Automation for Cryo-TEM: from Specimen Grid to 3D Map

2001 ◽  
Vol 7 (S2) ◽  
pp. 970-971
Author(s):  
B. Carragher ◽  
D. Fellmann ◽  
N. Kisseberth ◽  
R.A. Milligan ◽  
C.S. Potter ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Low Dose ◽  
Protein Complexes ◽  
Digital Camera ◽  
Single Session ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Data Collection And Analysis ◽  
Time Required ◽  
Automated Methods

Cryo-electron microscopy is becoming an increasingly powerful tool for solving the structure of protein complexes and has the potential to address problems that cannot be solved using other methods. The field however suffers from several major disadvantages related to the time required to acquire, process and analyze the data and the tedium of using the current prevailing methods. We have for some time been working towards the goal of developing a system that will result in a 3D map of a macromolecular structure automatically and within hours of inserting a specimen into a transmission electron microscope. We propose that these automated methods for data collection and analysis will have a significant impact in transferring the cryo-electron microscopy technology to the general biological community as well as in increasing the volume of data that can be collected during a single session at the microscope.The Leginon system that we have developed is designed to emulate all of the decisions and actions of a highly trained microscopist in collecting data from a vitreous ice specimen. These include identifying suitable areas of vitreous ice at low magnification, determining the presence and location of specimen on the grid, automatically adjusting imaging parameters (focus, astigmatism) under low dose conditions and acquiring images at high magnification to either film or a digital camera.

Search mode for rapid detection of virus particles

1991 ◽  
Vol 49 ◽  
pp. 286-287
Author(s):  
O. E. Bradfute
Keyword(s):  
Electron Microscopy ◽  
Rapid Detection ◽  
Plant Virus ◽  
Low Dose ◽  
Search Mode ◽  
Virus Diseases ◽  
Virus Particles ◽  
Focus Image ◽  
Time Required ◽  
Rapid Switching

Electron microscopy is frequently used in preliminary diagnosis of plant virus diseases by surveying negatively stained preparations of crude extracts of leaf samples. A major limitation of this method is the time required to survey grids when the concentration of virus particles (VPs) is low. A rapid survey of grids for VPs is reported here; the method employs a low magnification, out-of-focus Search Mode similar to that used for low dose electron microscopy of radiation sensitive specimens. A higher magnification, in-focus Confirm Mode is used to photograph or confirm the detection of VPs. Setting up the Search Mode by obtaining an out-of-focus image of the specimen in diffraction (K. H. Downing and W. Chiu, private communications) and pre-aligning the image in Search Mode with the image in Confirm Mode facilitates rapid switching between Modes.

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.

scholarly journals Structural Analysis of Jumbo Coliphage phAPEC6

2020 ◽  
Vol 21 (9) ◽  
pp. 3119 ◽  
Author(s):  
Jeroen Wagemans ◽  
Jessica Tsonos ◽  
Dominique Holtappels ◽  
Kiandro Fortuna ◽  
Jean-Pierre Hernalsteens ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Capsid Protein ◽  
Tem Analysis ◽  
Host Interaction ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Tail Sheath ◽  
Associated Proteins ◽  
Contractile Tail ◽  
Phage Capsid

The phAPEC6 genome encodes 551 predicted gene products, with the vast majority (83%) of unknown function. Of these, 62 have been identified as virion-associated proteins by mass spectrometry (ESI-MS/MS), including the major capsid protein (Gp225; present in 1620 copies), which shows a HK97 capsid protein-based fold. Cryo-electron microscopy experiments showed that the 350-kbp DNA molecule of Escherichia coli virus phAPEC6 is packaged in at least 15 concentric layers in the phage capsid. A capsid inner body rod is also present, measuring about 91 nm by 18 nm and oriented along the portal axis. In the phAPEC6 contractile tail, 25 hexameric stacked rings can be distinguished, built of the identified tail sheath protein (Gp277). Cryo-EM reconstruction reveals the base of the unique hairy fibers observed during an initial transmission electron microscopy (TEM) analysis. These very unusual filaments are ordered at three annular positions along the contractile sheath, as well as around the capsid, and may be involved in host interaction.

scholarly journals Application of Cryo-Electron Microscopy on Drug Discovery

2021 ◽  
Vol 27 (S1) ◽  
pp. 3250-3250
Author(s):  
Viswanath Vittaladevaram ◽  
Kranthi Kuruti
Keyword(s):  
Electron Microscopy ◽  
Protein Complexes ◽  
Lead Discovery ◽  
Biologically Relevant ◽  
Biological Targets ◽  
3 Dimensional ◽  
Drug Molecules ◽  
Cryo Electron Microscopy ◽  
Therapeutic Molecules ◽  
Novel Drug

