1.8 Å resolution structure of β-galactosidase with a 200 kV CRYO ARM electron microscope

We report the determination of the structure of Escherichia coli β-galactosidase at a resolution of ∼1.8 Å using data collected on a 200 kV CRYO ARM microscope equipped with a K3 direct electron detector. The data were collected in a single 24 h session by recording images from an array of 7 × 7 holes at each stage position using the automated data collection program SerialEM. In addition to the expected features such as holes in the densities of aromatic residues, the map also shows density bumps corresponding to the locations of hydrogen atoms. The hydrogen densities are useful in assigning absolute orientations for residues such as glutamine or asparagine by removing the uncertainty in the fitting of the amide groups, and are likely to be especially relevant in the context of structure-guided drug design. These findings validate the use of electron microscopes operating at 200 kV for imaging protein complexes at atomic resolution using cryo-EM.

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Sub-3 Å resolution structure of apoferritin using a multi-purpose TEM with a side-entry cryo-holder

10.1101/2020.03.24.006619 ◽

2020 ◽

Author(s):

Yoko Kayama ◽

Raymond N. Burton-Smith ◽

Chihong Song ◽

Naoya Terahara ◽

Takayuki Kato ◽

...

Keyword(s):

Protein Complexes ◽

Side Chain ◽

Particle Analysis ◽

Aromatic Side Chain ◽

Competitive Situation ◽

Cryo Electron Microscopy ◽

Electron Microscopes ◽

Imaging Conditions ◽

Biophysical Method ◽

Resolution Structure

SummaryThe structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single particle analysis (SPA) has had great impact as a biophysical method in recent years. Many results of cryo-EM SPA are based on state-of-the-art cryo-electron microscopes customized for SPA. These are currently only available in limited locations around the world, where securing machine time is highly competitive. One potential solution for this time-competitive situation is to reuse existing multi-purpose equipment. Here, we used a multi-purpose TEM with a side entry cryo-holder at our facility to evaluate the potential of high-resolution SPA. We report a 3 Å resolution map of apoferritin with local resolution extending to 2.6 Å. The map clearly showed two positions of an aromatic side chain. We also verified the optimal imaging conditions depending on different electron microscope and camera combinations. This study demonstrates the possibilities of more widely available and established electron microscopes, and their applications for cryo-EM SPA.

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Programming conventional electron microscopes for solving ultrahigh-resolution structures of small and macro-molecules

10.1101/557827 ◽

2019 ◽

Author(s):

Heng zhou ◽

Feng Luo ◽

Zhipu Luo ◽

Dan Li ◽

Cong Liu ◽

...

Keyword(s):

Organic Compounds ◽

Structure Determination ◽

Low Cost ◽

Ccd Camera ◽

Biological Macromolecules ◽

Hydrogen Atoms ◽

Ultrahigh Resolution ◽

Electron Microscopes ◽

Camera Synchronization

AbstractMicrocrystal electron diffraction (MicroED) is becoming a powerful tool in determining the crystal structures of biological macromolecules and small organic compounds. However, wide applications of this technique are still limited by the special requirement for radiation-tolerated movie-mode camera and the lacking of automated data collection method. Herein, we develop a stage-camera synchronization scheme to minimize the hardware requirements and enable the use of the conventional electron cryo-microscope with single-frame CCD camera, which ensures not only the acquisition of ultrahigh-resolution diffraction data but also low cost in practice. This method renders the structure determination of both peptide and small organic compounds at ultrahigh resolution up to ~0.60 Å with unambiguous assignment of nearly all hydrogen atoms. The present work provides a widely applicable solution for routine structure determination of MicroED, and demonstrates the capability of the low-end 120kV microscope with a CCD camera in solving ultra-high resolution structures of both organic compound and biological macromolecules.

