A New Generation of the IMAGIC Image Processing System

Heel, Marin van; Harauz, George; Orlova, Elena V.; Schmidt, Ralf; Schatz, Michael

doi:10.1006/jsbi.1996.0004

Cited by 1,170 publications

(940 citation statements)

References 47 publications

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“…A total of 14,918 particles were manually selected using XMIPP v3.0 (Scheres et al , 2008) and extracted from aligned, averaged and CTF corrected frames with a box size of 400 pixels. All subsequent operations were done using IMAGIC‐5 (van Heel et al , 1996). …”

Section: Methodsmentioning

confidence: 99%

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Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

et al. 2017

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Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA. T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1‐11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1‐11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein–protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.

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Section: Methodsmentioning

confidence: 99%

“…A total of 80 particles, representing complexes with and without a clear extra density corresponding to antibody, were manually picked from aligned, averaged and CTF corrected frames with a box size of 320 pixels. All subsequent operations were done using IMAGIC‐5 (van Heel et al , 1996). …”

Section: Methodsmentioning

confidence: 99%

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

et al. 2017

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“…EMAN (Ludtke et al 1999;Tang et al 2007) and Frealign (Grigorieff, 1998(Grigorieff, , 2007 were the first single particle computational approaches that explicitly included CTF correction at the heart of their operation, which is absolutely required to reach high resolution. The full range of point group symmetries was also embedded in both programs, a feature that had also been available in Imagic since the mid-1980s (van Heel et al 1996).…”

Section: Improved Computer Programsmentioning

confidence: 99%

Single particle electron cryomicroscopy: trends, issues and future perspective

Vinothkumar

Henderson

2016

Quart. Rev. Biophys.

175

115

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Abstract. There has been enormous progress during the last few years in the determination of three-dimensional biological structures by single particle electron cryomicroscopy (cryoEM), allowing maps to be obtained with higher resolution and from fewer images than required previously. This is due principally to the introduction of a new type of direct electron detector that has 2-to 3-fold higher detective quantum efficiency than available previously, and to the improvement of the computational algorithms for image processing. In spite of the great strides that have been made, quantitative analysis shows that there are still significant gains to be made provided that the problems associated with image degradation can be solved, possibly by minimising beam-induced specimen movement and charge build up during imaging. If this can be achieved, it should be possible to obtain near atomic resolution structures of smaller single particles, using fewer images and resolving more conformational states than at present, thus realising the full potential of the method. The recent popularity of cryoEM for molecular structure determination also highlights the need for lower cost microscopes, so we encourage development of an inexpensive, 100 keV electron cryomicroscope with a high-brightness field emission gun to make the method accessible to individual groups or institutions that cannot afford the investment and running costs of a state-of-the-art 300 keV installation. A key requisite for successful high-resolution structure determination by cryoEM includes interpretation of images and optimising the biochemistry and grid preparation to obtain nicely distributed macromolecules of interest. We thus include in this review a gallery of cryoEM micrographs that shows illustrative examples of single particle images of large and small macromolecular complexes.

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“…Among the mathematical operations to be carried out in the computer are alignment, determination of particle orientation (by random conical technique, common lines or reference to an existing density map), classification, reconstruction and correction of the contrast transfer function (CTF). It was earlier recognized that this kind of multistep processing, often with branching data flow, requires flexible, modular image processing software (SPIDER 3,4 , IMAGIC 5,6 , EMAN 7 and XMIPP 8 ; for a comprehensive overview, see the image processing software issue of the Journal of Structural Biology 9 ). Flexibility is desirable because of the need to explore different image processing and reconstruction strategies.…”

Section: Introductionmentioning

confidence: 99%

SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs

et al. 2008

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This protocol describes the reconstruction of biological molecules from the electron micrographs of single particles. Computation here is performed using the image-processing software SPIDER and can be managed using a graphical user interface, termed the SPIDER Reconstruction Engine. Two approaches are described to obtain an initial reconstruction: random-conical tilt and common lines. Once an existing model is available, reference-based alignment can be used, a procedure that can be iterated. Also described is supervised classification, a method to look for homogeneous subsets when multiple known conformations of the molecule may coexist.

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A New Generation of the IMAGIC Image Processing System

Cited by 1,170 publications

References 47 publications

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

Single particle electron cryomicroscopy: trends, issues and future perspective

SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs

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