AbstractThe key aspect for development of novel drug molecules is to perform structural determination of target molecule associated with its ligand. One such tool that provides insights towards structure of molecule is Cryo-electron microscopy which covers biological targets that are intractable. Examination of proteins can be carried out in native state, as the samples are frozen at -175 degree Celsius i.e. cryogenic temperatures. In addition to this, there were no limits for molecular and functional structures of proteins that can be imagined in 3-dimensional form. This includes ligands which unravel mechanisms that are biologically relevant. This will enable to better understand the mechanisms that are used for development of new therapeutics. Application of Cryo-electron microscopy is not limited to protein complexes and is considered as non-specific. Intervention of Cryo-EM would allow to analyse the structures and also able to dissect the interaction with therapeutic molecules. The study determines the usage of cryo-EM for providing resolutions that are acceptable for lead discovery. It also provides support for lead optimization and also for discovery of vaccines and therapeutics.

scholarly journals Practical Experience with Hole-Free Phase Plates for Cryo Electron Microscopy

2016 ◽  
Vol 22 (6) ◽  
pp. 1316-1328 ◽  
Author(s):  
Michael Marko ◽  
Chyongere Hsieh ◽  
Eric Leith ◽  
David Mastronarde ◽  
Sohei Motoki
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Practical Experience ◽  
Phase Plate ◽  
Native State ◽  
Automated Data Collection ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Phase Plates ◽  
Set Up

AbstractPhase plate (PP) imaging has proven to be valuable in transmission cryo electron microscopy of unstained, native-state biological specimens. Many PP types have been described, however until the recent implementation of the “hole-free” phase plate (HFPP), imaging has been challenging. We found the HFPP to be simple to construct and to set up in the transmission electron microscopy, but care in implementing automated data collection is needed. Performance may be variable, both initially and over time, thus it is important to monitor and evaluate image quality by observing the power spectrum. We found that while some HFPPs gave transfer to high resolution without CTF oscillation, most reached high resolution when operated with modest defocus.

scholarly journals Cryomesh™: A New Substrate for Cryo-Electron Microscopy

2010 ◽  
Vol 16 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Craig Yoshioka ◽  
Bridget Carragher ◽  
Clinton S. Potter
Keyword(s):  
Electron Microscopy ◽  
Semiconductor Industry ◽  
Three Dimensional ◽  
Quality Of Data ◽  
Temperature Conductivity ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Using Data ◽  
Collection Methods

AbstractHere we evaluate a new grid substrate developed by ProtoChips Inc. (Raleigh, NC) for cryo-transmission electron microscopy. The new grids are fabricated from doped silicon carbide using processes adapted from the semiconductor industry. A major motivating purpose in the development of these grids was to increase the low-temperature conductivity of the substrate, a characteristic that is thought to affect the appearance of beam-induced movement (BIM) in transmission electron microscope (TEM) images of biological specimens. BIM degrades the quality of data and is especially severe when frozen biological specimens are tilted in the microscope. Our results show that this new substrate does indeed have a significant impact on reducing the appearance and severity of beam-induced movement in TEM images of tilted cryo-preserved samples. Furthermore, while we have not been able to ascertain the exact causes underlying the BIM phenomenon, we have evidence that the rigidity and flatness of these grids may play a major role in its reduction. This improvement in the reliability of imaging at tilt has a significant impact on using data collection methods such as random conical tilt or orthogonal tilt reconstruction with cryo-preserved samples. Reduction in BIM also has the potential for improving the resolution of three-dimensional cryo-reconstructions in general.

scholarly journals Opportunities and challenges for assigning cofactors in cryo-EM density maps of chlorophyll-containing proteins

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Christopher J. Gisriel ◽  
Jimin Wang ◽  
Gary W. Brudvig ◽  
Donald A. Bryant
Keyword(s):  
Electron Microscopy ◽  
Electrostatic Potential ◽  
Protein Function ◽  
Protein Complexes ◽  
Chemical Environment ◽  
Cryo Electron Microscopy ◽  
Functional Differences ◽  
Density Maps ◽  
Structural Differences

AbstractThe accurate assignment of cofactors in cryo-electron microscopy maps is crucial in determining protein function. This is particularly true for chlorophylls (Chls), for which small structural differences lead to important functional differences. Recent cryo-electron microscopy structures of Chl-containing protein complexes exemplify the difficulties in distinguishing Chl b and Chl f from Chl a. We use these structures as examples to discuss general issues arising from local resolution differences, properties of electrostatic potential maps, and the chemical environment which must be considered to make accurate assignments. We offer suggestions for how to improve the reliability of such assignments.

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