on the outcome. Here, we present in-depth analysis of the influence of the main cryo-EM experimental factors on the performance for GPCR structure determination. We used a tandem experiment approach that combined real-world structural studies with parameter testing. We quantified the effects of using a Volta phase plate, zero-loss energy filtering, objective lens aperture, defocus magnitude, total exposure, and grid type. Through such systematic optimization of the experimental conditions, it has been possible to routinely determine class B1 GPCR structures at resolutions better than 2.5 Å. The improved fidelity of such maps helps to build higher confidence atomic models and will be crucial for the future expansion of cryo-EM into the structure-based drug design domain. The optimization guidelines drafted here are not limited to GPCRs and can be applied directly for the study of other challenging membrane protein targets.

scholarly journals High-resolution structure determination of sub-100 kilodalton complexes using conventional cryo-EM

2018 ◽  
Author(s):  
Mark A. Herzik ◽  
Mengyu Wu ◽  
Gabriel C. Lander
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Drug Target ◽  
Phase Plate ◽  
Biological Macromolecules ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
High Resolution Structure ◽  
Resolution Structure

Determining high-resolution structures of biological macromolecules with masses of less than 100 kilodaltons (kDa) has long been a goal of the cryo-electron microscopy (cryo-EM) community. While the Volta Phase Plate has enabled cryo-EM structure determination of biological specimens of this size range, use of this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine the high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.9 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity, indicating that these methodologies can be used to investigate the molecular details of drug-target interactions. Our ~2.8 Å and ~3.2 Å resolution structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the first sub-nanometer cryo-EM structure of a protein smaller than 50 kDa.

Frontiers in Cryo Electron Microscopy of Complex Macromolecular Assemblies

2019 ◽  
Vol 21 (1) ◽  
pp. 395-415 ◽  
Author(s):  
Jana Ognjenović ◽  
Reinhard Grisshammer ◽  
Sriram Subramaniam
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Specimen Preparation ◽  
Macromolecular Assemblies ◽  
Cryo Electron Microscopy ◽  
G Protein Coupled ◽  
Improved Methods

In recent years, cryo electron microscopy (cryo-EM) technology has been transformed with the development of better instrumentation, direct electron detectors, improved methods for specimen preparation, and improved software for data analysis. Analyses using single-particle cryo-EM methods have enabled determination of structures of proteins with sizes smaller than 100 kDa and resolutions of ∼2 Å in some cases. The use of electron tomography combined with subvolume averaging is beginning to allow the visualization of macromolecular complexes in their native environment in unprecedented detail. As a result of these advances, solutions to many intractable challenges in structural and cell biology, such as analysis of highly dynamic soluble and membrane-embedded protein complexes or partially ordered protein aggregates, are now within reach. Recent reports of structural studies of G protein–coupled receptors, spliceosomes, and fibrillar specimens illustrate the progress that has been made using cryo-EM methods, and are the main focus of this review.

scholarly journals Near-Atomic Resolution Structure Determination in Over-Focus with Volta Phase Plate by Cs-corrected Cryo-EM

2017 ◽  
Author(s):  
Xiao Fan ◽  
Lingyun Zhao ◽  
Chuan Liu ◽  
Jin-Can Zhang ◽  
Kelong Fan ◽  
...  
Keyword(s):  
Optical Properties ◽  
High Resolution ◽  
Spherical Aberration ◽  
Atomic Resolution ◽  
Objective Lens ◽  
Phase Plate ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Imaging Strategy ◽  
Imaging Conditions

