Delivering a toxic metal to the active site of urease.

Urease is a nickel (Ni) enzyme that is essential for the colonization of Helicobacter pylori in the human stomach. To solve the problem of delivering the toxic Ni ion to the active site without diffusing into the cytoplasm, cells have evolved metal carrier proteins, or metallochaperones, to deliver the toxic ions to specific protein complexes. Ni delivery requires urease to form an activation complex with the urease accessory proteins UreFD and UreG. Here, we determined the cryo-electron microscopy structures of H. pylori UreFD/urease and Klebsiella pneumoniae UreD/urease complexes at 2.3- and 2.7-angstrom resolutions, respectively. Combining structural, mutagenesis, and biochemical studies, we show that the formation of the activation complex opens a 100-angstrom-long tunnel, where the Ni ion is delivered through UreFD to the active site of urease.

Approaches to Using the Chameleon: Robust, Automated, Fast-Plunge cryoEM Specimen Preparation.

The specimen preparation process is a key determinant in the success of any cryo electron microscopy (cryoEM) structural study and until recently had remained largely unchanged from the initial designs of Jacques Dubochet and others in the 1980s. The process has transformed structural biology, but it is largely manual and can require extensive optimisation for each protein sample. The chameleon instrument with its self-wicking grids and fast-plunge freezing represents a shift towards a robust, automated, and highly controllable future for specimen preparation. However, these new technologies require new workflows and an understanding of their limitations and strengths. As early adopters of the chameleon technology, we report on our experiences and lessons learned through case studies. We use these to make recommendations for the benefit of future users of the chameleon system and the field of cryoEM specimen preparation generally.

Effects of chameleon dispense-to-plunge speed on particle concentration, complex formation, and final resolution: A case study using the Neisseria gonorrhoeae ribonucleotide reductase inactive complex.

Ribonucleotide reductase (RNR) is an essential enzyme that converts ribonucleotides to deoxyribonucleotides and is a promising antibiotic target, but few RNRs have been structurally characterized. We present the use of the chameleon, a commercially-available piezoelectric cryogenic electron microscopy plunger, to address complex denaturation in the Neisseria gonorrhoeae class Ia RNR. Here, we characterize the extent of denaturation of the ring-shaped complex following grid preparation using a traditional plunger and using a chameleon with varying dispense-to-plunge times. We also characterize how dispense-to-plunge time influences the amount of protein sample required for grid preparation and preferred orientation of the sample. We demonstrate that the fastest dispense-to-plunge time of 54 ms is sufficient for generation of a data set that produces a high quality structure, and that a traditional plunging technique or slow chameleon dispense-to-plunge times generate data sets limited in resolution by complex denaturation. The 4.3 Å resolution structure of Neisseria gonorrhoeae class Ia RNR in the inactive α4ß4 oligomeric state solved using the chameleon with a fast dispense-to-plunge time yields molecular information regarding similarities and differences to the well studied Escherichia coli class Ia RNR α4ß4 ring.

RAG1/2 re-expression causes receptor revision in a model B cell line.

The expression of RAG1 and RAG2 is essential for V(D)J rearrangement of the immunoglobulin (Ig) locus in developing B cells. In mature B cells further V(D)J rearrangement is suppressed and RAG1/2 proteins decline to undetectable levels. However, there is evidence that mature B cells in the periphery may re-express RAG1/2. In humans evidence of RAG1/2 re-expression is often linked with an autoimmune state, indicating that further understanding of re-expression may be crucial to understanding immune disorders. We have investigated the molecular consequences of RAG1/2 expression in mature lymphocytes using a cell culture system (M12 and DR3). M12 (IgG+, Igkappa+ and RAG-) is a mouse B cell lymphoma. DR3 is a RAG1+/RAG2+ line derived from M12 by introduction of stable plasmids carrying RAG1 and RAG2 cDNAs. RAG1/2 mediated receptor revision occurs in the DR3 line, as evidenced by both the deletion of the endogenous rearranged Igkappa gene segment (present in the parent M12 lines) and the presence of a new Iglambda rearrangement. Gene expression profiles obtained through microarray analysis and RT-PCR found differences in expression levels between the two lines for: fibronectin, lysyl oxidase, TAP2, B220, Igkappa, TIS11B, HMG2 and DNAPKcs. Thus, the expression of RAG1/2 in a previously RAG- cell line results in multiple changes to the gene expression profile as well as receptor revision. The significance of the changes found in this model of RAG re-expression is discussed.