Computer controlled cryo-electron microscopy – TOM2 a software package for high-throughput applications

Korinek, Andreas; Beck, Florian; Baumeister, Wolfgang; Nickell, Stephan; Plitzko, Jürgen M.

doi:10.1016/j.jsb.2011.06.003

Cited by 48 publications

(33 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Tilt series typically covering an angular range from −60°to 60°with 1.5°or 2°tilt increments were recorded automatically (29,30) under low-dose conditions using a Tecnai G2 Polara microscope (FEI) equipped with a GIF 2002 postcolumn energy filter and slow-scan CCD camera with 2; 048 × 2; 048 pixels (Gatan). The TEM was operated at an accelerating voltage of 300 kV; the pixel size in the object-plane was between 0.46 nm and 0.71 nm.…”

Section: Methodsmentioning

confidence: 99%

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

Rigort

Bäuerlein

Villa

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

399

306

View full text Add to dashboard Cite

Cryoelectron tomography provides unprecedented insights into the macromolecular and supramolecular organization of cells in a close-to-living state. However because of the limited thickness range (< 0.5–1 μm) that is accessible with today’s intermediate voltage electron microscopes only small prokaryotic cells or peripheral regions of eukaryotic cells can be examined directly. Key to overcoming this limitation is the ability to prepare sufficiently thin samples. Cryosectioning can be used to prepare thin enough sections but suffers from severe artefacts, such as substantial compression. Here we describe a procedure, based upon focused ion beam (FIB) milling for the preparation of thin (200–500 nm) lamellae from vitrified cells grown on electron microscopy (EM) grids. The self-supporting lamellae are apparently free of distortions or other artefacts and open up large windows into the cell’s interior allowing tomographic studies to be performed on any chosen part of the cell. We illustrate the quality of sample preservation with a structure of the nuclear pore complex obtained from a single tomogram.

show abstract

Section: Methodsmentioning

confidence: 99%

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

Rigort

Bäuerlein

Villa

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

399

306

View full text Add to dashboard Cite

show abstract

“…Indeed, the AMP-PNP density shows a well-defined density segment (Fig. 3B), for which the fitted atomic model suggests that it corresponds to the very N-terminal residues (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. These N-terminal 20 residues are absent from all p97 crystal structures but one protomer of the ATPcS-bound IBMPFD mutant A232E, which includes residues 12-20 [10].…”

Section: Conformational Changes Of the N-d1 Segmentmentioning

confidence: 99%

“…TOM 2 automated acquisition software [19] was used to collect a total of 2464 (p97-AMP-PNP), 3276 (p97-ADP) and 1728 (p97-ATP-cS) micrographs.…”

mentioning

confidence: 99%

Nucleotide‐dependent conformational changes of the AAA+ ATPase p97 revisited

et al. 2016

Self Cite

View full text Add to dashboard Cite

Edited by Peter BrzezinskiThe ubiquitous AAA-ATPase p97 segregates ubiquitylated proteins from their molecular environment. Previous studies of the nucleotide-dependent conformational changes of p97 were inconclusive. Here, we determined its structure in the presence of ADP, AMP-PNP, or ATP-cS at 6.1-7.4 A resolution using single particle cryo-electron microscopy. Both AAA domains, D1 and D2, assemble into essentially six-fold symmetrical rings. The pore of the D1-ring remains essentially closed under all nucleotide conditions, whereas the D2-ring shows an iris-like opening for ADP. The largest conformational changes of p97 are 'swinging motions' of the N-terminal domains, which may enable segregation of ubiquitylated substrates from their environment.

show abstract

“…5), good squares need to be unbroken, not too thickly covered by ice (which will adversely affect contrast and result in excessive variation in Z-height of particles), and not so thin as to exclude the particles completely [47]. Automated picking of good squares for cryo-EM entails checking ice thickness by estimating the transmittance of electrons [18,20,23,26]. In order to eliminate holes containing uneven ice or some contamination, some programs such as AutoEMation and Leginon also offer the option to reject holes that have a measured transmittance beyond a certain mean or standard deviation threshold [18,23].…”

Section: Target Acquisitionmentioning

confidence: 99%

“…In TOM 2 [20], the hardware abstraction layer (HAL) deals with individual hardware components. Leginon [18] and SerialEM [15] have also been built with this approach, allowing them to be compatible with multiple brands of microscopes and detectors (Table 1).…”

Section: Hardware Compatibilitymentioning

confidence: 99%

Automated data collection in single particle electron microscopy

et al. 2015

View full text Add to dashboard Cite

Automated data collection is an integral part of modern workflows in single particle electron microscopy (EM) research. This review surveys the software packages available for automated single particle EM data collection. The degree of automation at each stage of data collection is evaluated, and the capabilities of the software packages are described. Finally, future trends in automation are discussed.

show abstract

Computer controlled cryo-electron microscopy – TOM2 a software package for high-throughput applications

Cited by 48 publications

References 51 publications

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

Nucleotide‐dependent conformational changes of the AAA+ ATPase p97 revisited

Automated data collection in single particle electron microscopy

Contact Info

Product

Resources

About