SummaryVolta phase plate (VPP) is a recently developed transmission electron microscope (TEM) apparatus that can significantly enhance the image contrast of biological samples in cryo-electron microscopy (cryo-EM) therefore impose the possibility to solve structures of relatively small macromolecules at high resolution. In this work, we performed theoretical analysis and found that using phase plate on objective lens spherical aberration (Cs)-corrected TEM may gain some interesting optical properties, including the over-focus imaging of macromolecules. We subsequently evaluated the imaging strategy of frozen-hydrated apo-ferritin with VPP on a Cs-corrected TEM and obtained the structure of apo-ferritin at near atomic resolution from both under- and over-focused dataset, illustrating the feasibility and new potential of combining VPP with Cs-corrected TEM for high resolution cryo-EM.HighlightsThe successful combination of volta phase plate and Cs-corrector in single particle cryo-EM.Near-atomic structure determined from over-focused images by cryo-EM. VPP-Cs-corrector coupled EM provides interesting optical properties.In BriefWe took the unique advantage of the optical system by combining the volta phase plate and Cs-corrector in a modern TEM to collect high resolution micrographs of frozen-hydrated apo-ferritin in over-focus imaging conditions and determined the structure of apo-ferritin at 3.0 Angstrom resolution.

scholarly journals Toward G protein-coupled receptor structure-based drug design using X-ray lasers

IUCrJ ◽  
2019 ◽  
Vol 6 (6) ◽  
pp. 1106-1119 ◽  
Author(s):  
Andrii Ishchenko ◽  
Benjamin Stauch ◽  
Gye Won Han ◽  
Alexander Batyuk ◽  
Anna Shiriaeva ◽  
...  
Keyword(s):  
Drug Design ◽  
Crystal Structures ◽  
G Protein ◽  
Rapid Determination ◽  
Binding Pocket ◽  
G Protein Coupled Receptors ◽  
Structure Based Drug Design ◽  
G Protein Coupled ◽  
Serial Femtosecond Crystallography

Rational structure-based drug design (SBDD) relies on the availability of a large number of co-crystal structures to map the ligand-binding pocket of the target protein and use this information for lead-compound optimization via an iterative process. While SBDD has proven successful for many drug-discovery projects, its application to G protein-coupled receptors (GPCRs) has been limited owing to extreme difficulties with their crystallization. Here, a method is presented for the rapid determination of multiple co-crystal structures for a target GPCR in complex with various ligands, taking advantage of the serial femtosecond crystallography approach, which obviates the need for large crystals and requires only submilligram quantities of purified protein. The method was applied to the human β2-adrenergic receptor, resulting in eight room-temperature co-crystal structures with six different ligands, including previously unreported structures with carvedilol and propranolol. The generality of the proposed method was tested with three other receptors. This approach has the potential to enable SBDD for GPCRs and other difficult-to-crystallize membrane proteins.

Characteristics of an Electrostatic Phase Plate

1972 ◽  
Vol 30 ◽  
pp. 618-619
Author(s):  
William Krakow ◽  
Benjamin Siegel
Keyword(s):  
Phase Contrast ◽  
Objective Lens ◽  
Phase Plate ◽  
Net Charge ◽  
Phase Plates ◽  
Beam Currents ◽  
Contrast Image ◽  
Bright Phase ◽  
Additional Phase Shift ◽  
Additional Phase

Unwin has used a metallized non-conducting thread in the back focal plane of the objective lens that stops out a portion of the unscattered beam, takes on a localized positive charge and thus produces an additional phase shift to give a different transfer function of the lens. Under the particular conditions Unwin used, the phase contrast image was shifted to bright phase contrast for optimum focus.We have investigated the characteristics of this type of electrostatic phase plate, both analytically and experimentally, as functions of the magnitude of charge and defocus. Phase plates have been constructed by using Wollaston wire to mount 0.25μ diameter platinum wires across apertures ranging from 50 to 200μ diameter and vapor depositing SiO and gold on the mounted wires to give them the desired charging characteristics. The net charge was varied by adjusting only the bias on the Wehnelt shield of the gun, and hence the beam currents and effective size of the source.

Enhanced information from biological materials through technological improvements in cryo-electron microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 788-789
Author(s):  
Marc J.C. de Jong ◽  
Wim M. Busing ◽  
Max T. Otten
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Biological Materials ◽  
Electron Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
The Many ◽  
Technological Improvements ◽  
Beam Damage

Biological materials damage rapidly in the electron beam, limiting the amount of information that can be obtained in the transmission electron microscope. The discovery that observation at cryo temperatures strongly reduces beam damage (in addition to making it unnecessaiy to use chemical fixatives, dehydration agents and stains, which introduce artefacts) has given an important step forward to preserving the ‘live’ situation and makes it possible to study the relation between function, chemical composition and morphology.Among the many cryo-applications, the most challenging is perhaps the determination of the atomic structure. Henderson and co-workers were able to determine the structure of the purple membrane by electron crystallography, providing an understanding of the membrane's working as a proton pump. As far as understood at present, the main stumbling block in achieving high resolution appears to be a random movement of atoms or molecules in the specimen within a fraction of a second after exposure to the electron beam, which destroys the highest-resolution detail sought.

