The Resolution Revolution

Kühlbrandt, Werner

doi:10.1126/science.1251652

Cited by 1,101 publications

(705 citation statements)

References 10 publications

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“…Although good studies with beautiful TEM can be found where cryo‐electron microscopy47 and 3D methods such as cryo‐electron tomography19, 48 and 2D49 and 3D single‐particle analyses50 have been put to use on soft matter structures, too many misapplications and misinterpretations are present in the current literature.…”

Section: Quality and Outlookmentioning

confidence: 99%

“…Other research fields that use cryo‐TEM are making more and more use of new technical developments such as the volta‐phase plate56 that enhances image contrast and allows imaging in focus. Another great and recent development is the direct detection camera57 that induced a true resolution revolution47 in the field of structural biology because of its ability to record movies that allow the correction for drift and the detection of radiation damage that accumulates with exposure time.…”

Section: Quality and Outlookmentioning

confidence: 99%

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Transmission Electron Microscopy as a Tool for the Characterization of Soft Materials: Application and Interpretation

2017

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Transmission electron microscopy (TEM) provides direct structural information on nano‐structured materials and is popular as a characterization tool in soft matter and supramolecular chemistry. However, technical aspects of sample preparation are overlooked and erroneous image interpretations are regularly encountered in the literature. There are three most commonly used TEM methods as we derived from literature: drying, staining and cryo‐TEM, which are explained here with respect to their application, limitations and interpretation. Since soft matter chemistry relies on a lot of indirect evidence, the role of TEM for the correct evaluation of the nature of an assembly is very large. Mistakes in application and interpretation can therefore have enormous impact on the quality of present and future studies. We provide helpful background information of these three techniques, the information that can and cannot be derived from them and provide assistance in selecting the right technique for soft matter imaging. This essay warns against the use of drying and explains why. In general cryo‐TEM is by far the best suited method and many mistakes and over‐interpretations can be avoided by the use of this technique.

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Section: Quality and Outlookmentioning

confidence: 99%

Section: Quality and Outlookmentioning

confidence: 99%

Transmission Electron Microscopy as a Tool for the Characterization of Soft Materials: Application and Interpretation

2017

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“…The wide application of direct electron detection device (DDD) cameras enabled a 'resolution revolution' in singleparticle cryo-EM [10]. All DDD cameras have significantly higher DQEs than photographic film and scintillator-based cameras [4,5,11].…”

Section: Image Acquisition Using Direct Electron Detection Camerasmentioning

confidence: 99%

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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“…This makes it very difficult to be purified in abundant and homogenous form, to be crystallized, therefore to be solved structurally using X-ray crystallography. The most recent technique revolution in cryo-electron microscopy made it possible to solve macromolecules at atomic resolution without the crystallization step [4]. The even more powerful capability of the new technology is to separate compositional or conformational heterogeneous molecule structures by computation after taking images of them in frozenhydrated specimen in an electron microscope.…”

mentioning

confidence: 99%

Opening new doors for understanding eukaryotic RNA splicing

Wang

2015

Sci. China Life Sci.

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Since the central dogma of molecular biology was proposed by Francis Crick in 1956, many fundamental discoveries have been made to strengthen or expand the theory for gene expression. Nowadays, we know that genetic information flow in a cell is a far more complicated process than the original succinct but explicit version. We now know that besides DNA duplication, DNA to RNA transcription, RNA to protein translation, there are also RNA to DNA transcription (reverse transcription), RNA to RNA replication, and RNA splicing and processing between the transcription and translation steps. Modifications and other processes to regulate and fine-tune gene expression have also been discovered in most recent years. All of these events are executed by multiple supramolecular machineries such as DNA and RNA polymerases, ribosomes, and spliceosomes. These machineries are normally multi-functional complexes composed of dozens of protein and RNA molecules and their activities are regulated by many other co-factor proteins or RNA molecules.In the past few decades, structural biology, as a tool to decipher the precise atomic coordinates within a molecule, has played essential roles in elucidating most of the critical steps in gene expression. The atomic models of DNA polymerases revealed how DNA is duplicated; the atomic model of RNA polymerase complex explained in great detail of how RNA is transcribed from its DNA gene; the structures of ribosome in its various states described vividly how proteins are translated from messenger RNAs. Not only were the structures of the major molecular machines involved in the aforementioned processes solved, but also their different working states and regulatory co-factors have been revealed in great structural details. These structures brought major leaps for our understanding of central dogma. In contrast, a major step of the gene expression, RNA splicing, is still largely a jigsaw puzzle in fog, due to the lack of structural information of the major machinery, the spliceosome. More than 30 years after its first component was discovered, the highest resolution of spliceosome complex was only solved at about 20 Å using electron microscopy, far away for us to understand the detailed mechanism of how RNA splicing is performed. This embarrassing situation now gets to a turning point to catch up soon with other central dogma steps. The most recently solved spliceosome complex structure at near atomic resolution by professor Yigong Shi and his colleagues in Tsinghua University wipes off the fog and opens new doors for understanding RNA splicing in a new foreground [1,2].Almost all RNA transcripts in eukaryotic cells need additional processing steps before their maturation. The most critical processing step for messenger RNAs (mRNA) is the splicing, which removes the non-coding region (intron) and ligates the coding regions (exon) from a newly transcribed RNA to generate a mature RNA for translation. In splicing, the two ends of an intron are brought to spatial proximity for two successive trans-es...

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The Resolution Revolution

Abstract: Advances in detector technology and image processing are yielding high-resolution electron cryo-microscopy structures of biomolecules. [Also see Report by Amunts et al. ]

Cited by 1,101 publications

References 10 publications

Transmission Electron Microscopy as a Tool for the Characterization of Soft Materials: Application and Interpretation

Transmission Electron Microscopy as a Tool for the Characterization of Soft Materials: Application and Interpretation

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

Opening new doors for understanding eukaryotic RNA splicing

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