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Resolving Individual-Atom of Protein Complex using Commonly Available 300-kV Cryo-electron Microscopes

10.1101/2020.08.19.256909 ◽

2020 ◽

Cited By ~ 2

Author(s):

Kaiming Zhang ◽

Grigore D. Pintilie ◽

Shanshan Li ◽

Michael F. Schmid ◽

Wah Chiu

Keyword(s):

Structure Determination ◽

Single Particle ◽

Heavy Atom ◽

Atomic Resolution ◽

Water Molecules ◽

Hydrogen Atoms ◽

Cryo Electron Microscopy ◽

Resolution Model ◽

Electron Microscopes ◽

Resolution Structure

AbstractBreakthroughs in single-particle cryo-electron microscopy (cryo-EM) technology have made near-atomic resolution structure determination possible. Here, we report a ∼1.35-Å structure of apoferritin reconstructed from images recorded on a Gatan K3 or a Thermo Fisher Falcon 4 detector in a commonly available 300-kV Titan Krios microscope (G3i) equipped with or without a Gatan post-column energy filter. Our results demonstrate that the atomic-resolution structure determination can be achieved by single-particle cryo-EM with a fraction of a day of automated data collection. These structures resolve unambiguously each heavy atom (C, N, O, and S) in the amino acid side chains with an indication of hydrogen atoms’ presence and position, as well as the unambiguous existence of multiple rotameric configurations for some residues. We also develop a statistical and chemical based protocol to assess the positions of the water molecules directly from the cryo-EM map. In addition, we have introduced a B’ factor equivalent to the conventional B factor traditionally used by crystallography to annotate the atomic resolution model for determined structures. Our findings will be of immense interest among protein and medicinal scientists engaging in both basic and translational research.

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Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins

FEBS Letters ◽

10.1016/j.febslet.2010.04.015 ◽

2010 ◽

Vol 584 (12) ◽

pp. 2539-2547 ◽

Cited By ~ 38

Author(s):

Yo Sonoda ◽

Alex Cameron ◽

Simon Newstead ◽

Hiroshi Omote ◽

Yoshinori Moriyama ◽

...

Keyword(s):

High Resolution ◽

Membrane Proteins ◽

Structure Determination ◽

High Resolution Structure ◽

Resolution Structure

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Ab Initio Determination of a New Complex Structure (55 Non-Hydrogen Atoms) from Synchrotron Powder Data: An Uncommon Nickel Succinate, Ni7(C4H4O4)4(OH)6(H2O)3•7H2O

Materials Science Forum ◽

10.4028/www.scientific.net/msf.443-444.333 ◽

2004 ◽

Vol 443-444 ◽

pp. 333-336

Author(s):

N. Guillou ◽

C. Livage ◽

W. van Beek ◽

G. Férey

Keyword(s):

Ab Initio ◽

Complex Structure ◽

Three Dimensional ◽

Free Water ◽

Hydrogen Atoms ◽

Monoclinic System ◽

Synchrotron Powder Diffraction ◽

Chloride Hexahydrate ◽

Autogenous Pressure

Ni7(C4H4O4)4(OH)6(H2O)3. 7H2O, a new layered nickel(II) succinate, was prepared hydrothermally (180°C, 48 h, autogenous pressure) from a 1:1.5:4.1:120 mixture of nickel (II) chloride hexahydrate, succinic acid, potassium hydroxide and water. It crystallizes in the monoclinic system (space group P21/c, Z = 4) with the following parameters a = 7.8597(1) Å, b = 18.8154(3)Å, c = 23.4377(4) Å,ϐ = 92.0288(9)°, and V = 3463.9(2) Å3. Its structure, which contains 55 non-hydrogen atoms, was solved ab initio from synchrotron powder diffraction data. It can be described from hybrid organic-inorganic layers, constructed from nickel oxide corrugated chains. These chains are built up from NiO6hexameric units connected via a seventh octahedron. Half of the succinates decorate the chains, and the others connect them to form the layers. The three dimensional arrangement is ensured by hydrogen bonds directly between two adjacent layers and via free water molecules.

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Biofilm Localization in the Vertical Wall of Shaking 96-Well Plates

Scientifica ◽

10.1155/2014/231083 ◽

2014 ◽

Vol 2014 ◽

pp. 1-6 ◽

Cited By ~ 3

Author(s):

Luciana C. Gomes ◽

Joana M. R. Moreira ◽

Manuel Simões ◽

Luís F. Melo ◽

Filipe J. Mergulhão

Keyword(s):