Trial production of γ-type energy filtering TEM

1995 ◽  
Vol 53 ◽  
pp. 604-605
Author(s):  
Y. Taniguchi ◽  
E. Nakazawa ◽  
S. Taya
Keyword(s):  
Magnetic Energy ◽  
Electron Optics ◽  
Electron Microscopic ◽  
Mass Spectrometers ◽  
Ion Optics ◽  
Energy Filtering ◽  
New Information ◽  
New Type ◽  
Fringing Fields

Imaging energy filters can add new information to electron microscopic images with respect to energy-axis, so-called electron spectroscopic imaging (ESI). Recently, many good results have been reported using this imaging technique. ESI also allows high-contrast observation of unstained biological samples, becoming a trend of the field of morphology. We manufactured a new type of energy filter as a trial production. This energy filter consists of two magnets, and we call γ-filter since the trajectory of electrons shows ‘γ’-shape inside the filter. We evaluated the new energyγ-filter TEM with the γ-filter.Figure 1 shows schematic view of the electron optics of the γ-type energy filter. For the determination of the electron-optics of the γ-type energy filter, we used the TRIO (Third Order Ion Optics) program which has been developed for the design of high resolution mass spectrometers. The TRIO takes the extended fringing fields (EFF) into consideration. EFF makes it difficult to design magnetic energy filters with magnetic sector fields.

Computer-Assisted High-Re Solution TEM

1990 ◽  
Vol 48 (1) ◽  
pp. 162-163
Author(s):  
F.A. Ponce ◽  
H. Hikashi
Keyword(s):  
Signal To Noise Ratio ◽  
Accurate Determination ◽  
Computer Software ◽  
Video Camera ◽  
Image Contrast ◽  
Objective Lens ◽  
Computer Assisted ◽  
Optical Parameters ◽  
Astigmatism Correction

The determination of the atomic positions from HRTEM micrographs is only possible if the optical parameters are known to a certain accuracy, and reliable through-focus series are available to match the experimental images with calculated images of possible atomic models. The main limitation in interpreting images at the atomic level is the knowledge of the optical parameters such as beam alignment, astigmatism correction and defocus value. Under ordinary conditions, the uncertainty in these values is sufficiently large to prevent the accurate determination of the atomic positions. Therefore, in order to achieve the resolution power of the microscope (under 0.2nm) it is necessary to take extraordinary measures. The use of on line computers has been proposed [e.g.: 2-5] and used with certain amount of success.We have built a system that can perform operations in the range of one frame stored and analyzed per second. A schematic diagram of the system is shown in figure 1. A JEOL 4000EX microscope equipped with an external computer interface is directly linked to a SUN-3 computer. All electrical parameters in the microscope can be changed via this interface by the use of a set of commands. The image is received from a video camera. A commercial image processor improves the signal-to-noise ratio by recursively averaging with a time constant, usually set at 0.25 sec. The computer software is based on a multi-window system and is entirely mouse-driven. All operations can be performed by clicking the mouse on the appropiate windows and buttons. This capability leads to extreme friendliness, ease of operation, and high operator speeds. Image analysis can be done in various ways. Here, we have measured the image contrast and used it to optimize certain parameters. The system is designed to have instant access to: (a) x- and y- alignment coils, (b) x- and y- astigmatism correction coils, and (c) objective lens current. The algorithm is shown in figure 2. Figure 3 shows an example taken from a thin CdTe crystal. The image contrast is displayed for changing objective lens current (defocus value). The display is calibrated in angstroms. Images are stored on the disk and are accessible by clicking the data points in the graph. Some of the frame-store images are displayed in Fig. 4.

