Estimating contrast transfer function and associated parameters by constrained non‐linear optimization

Yang, Chao; Jiang, Wen; Chen, Donghua; Adiga, Umesh; Ng, Esmond G.; Chiu, Wah

doi:10.1111/j.1365-2818.2009.03137.x

Cited by 31 publications

(29 citation statements)

References 36 publications

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“…Particles were selected from scanned micrograph images, first automatically by the ETHAN method (58) and then by manual screening with the boxer program in EMAN (59). The TEM instrument contrast transfer function parameters were determined automatically using fitctf2.py (60,61) and were then visually validated using EMAN ctfit program. For 3D reconstructions, whole datasets were divided into even-odd halves.…”

Section: Methodsmentioning

confidence: 99%

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

Guo

Liu

Fang

et al. 2014

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

128

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Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.bacteriophage T7 maturation | DNA packaging intermediates | noncovalent topological linking | procapsid | single-particle cryo-EM M any dsDNA viruses, including tailed phages and herpes viruses, initially assemble a DNA-free procapsid with assistance of a network of scaffold proteins. Accompanying the exit of scaffolding proteins during subsequent ATP-driven DNA packaging, the icosahedral shell of the procapsid undergoes dramatic conformational changes and matures into a typically larger and more angular shell of the infectious phage (1-6). However, structural details, including those of capsid intermediates, are limited to the phage HK97 system (5, 7-9), for which recombinantly produced procapsid and nonphysiological conversion products were analyzed.The packaging of the 39.937-kbp DNA genome of the shorttail Escherichia coli bacteriophage, T7, is a model for understanding basic principles common to dsDNA tailed phages and herpes viruses. The T7 system is also of interest because it has been used for popular biotechnologies, such as recombinant protein expression (10) and protein display on the capsid surface (11). The T7 capsid contains 415 copies of the major shell protein gp10 (12) that form a T = 7L icosahedral lattice. From lowresolution cryo-EM 3D reconstructions the tertiary topology of gp10 can be divided into four regions: N-...

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Section: Methodsmentioning

confidence: 99%

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

Guo

Liu

Fang

et al. 2014

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

128

View full text Add to dashboard Cite

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“…The astigmatism is an essential component of the contrast transfer function (CTF) of TEM images. Many approaches have been proposed for the estimation of CTF parameters based on the power spectra of images (Fernando, 2008; Huang et al, 2003; Jiang et al, 2012; Mallick et al, 2005; Mindell and Grigorieff, 2003; Sander et al, 2003; Sorzano et al, 2007; Vulović et al, 2012; Yang et al, 2009). The CTF parameters are typically determined by iterative fitting simulated power spectra to the experimental power spectra by varying the parameters in the CTF model.…”

Section: Introductionmentioning

confidence: 99%

Real-time detection and single-pass minimization of TEM objective lens astigmatism

Yan

Jiang

2017

Journal of Structural Biology

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Minimization of the astigmatism of the objective lens is a critical daily instrument alignment task essential for high resolution TEM imaging. Fast and sensitive detection of astigmatism is needed to provide real-time feedback and adjust the stigmators to efficiently reduce astigmatism. Currently the method used by many microscopists is to visually examine the roundness of a diffractogram (Thon rings) and iteratively adjust the stigmators to make the Thon rings circular. This subjective method is limited by poor sensitivity and potentially biased by the astigmatism of human eyes. In this study, an s2 power spectra based method, s2stigmator, was developed to allow fast and sensitive detection of the astigmatism in TEM live images. The “radar”-style display provides real-time feedback to guide the adjustment of the objective lens stigmators. Such unique capability allowed us to discover the mapping of the two stigmators to the astigmatism amplitude and angle, which led us to develop a single-pass tuning strategy capable of significantly quicker minimization of the objective lens astigmatism.

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“…Particles were selected manually with the boxer program in EMAN (20,21). The microscope contrast transfer function parameters for each micrograph were first determined using an automated fitting method (22) and then manually verified and corrected using the EMAN ctfit graphic program. To avoid model bias, 5 "random" initial models per data set were generated by random particle orientation assignment.…”

Section: Methodsmentioning

confidence: 99%

Obstruction of Dengue Virus Maturation by Fab Fragments of the 2H2 Antibody

Wang

Pennington

et al. 2013

J Virol

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The 2H2 monoclonal antibody recognizes the precursor peptide on immature dengue virus and might therefore be a useful tool for investigating the conformational change that occurs when the immature virus enters an acidic environment. During dengue virus maturation, spiky, immature, noninfectious virions change their structure to form smooth-surfaced particles in the slightly acidic environment of the trans-Golgi network, thereby allowing cellular furin to cleave the precursor-membrane proteins. The dengue virions become fully infectious when they release the cleaved precursor peptide upon reaching the neutral-pH environment of the extracellular space. Here we report on the cryo-electron microscopy structures of the immature virus complexed with the 2H2 antigen binding fragments (Fab) at different concentrations and under various pH conditions. At neutral pH and a high concentration of Fab molecules, three Fab molecules bind to three precursor-membrane proteins on each spike of the immature virus. However, at a low concentration of Fab molecules and pH 7.0, only two Fab molecules bind to each spike. Changing to a slightly acidic pH caused no detectable change of structure for the sample with a high Fab concentration but caused severe structural damage to the low-concentration sample. Therefore, the 2H2 Fab inhibits the maturation process of immature dengue virus when Fab molecules are present at a high concentration, because the three Fab molecules on each spike hold the precursor-membrane molecules together, thereby inhibiting the normal conformational change that occurs during maturation. Dengue virus (DENV) is a lipid-enveloped, positive-strand RNA virus that is a member of the Flaviviridae. Mosquitos are the major vector for DENV transmission to humans. Symptoms of primary infection are febrile and nonfatal, whereas secondary infections lead to life-threatening symptoms such as hemorrhagic fever or dengue shock syndrome (http://www.cdc.gov/dengue /clinicalLab/). The severity of the secondary DENV infection may be associated with antibody-dependent enhancement of infection (ADE) (1, 2).DENV is initially assembled on the endoplasmic reticulum of cells in an immature noninfectious form. The fully infectious mature virus is not formed until it is released from its host (3, 4). Both immature (5) and mature (6) DENV particles have icosahedral symmetry, with diameters of about 600 Å and 500 Å and with spiky and smooth surfaces, respectively. The structural proteins of DENV are the capsid protein, the precursor-membrane (prM) protein, and the envelope (E) protein. The latter two are membrane anchored and are involved in structural rearrangements during maturation and fusion. The prM molecule is a chaperone protein that helps E to fold and to form a heterodimer with prM. The 180 copies of prM-E heterodimers then assemble into 60 trimeric spikes of the immature virus (5). In this form, the pr peptide is located on top of each trimeric spike, burying much of the fusion loop underneath it (7). Immature DENV particles are ...

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Estimating contrast transfer function and associated parameters by constrained non‐linear optimization

Cited by 31 publications

References 36 publications

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

Real-time detection and single-pass minimization of TEM objective lens astigmatism

Obstruction of Dengue Virus Maturation by Fab Fragments of the 2H2 Antibody

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