Low Cost ◽

Vertical Wall ◽

Spatial Location ◽

Strain Rates ◽

Microtiter Plates ◽

Equipment Availability ◽

Cell Accumulation ◽

Electron Microscopes ◽

Air Liquid Interface

Microtiter plates with 96 wells are being increasingly used for biofilm studies due to their high throughput, low cost, easy handling, and easy application of several analytical methods to evaluate different biofilm parameters. These methods provide bulk information about the biofilm formed in each well but lack in detail, namely, regarding the spatial location of the biofilms. This location can be obtained by microscopy observation using optical and electron microscopes, but these techniques have lower throughput and higher cost and are subjected to equipment availability. This work describes a differential crystal violet (CV) staining method that enabled the determination of the spatial location ofEscherichia colibiofilms formed in the vertical wall of shaking 96-well plates. It was shown that the biofilms were unevenly distributed on the wall with denser cell accumulation near the air-liquid interface. The results were corroborated by scanning electron microscopy and a correlation was found between biofilm accumulation and the wall shear strain rates determined by computational fluid dynamics. The developed method is quicker and less expensive and has a higher throughput than the existing methods available for spatial location of biofilms in microtiter plates.

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Determination of hydrogen ordering within the β-RH2+xphase (R= Ho, Y) using electron diffraction techniques

Journal of Applied Crystallography ◽

10.1107/s0021889800009523 ◽

2000 ◽

Vol 33 (5) ◽

pp. 1246-1252 ◽

Cited By ~ 3

Author(s):

Elizabeth J. Grier ◽

Amanda K. Petford-Long ◽

Roger C. C. Ward

Keyword(s):

Electron Diffraction ◽

Neutron Scattering ◽

Diffraction Pattern ◽

Computer Simulations ◽

Fluorite Structure ◽

Long Range Order ◽

Hydrogen Atoms ◽

Ordered Structures ◽

Diffraction Patterns

Computer simulations of the electron diffraction patterns along the [\bar{1}10] zone axes of four ordered structures within the β-RH2+xphase, withR= Ho or Y, and 0 ≤x≤ 0.25, have been performed to establish whether or not the hydrogen ordering could be detected using electron diffraction techniques. Ordered structures within otherRH2+x(R= Ce, Tb) systems have been characterized with neutron scattering experiments; however, for HoH(D)2+x, neutron scattering failed to characterize the superstructure, possibly because of the lowxconcentration or lack of long-range order within the crystal. This paper aims to show that electron diffraction could overcome both of these problems. The structures considered were the stoichiometric face-centred cubic (f.c.c.) fluorite structure (x= 0), theD1 structure (x= 0.125), theD1astructure (x= 0.2) and theD022structure (x= 0.25). In the stoichiometric structure, with all hydrogen atoms located on the tetrahedral (t) sites, only the diffraction pattern from the f.c.c. metal lattice was seen; however, for the superstoichiometric structures, with the excess hydrogen atoms ordered on the octahedral (o) sites, extra reflections were visible. All the superstoichiometric structures showed extra reflections at the (001)f.c.c.and (110)f.c.c.type positions, with structureD1 also showing extra peaks at (½ ½ ½)f.c.c.. These reflections are not seen in the simulations at similar hydrogen concentrations with the hydrogen atoms randomly occupying theovacancies.

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Sport Concussion Education and Prevention

Journal of Clinical Sport Psychology ◽

10.1123/jcsp.6.3.293 ◽

2012 ◽

Vol 6 (3) ◽

pp. 293-301 ◽

Cited By ~ 25

Author(s):

Charles H. Tator

Keyword(s):

Health Care ◽

Health Care Professionals ◽

Educational Strategies ◽

Short Term ◽

Sports Organizations ◽

Sport Concussion ◽

Data Collection Program ◽

Collection Program ◽

Concussion Education

There has been a remarkable increase in the past 10 years in the awareness of concussion in the sports and recreation communities. Just as sport participants, their families, coaches, trainers, and sports organizations now know more about concussions, health care professionals are also better prepared to diagnose and manage concussions. As has been stated in the formal articles in this special issue on sport-related concussion, education about concussion is one of the most important aspects of concussion prevention, with the others being data collection, program evaluation, improved engineering, and introduction and enforcement of rules. Unfortunately, the incidence of concussion appears to be rising in many sports and thus, additional sports-specific strategies are required to reduce the incidence, short-term effects, and long term consequences of concussion. Enhanced educational strategies are required to ensure that individual participants, sports organizations, and health care professionals recognize concussions and manage them proficiently according to internationally recognized guidelines. Therefore, this paper serves as a “brief report” on a few important aspects of concussion education and prevention.