ANALYSIS OF ACCOMPANYING PATHOLOGIES IN WOMEN WITH VARIOUS DISORDERS OF THE MENSTRUAL CYCLE

2020 ◽  
pp. 33-43
Author(s):  
Ольга Александровна Судакова ◽  
Михаил Вадимович Фролов ◽  
Алина Сергеевна Позднякова ◽  
Евгений Владимирович Белов ◽  
Данаил Красимирович Назлиев
Keyword(s):  
Antenatal Clinic ◽  
Reproductive Age ◽  
General Will ◽  
Organ Systems ◽  
Depth Analysis ◽  
Menstrual Irregularities ◽  
Positive Effect ◽  
Female Genital ◽  
Number Of Authors

Статья посвящена изучению сопутствующей патологии, у женщин репродуктивного возраста, обращающихся в стационар с жалобами на нарушения менструального цикла (НМЦ). Актуальность данной тематики не вызывает сомнения, так как с каждым годом в России и во всем мире регистрируется все большее количество случаев НМЦ. По мнению ряда авторов, данные нарушения могут составлять до 50% всех патологий женской половой сферы. Большой интерес представляет и изучение ряда сопутствующих заболеваний, которые могут отягощать течение НМЦ или наоборот, приводить к их развитию. Целью работы стал анализ разнообразной сопутствующей патологии при НМЦ, с выявлением основных причин нарушений менструального цикла у женщин фертильного возраста. Объектами исследования стали 300 пациенток, с диагнозом НМЦ, которые были разделены на 3 группы, в зависимости от уровня лечебного учреждения, где они проходили обследование - по 100 пациенток: проходивших обследование в больнице скорой медицинской помощи, обследующиеся в женской консультации и проходящие лечение сопутствующей онкопатологии в областном онкологическом диспансере. В дальнейшем проводилась дополнительное деление в каждой группе на 2 подгруппы, в зависимости от того был ли НМЦ впервые выявленным или повторно выявленным. В самой работе проводился подробный анализ сопутствующей патологии у женщин в зависимости от группы и их возраста. Определялись не только «пораженные» системы органов, но и проводился углубленный анализ по нозологиям. Работа интересна еще и тем, что в ней у всех пациенток на протяжении исследования определялся уровень стресса и наличие возможных депрессивных состояний. Определение наиболее вероятных причин НМЦ стало завершающим этапом исследования. Полученные данные могут приблизить практикующих акушеров-гинекологов к более полному пониманию различных нарушений менструального цикла, что в целом, положительно скажется на качестве и эффективности оказываемой медицинской помощи The article is devoted to the study of concomitant pathology in women of reproductive age who go to the hospital with complaints of menstrual irregularities (NMC). The relevance of this topic is beyond doubt, since every year in Russia and around the world an increasing number of cases of NMC are registered. According to a number of authors, these violations can account for up to 50% of all pathologies of the female genital area. Of great interest is the study of a number of concomitant diseases that can aggravate the course of NMC or, conversely, lead to their development. The aim of the work was to analyze a variety of concomitant pathologies in NMC, with the identification of the main causes of menstrual irregularities in women of fertile age. The objects of the study were 300 patients diagnosed with NMC, who were divided into 3 groups, depending on the level of the medical institution where they were examined - 100 patients each: who were examined in an emergency hospital, examined in an antenatal clinic and undergoing treatment for concomitant oncopathology in the regional oncological dispensary. Subsequently, an additional division was carried out in each group into 2 subgroups, depending on whether the NMC was newly detected or re-identified. In the work itself, a detailed analysis of comorbidities in women was carried out, depending on the group and their age. Not only the "affected" organ systems were identified, but an in-depth analysis of nosologies was also carried out. The work is also interesting in that during the study the level of stress and the presence of possible depressive states were determined in all patients. Determination of the most probable causes of NMC was the final stage of the study. The data obtained can bring practicing obstetricians and gynecologists closer to a more complete understanding of various menstrual irregularities, which, in general, will have a positive effect on the quality and effectiveness of medical care

Sign in / Sign up

Export Citation Format

Share Document