2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor

Bartesaghi, Alberto; Merk, Alan; Banerjee, Soojay; Matthies, Doreen; Wu, Xiongwu; Milne, Jacqueline L.S.; Subramaniam, Sriram

doi:10.1126/science.aab1576

Cited by 453 publications

(462 citation statements)

References 34 publications

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“…Careful selection of images taken from holes with the thinnest possible ice helps to improve the Thon ring visibility at high frequencies, as well as the resolution of the final 3D reconstruction [18]. However, this is not a generally applicable approach.…”

Section: Potential Limitations and Possible Approaches To Overcomementioning

confidence: 99%

“…Furthermore, tools that are used to align tilt series from tomographic data can be repurposed to align movie frames of single-particle data recorded with DDDs [18]. Each tool has its unique approach and features that are specifically designed to deal with either global or local motion (such as correcting motion per particle).…”

Section: Processing Image Stacksmentioning

confidence: 99%

“…For the K2 Summit operating in counting mode, it is important to optimize the dose rate on the camera. Typically, images are recorded at a dose rate of 10e − pixel −1 s −1 [4,17] or less [18]. Using lower dose rate can improve DQE, but at the expense of a longer exposure time to accumulate the same total dose, which may lead to larger file sizes.…”

Section: Image Acquisition Using Direct Electron Detection Camerasmentioning

confidence: 99%

“…For example, after image motion correction, a simple average of the entire stack usually has strong image contrast, essential for particle picking and facilitating accurate particle alignment. After obtaining a good alignment, the user may discard the first few frames, which usually suffer a large uncorrectable motion, and the later ones that suffer significant radiation damage and only use frames that have well-preserved highresolution signals to calculate the final reconstruction [4,17,18]. Instead of discarding the frames that are deteriorated by motion or radiation damage, a better approach is to apply a low-pass filter to each frame, down-weighting compromised high-resolution signal in frames before averaging them.…”

Section: Processing Image Stacksmentioning

confidence: 99%

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Single-particle cryo-EM data acquisition by using direct electron detection camera

Armache

Cheng

2015

Microscopy (Tokyo)

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Recent advances in single-particle electron cryo-microscopy (cryo-EM) were largely facilitated by the application of direct electron detection cameras. These cameras feature not only a significant improvement in detective quantum efficiency but also a high frame rate that enables images to be acquired as 'movies' made of stacks of many frames. In this review, we discuss how the applications of direct electron detection cameras in cryo-EM have changed the way the data are acquired.

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Section: Potential Limitations and Possible Approaches To Overcomementioning

confidence: 99%

Section: Processing Image Stacksmentioning

confidence: 99%

Section: Image Acquisition Using Direct Electron Detection Camerasmentioning

confidence: 99%

Section: Processing Image Stacksmentioning

confidence: 99%

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Single-particle cryo-EM data acquisition by using direct electron detection camera

Armache

Cheng

2015

Microscopy (Tokyo)

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“…With the advent of direct-electron-detection cameras, which offer improved low-dose imaging and which facilitate compensation for specimen drift and beam-induced motion, single-particle reconstruction now yields maps with resolutions below 3Å [6]. Recognizing that efficient automated data collection is essential for this work, we have implemented JDAS [7] on the current-generation cryo-TEM.…”

mentioning

confidence: 99%

Development of High-Resolution Transmission Electron Microscopes for Analysis of Biomolecular Structure

et al. 2017

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Determining the structure of biological macromolecules, such as proteins and nucleic acids, is important for understanding their function. This leads to a mechanistic understanding of biological systems, and suggests pathways for discovery of therapeutic drugs. Structural analysis of biological macromolecules was first demonstrated in 1968 by DeRosier and Klug, with their three-dimensional reconstruction of negatively stained bacteriophage T4 [1]. Since liquid water is not compatible with the vacuum of an electron microscope, yet water retention is essential to maintain high-resolution molecular structure, methods of freezing the water in a vitreous state were eventually developed. This enabled the molecules to be imaged in a near-native state in the electron microscope. Yet, the high-resolution structure of these samples is easily compromised by electron-beam irradiation. As expected, lowering the temperature of the sample in the electron microscope was shown to reduce radiation sensitivity, and the lowest temperatures provided the most protection [2]. Thus, an electron microscope with a cryostage to maintain the low temperature, and a cryo-transfer system to avoid frosting or devitrification during specimen insertion, was called for.In 1983, development of a dedicated cryo-transmission electron microscope (cryo-TEM) was started jointly by Y. Fujiyoshi and JEOL. Membrane proteins were chosen as the first specimens to be imaged, since they play important functions in the cell for trans-membrane transport and signaling, and knowledge of the structure and function of membrane proteins at the molecular level may clarify disease mechanisms. The three-dimensional structure of macromolecules can be obtained by several methods, including electron tomography, single-particle analysis, and electron diffraction. We chose electron diffraction, optimized for cryo-TEM. Either x-ray or electron diffraction can yield atomic or nearatomic results. Previously, x-ray diffraction was favored for obtaining the best resolution, but a threedimensional crystal is required. Electron diffraction is carried out with two-dimensional crystals, which are often easier to produce from biological macromolecules. By embedding two-dimensional crystals in lipid layers, followed by vitreous freezing, we were able to use electron diffraction to study membrane proteins at near-atomic resolution in a near-native state.We used a liquid-helium reservoir centrally located in the TEM column to cool the specimen stage. The helium reservoir was surrounded by a liquid-nitrogen tank to suppress thermal radiation from the surrounding room environment. In order to avoid vibration from bubbling of the liquid helium, we made use of super-fluidity to cool the specimen, by which we could reach a temperature close to 2K. In keeping with best practices at the time, we employed a top-entry stage design for optimum stability. After much trial-and-error, the first-generation cryo-TEM was completed in 1986, which enabled highresolution imaging at 4.2K. Since then, the c...

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Structural biology data archiving – where we are and what lies ahead

2018

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For almost 50 years, structural biology has endeavoured to conserve and share its experimental data and their interpretations (usually, atomistic models) through global public archives such as the Protein Data Bank, Electron Microscopy Data Bank and Biological Magnetic Resonance Data Bank (BMRB). These archives are treasure troves of freely accessible data that document our quest for molecular or atomic understanding of biological function and processes in health and disease. They have prepared the field to tackle new archiving challenges as more and more (combinations of) techniques are being utilized to elucidate structure at ever increasing length scales. Furthermore, the field has made substantial efforts to develop validation methods that help users to assess the reliability of structures and to identify the most appropriate data for their needs. In this Review, we present an overview of public data archives in structural biology and discuss the importance of validation for users and producers of structural data. Finally, we sketch our efforts to integrate structural data with bioimaging data and with other sources of biological data. This will make relevant structural information available and more easily discoverable for a wide range of scientists.

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2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor

Cited by 453 publications

References 34 publications

Single-particle cryo-EM data acquisition by using direct electron detection camera

Single-particle cryo-EM data acquisition by using direct electron detection camera

Development of High-Resolution Transmission Electron Microscopes for Analysis of Biomolecular Structure

Structural biology data archiving – where we are and what lies ahead

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