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Remote Sensing Is Changing Our View of the Coast: Insights from 40 Years of Monitoring at Narrabeen-Collaroy, Australia

Remote Sensing ◽

10.3390/rs10111744 ◽

2018 ◽

Vol 10 (11) ◽

pp. 1744 ◽

Cited By ~ 28

Author(s):

Kristen Splinter ◽

Mitchell Harley ◽

Ian Turner

Keyword(s):

Remote Sensing ◽

Data Collection ◽

Temporal Resolution ◽

Monitoring Program ◽

Google Earth ◽

Airborne Lidar ◽

Data Collection Program ◽

Insight Into ◽

Collection Program

Narrabeen-Collaroy Beach, located on the Northern Beaches of Sydney along the Pacific coast of southeast Australia, is one of the longest continuously monitored beaches in the world. This paper provides an overview of the evolution and international scientific impact of this long-term beach monitoring program, from its humble beginnings over 40 years ago using the rod and tape measure Emery field survey method; to today, where the application of remote sensing data collection including drones, satellites and crowd-sourced smartphone images, are now core aspects of this continuing and much expanded monitoring effort. Commenced in 1976, surveying at this beach for the first 30 years focused on in-situ methods, whereby the growing database of monthly beach profile surveys informed the coastal science community about fundamental processes such as beach state evolution and the role of cross-shore and alongshore sediment transport in embayment morphodynamics. In the mid-2000s, continuous (hourly) video-based monitoring was the first application of routine remote sensing at the site, providing much greater spatial and temporal resolution over the traditional monthly surveys. This implementation of video as the first of a now rapidly expanding range of remote sensing tools and techniques also facilitated much wider access by the international research community to the continuing data collection program at Narrabeen-Collaroy. In the past decade the video-based data streams have formed the basis of deeper understanding into storm to multi-year response of the shoreline to changing wave conditions and also contributed to progress in the understanding of estuary entrance dynamics. More recently, ‘opportunistic’ remote sensing platforms such as surf cameras and smartphones have also been used for image-based shoreline data collection. Commencing in 2011, a significant new focus for the Narrabeen-Collaroy monitoring program shifted to include airborne lidar (and later Unmanned Aerial Vehicles (UAVs)), in an enhanced effort to quantify the morphological impacts of individual storm events, understand key drivers of erosion, and the placing of these observations within their broader regional context. A fixed continuous scanning lidar installed in 2014 again improved the spatial and temporal resolution of the remote-sensed data collection, providing new insight into swash dynamics and the often-overlooked processes of post-storm beach recovery. The use of satellite data that is now readily available to all coastal researchers via Google Earth Engine continues to expand the routine data collection program and provide key insight into multi-decadal shoreline variability. As new and expanding remote sensing technologies continue to emerge, a key lesson from the long-term monitoring at Narrabeen-Collaroy is the importance of a regular re-evaluation of what data is most needed to progress the science.

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Breaking the next Cryo-EM resolution barrier – Atomic resolution determination of proteins!

10.1101/2020.05.21.106740 ◽

2020 ◽

Cited By ~ 12

Author(s):

Ka Man Yip ◽

Niels Fischer ◽

Elham Paknia ◽

Ashwin Chari ◽

Holger Stark

Keyword(s):

Protein Function ◽

Model Building ◽

Technological Development ◽

Drug Binding ◽

Single Atom ◽

Biological Macromolecules ◽

Direct Visualization ◽

Hydrogen Atoms ◽

Structure Based Drug Design ◽

Electron Microscopes

SummarySingle particle cryo-EM is a powerful method to solve the three-dimensional structures of biological macromolecules. The technological development of electron microscopes, detectors, automated procedures in combination with user friendly image processing software and ever-increasing computational power have made cryo-EM a successful and largely expanding technology over the last decade. At resolutions better than 4 Å, atomic model building starts becoming possible but the direct visualization of true atomic positions in protein structure determination requires significantly higher (< 1.5 Å) resolution, which so far could not be attained by cryo-EM. The direct visualization of atom positions is essential for understanding protein-catalyzed chemical reaction mechanisms and to study drug-binding and -interference with protein function. Here we report a 1.25 Å resolution structure of apoferritin obtained by cryo-EM with a newly developed electron microscope providing unprecedented structural details. Our apoferritin structure has almost twice the 3D information content of the current world record reconstruction (at 1.54 Å resolution 1). For the first time in cryo-EM we can visualize individual atoms in a protein, see density for hydrogen atoms and single atom chemical modifications. Beyond the nominal improvement in resolution we can also show a significant improvement in quality of the cryo-EM density map which is highly relevant for using cryo-EM in structure-based drug design.

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