An Automatic Particle Pickup Method Using a Neural Network Applicable to Low-Contrast Electron Micrographs

Tong

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

et al. 2010

Self Cite

202

154

Keap1 is a substrate adaptor of a Cullin 3-based E3 ubiquitin ligase complex that recognizes Nrf2, and also acts as a cellular sensor for xenobiotics and oxidative stresses. Nrf2 is a transcriptional factor regulating the expression of cytoprotective enzyme genes in response to such stresses. Under unstressed conditions Keap1 binds Nrf2 and results in rapid degradation of Nrf2 through the proteasome pathway. In contrast, upon exposure to oxidative and electrophilic stress, reactive cysteine residues in intervening region (IVR) and Broad complex, Tramtrack, and Bric-à-Brac domains of Keap1 are modified by electrophiles. This modification prevents Nrf2 from rapid degradation and induces Nrf2 activity by repression of Keap1. Here we report the structure of mouse Keap1 homodimer by single particle electron microscopy. Three-dimensional reconstruction at 24-Å resolution revealed two large spheres attached by short linker arms to the sides of a small forked-stem structure, resembling a cherry-bob. Each sphere has a tunnel corresponding to the central hole of the β-propeller domain, as determined by x-ray crystallography. The IVR domain appears to surround the core of the β-propeller domain. The unexpected proximity of IVR to the β-propeller domain suggests that any distortions generated during modification of reactive cysteine residues in the IVR domain may send a derepression signal to the β-propeller domain and thereby stabilize Nrf2. This study thus provides a structural basis for the two-site binding and hinge-latch model of stress sensing by the Nrf2-Keap1 system.

show abstract

Section: Resultsmentioning

confidence: 99%

“…Image analysis was performed with our SPINNS program (33)(34)(35)(36) and IMAGIC V (37). Twohundred and sixteen Keap1 projections were selected (34) and used to train a neural network (NN) (33,42). With the trained NN, 12,651 particles were selected, aligned (32,43), and sorted (35).…”

Section: Methodsmentioning

confidence: 99%

Keap1 is a forked-stem dimer structure with two large spheres enclosing the intervening, double glycine repeat, and C-terminal domains

Tong

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

et al. 2010

Self Cite

202

154

show abstract

“…The projections of CFTR were picked up by a combination of two automatic programs: the autoaccumulation method using SA (28) and the three-layered neural network method (25,26). The three-dimensional structure was reconstructed with echo- correlated reconstruction methods using SA assuming C2 symmetry in our SPINNS (25)(26)(27)(28)(29)(30) and other algorithms in the IMAGIC V software (31) (see "Experimental Procedures" for details).…”

Section: Resultsmentioning

confidence: 99%

“…Automated Particle Selection and Image Analysis-We have developed a single particle image analysis method using neural network (25)(26)(27) and simulated annealing (28,29) named SPINNS (30). The following image analysis was performed using our SPINNS and IMAGIC V (31).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Three-dimensional Reconstruction of Human Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Revealed an Ellipsoidal Structure with Orifices beneath the Putative Transmembrane Domain

Journal of Biological Chemistry

et al. 2008

Self Cite

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to an ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. Here we expressed wild-type human CFTR as a FLAG-fused protein in HEK293 cells heterologously and purified it in three steps: anti-FLAG and wheat germ agglutinin affinity chromatographies and size exclusion chromatography. The stoichiometry of the protein was analyzed using various biochemical approaches, including chemical cross-linking, blue-native PAGE, size exclusion chromatography, and electron microscopy (EM) observation of antibody-decorated CFTR. All these data support a dimeric assembly of CFTR. Using 5,039 automatically selected particles from negatively stained EM images, the three-dimensional structure of CFTR was reconstructed at 2-nm resolution assuming a 2-fold symmetry. CFTR, presumably in a closed state, was shown to be an ellipsoidal particle with dimensions of 120 ؋ 106 ؋ 162 Å . It comprises a small dome-shaped extracellular and membrane-spanning domain and a large cytoplasmic domain with orifices beneath the putative transmembrane domain. EM observation of CFTR⅐anti-regulatory domain antibody complex confirmed that two regulatory domains are located around the bottom end of the larger oval cytoplasmic domain.The cystic fibrosis transmembrane conductance regulator (CFTR) 3 (also termed ABCC7) is a unique member of the ATPbinding cassette (ABC) superfamily in that CFTR functions as an anion channel, whereas most other members function as active transporters. CFTR is expressed in the luminal membranes of secreting and absorbing epithelia and plays a critical role in transepithelial salt and water transport. Dysfunction of CFTR leads to cystic fibrosis, the most common lethal autosomal recessive disorder in Caucasians (1-3). On the other hand, extremely high activity of CFTR, usually caused by bacterial toxins, results in secretory diarrhea (4, 5), killing millions of infants in developing countries every year (6). Understanding the structure/function relationship and the underlying mechanisms of CFTR are essential for developing novel therapeutics for CFTR-mediated diseases.Like other ABC transporters, CFTR is formed by two repeated motifs, each of which has a membrane-spanning domain (MSD) and a cytoplasmic nucleotide-binding domain (NBD). However, a regulatory domain (R domain) located between the first NBD (NBD1) and the second MSD (MSD2) is unique in CFTR among ABC transporters. This domain contains several phosphorylation sites for protein kinase A and protein kinase C, and the level of their phosphorylation controls CFTR channel activity. Once they are phosphorylated, opening and closing (gating) of the CFTR channel is controlled by ATP binding and hydrolysis at its two NBDs (7). Each NBD contains the Walker A and Walker B nucleotidebinding motifs and the "signature sequence" LSGGQ, which defines the...

show abstract

Nano-Microscopy of Therapeutic Antibody Aggregates in Solution

Senga

Imamura

et al. 2021

Therapeutic Antibodies