Use of phase plate cryo-EM reveals conformation diversity of therapeutic IgG with 50 kDa Fab fragment resolved below 6 Å.

While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of small domains like immunoglobulin (IgG) remain challenging. Here, we applied single particle cryo-EM to Rituximab, a therapeutic IgG mediating anti-tumor toxicity, to explore its solution conformations. We found Rituximab molecules exhibited aggregates in cryo-EM specimens contrary to its solution behavior, and utilized a non-ionic detergent to successfully disperse them as isolated particles amenable to single particle analysis. As the detergent adversely reduced the protein-to-solvent contrast, we employed phase plate contrast to mitigate the impaired protein visibility. Assisted by phase plate imaging, we obtained a canonical three-arm IgG structure with other structures displaying variable arm densities co-existing in solution, affirming high flexibility of arm-connecting linkers. Furthermore, we showed phase plate imaging enables reliable structure determination of Fab to sub-nanometer resolution from ab initio, yielding a characteristic two-lobe structure that could be unambiguously docked with crystal structure. Our findings revealed conformation diversity of IgG and demonstrated phase plate was viable for cryo-EM analysis of small proteins without symmetry. This work helps extend cryo-EM boundaries, providing a valuable imaging and structural analysis framework for macromolecules with similar challenging features.

A RAD51-ADP double filament structure unveils the mechanism of filament dynamics in homologous recombination.

ATP-dependent RAD51 recombinases play an essential role in eukaryotic homologous recombination by catalyzing a four-step process: 1) formation of a RAD51 single-filament assembly on ssDNA in the presence of ATP, 2) complementary DNA strand-exchange, 3) ATP hydrolysis transforming the RAD51 filament into an ADP-bound disassembly-competent state, and 4) RAD51 disassembly to provide access for DNA repairing enzymes. Of these steps, filament dynamics between the ATP- and ADP-bound states, and the RAD51 disassembly mechanism, are poorly understood due to the lack of near-atomic-resolution information of the ADP-bound RAD51-DNA filament structure. We report the cryo-EM structure of ADP-bound RAD51-DNA filaments at 3.1 Å resolution, revealing a unique RAD51 double-filament that wraps around ssDNA. Structural analysis, supported by ATP-chase and time-resolved cryo-EM experiments, reveals a collapsing mechanism involving two four-protomer movements along ssDNA for mechanical transition between RAD51 single- and double-filament without RAD51 dissociation. This mechanism enables elastic change of RAD51 filament length during structural transitions between ATP- and ADP-states.

Log-linear, logistic model fitting and local score statistics for cluster detection with covariate adjustments.

The standard method for p-value computation of spatial scan statistics, with adjustments for covariate effects, is to conduct Monte Carlo simulations with these effects estimated under the null hypothesis of no clustering. However when the covariates are geographically unbalanced, the proposed Monte Carlo p-value estimates are too conservative, with corresponding loss of power, due to excessive adjustments for confounding between covariates and location. We show that the use of an alternative procedure that involves local score statistics, with parameters fitted on a log-linear or logistic model, addresses this problem. We also discuss extensions of the procedure when there are multiple or continuous covariates.

Comprehensive analysis of 19q12 amplicon in human gastric cancers.

Amplification at 19q12 has been observed in multiple tumor types, while cyclin E1 (CCNE1) has been considered to be the key oncogene within this amplicon. We have previously applied cDNA microarray analysis to systematically characterize gene expression patterns of gastric tumor and nontumor samples. We identified a cluster of five tightly coregulated genes all located at chromosome 19q12, including CCNE1. We found that the 19q12 gene cluster is highly expressed in gastric tumors compared to nontumor gastric samples. Array based comparative genomic hybridization and real-time PCR was used to define the boundary of the 19q12 amplicon to a region of approximately 200 kb. Interestingly, we found that in some cases amplification at 19q12 was not associated with DNA copy number gain at CCNE1, suggesting that some other genes within the 19q12 amplicon may also have important function during gastric tumorigenesis. We found high expression of the 19q12 gene cluster to be statistically correlated with the cell proliferation gene signature. Using the SAM software, we identified a set of 577 genes whose expression levels positively correlated with the 19q12 gene cluster. GO term analysis revealed that this genelist is enriched with genes involved in cell cycle regulation and cell proliferation. In conclusion, expression array analysis combined with array comparative genomic hybridization and real-time PCR provides a new and powerful tool to identify clusters of genes which may be regulated by genomic DNA aberrations. In addition, our study indicates that amplification at 19q12 is associated with cell proliferation in vivo.

A method for detecting and correcting feature misidentification on expression microarrays.

BACKGROUND: Much of the microarray data published at Stanford is based on mouse and human arrays produced under controlled and monitored conditions at the Brown and Botstein laboratories and at the Stanford Functional Genomics Facility (SFGF). Nevertheless, as large datasets based on the Stanford Human array began to accumulate, a small but significant number of discrepancies were detected that required a serious attempt to track down the original source of error. Due to a controlled process environment, sufficient data was available to accurately track the entire process leading to up to the final expression data. In this paper, we describe our statistical methods to detect the inconsistencies in microarray data that arise from process errors, and discuss our technique to locate and fix these errors. RESULTS: To date, the Brown and Botstein laboratories and the Stanford Functional Genomics Facility have together produced 40,000 large-scale (10-50,000 feature) cDNA microarrays. By applying the heuristic described here, we have been able to check most of these arrays for misidentified features, and have been able to confidently apply fixes to the data where needed. Out of the 265 million features checked in our database, problems were detected and corrected on 1.3 million of them. CONCLUSION: Process errors in any genome scale high throughput production regime can lead to subsequent errors in data analysis. We show the value of tracking multi-step high throughput operations by using this knowledge to detect and correct misidentified data on gene expression microarrays.

Gene expression patterns in ovarian carcinomas.

We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.

Phospholipase A2 group IIA expression in gastric adenocarcinoma is associated with prolonged survival and less frequent metastasis.

We analyzed gene expression patterns in human gastric cancers by using cDNA microarrays representing approximately equal 30,300 genes. Expression of PLA2G2A, a gene previously implicated as a modifier of the Apc(Min/+) (multiple intestinal neoplasia 1) mutant phenotype in the mouse, was significantly correlated with patient survival. We confirmed this observation in an independent set of patient samples by using quantitative RT-PCR. Beyond its potential diagnostic and prognostic significance, this result suggests the intriguing possibility that the activity of PLA2G2A may suppress progression or metastasis of human gastric cancer.