CTF Determination of Images of Ice-Embedded Single Particles Using a Graphics Interface

Zhou, Zhan; Hardt, Steve; Wang, Bin; Sherman, Michael B; Jakana, Joanita; Chiu, Wah

doi:10.1006/jsbi.1996.0033

Cited by 91 publications

(67 citation statements)

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“…The final reconstruction was calculated by merging data to 25-Å resolution (for tE 2 ) or 30-Å resolution (for mE 2 ) from particle images with defocus values around 2-3 m, which were determined from the incoherently averaged Fourier transforms of particle images in each micrograph (23). The final reconstructions were corrected for the contrast transfer function of the microscope (24).…”

Section: Methodsmentioning

confidence: 99%

Direct Evidence for the Size and Conformational Variability of the Pyruvate Dehydrogenase Complex Revealed by Three-dimensional Electron Microscopy

Zhou¹,

Liao²,

Cheng

et al. 2001

Journal of Biological Chemistry

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Structural studies by three-dimensional electron microscopy of the Saccharomyces cerevisiae truncated dihydrolipoamide acetyltransferase (tE 2 ) component of the pyruvate dehydrogenase complex reveal an extraordinary example of protein dynamics. The tE 2 forms a 60-subunit core with the morphology of a pentagonal dodecahedron and consists of 20 cone-shaped trimers interconnected by 30 bridges. Frozen-hydrated and stained molecules of tE 2 in the same field vary in size ϳ20%. Analyses of the data show that the size distribution is bell-shaped, and there is an approximately 40-Å difference in the diameter of the smallest and largest structures that corresponds to ϳ14 Å of variation in the length of the bridge between interconnected trimers. Companion studies of mature E 2 show that the complex of the intact subunit exhibits a similar size variation. The x-ray structure of Bacillus stearothermophilus tE 2 shows that there is an ϳ10-Å gap between adjacent trimers and that the trimers are interconnected by the potentially flexible C-terminal ends of two adjacent subunits. We propose that this springlike feature is involved in a thermally driven expansion and contraction of the core and, since it appears to be a common feature in the phylogeny of pyruvate dehydrogenase complexes, protein dynamics is an integral component of the function of these multienzyme complexes.The pyruvate dehydrogenase complexes (PDCs) 1 are among the largest (M r ϳ10 6 to 10 7 ) and most complex multienzyme structures known. A central feature of these complexes is a 24-mer (Escherichia coli) or 60-mer (eukaryotes and some Gram-positive bacteria) dihydrolipoamide acetyltransferase (E 2 ) core with the morphologies of a cube or a pentagonal dodecahedron, respectively (1-4). The cores have both functional and structural roles in organizing the multienzyme complex; the E 2 activity is associated with the scaffold to which the other components are attached. These include the pyruvate dehydrogenase (E 1 ) and dihydrolipoamide dehydrogenase (E 3 ), which requires a binding protein (BP) to anchor it to the core of the yeast and mammalian PDCs, although, in E. coli and Bacillus stearothermophilus PDCs, BP is not required (1-4).The E 2 subunits have multidomain structures consisting of one, two, or three amino-terminal lipoyl domains, followed by an E 1 and/or E 3 binding domain, and a carboxyl-terminal catalytic domain (1-4). X-ray crystallography (5-9) and threedimensional electron microscopy (10, 11) show that the E 2 catalytic domains are arranged in cone-shaped trimers at each of the 8 or 20 vertices of the cubic or dodecahedral structures, respectively (7,8,10,11). The trimers are interconnected by bridges to form an empty cage-like complex with the tip of the trimer directed toward the center of the structure.Examination of the 4-Å resolution crystal structures of dodecahedral truncated E 2 (tE 2 ) cores from Enterococcus faecalis and B. stearothermophilus and the 2-Å resolution crystal structure of a cubic tE 2 core from Azotobacter vinelandi...

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Section: Methodsmentioning

confidence: 99%

Direct Evidence for the Size and Conformational Variability of the Pyruvate Dehydrogenase Complex Revealed by Three-dimensional Electron Microscopy

Zhou¹,

Liao²,

Cheng

et al. 2001

Journal of Biological Chemistry

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“…For those images that revealed Thon rings, 44 the CTF was determined using ICE. 45 For those that did not, we utilized a grid search algorithm which applied a test value of the CTF to a reference obtained from previously merged images that had been CTF corrected. 24,46 All of the CTF corrected images were merged together at a common phase origin in the two-sided plane group p2.…”

Section: Image Processing and 3-d Reconstructionmentioning

confidence: 99%

A 3-D Reconstruction of Smooth Muscle α-Actinin by CryoEm Reveals Two Different Conformations at the Actin-binding Region

Taylor

2004

Journal of Molecular Biology

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“…Most CTF parameters can be estimated directly from a plot of M 2 (s) (Zhou et al, 1996;Ludtke et al, 1999). However, while methods for determining the parameters of different envelope functions of the microscope have been developed (Frank, 1969(Frank, , 1976, they have not been routinely applied as part of the image reconstruction procedure for ice-embedded particles.…”

Section: Theoretical Backgroundmentioning

confidence: 99%

Fourier Amplitude Decay of Electron Cryomicroscopic Images of Single Particles and Effects on Structure Determination

Saâd

Ludtke

Jakana

et al. 2001

Journal of Structural Biology

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102

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Several factors, including spatial and temporal coherence of the electron microscope, specimen movement, recording medium, and scanner optics, contribute to the decay of the measured Fourier amplitude in electron image intensities. We approximate the combination of these factors as a single Gaussian envelope function, the width of which is described by a single experimental B-factor. We present an improved method for estimating this Bfactor from individual micrographs by combining the use of X-ray solution scattering and numerical fitting to the average power spectrum of particle images. A statistical estimation from over 200 micrographs of herpes simplex virus type-1 capsids was used to estimate the spread in the experimental B-factor of the data set. The B-factor is experimentally shown to be dependent on the objective lens defocus setting of the microscope. The average Bfactor, the X-ray scattering intensity of the specimen, and the number of particles required to determine the structure at a lower resolution can be used to estimate the minimum fold increase in the number of particles that would be required to extend a single particle reconstruction to a specified higher resolution. We conclude that microscope and imaging improvements to reduce the experimental Bfactor will be critical for obtaining an atomic resolution structure.

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CTF Determination of Images of Ice-Embedded Single Particles Using a Graphics Interface

Cited by 91 publications

References 0 publications

Direct Evidence for the Size and Conformational Variability of the Pyruvate Dehydrogenase Complex Revealed by Three-dimensional Electron Microscopy

Direct Evidence for the Size and Conformational Variability of the Pyruvate Dehydrogenase Complex Revealed by Three-dimensional Electron Microscopy

A 3-D Reconstruction of Smooth Muscle α-Actinin by CryoEm Reveals Two Different Conformations at the Actin-binding Region

Fourier Amplitude Decay of Electron Cryomicroscopic Images of Single Particles and Effects on Structure Determination

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