The structures of secretory and dimeric immunoglobulin A

Secretory (S) Immunoglobulin (Ig) A is the predominant mucosal antibody, which binds pathogens and commensal microbes. SIgA is a polymeric antibody, typically containing two copies of IgA that assemble with one joining-chain (JC) to form dimeric (d) IgA that is bound by the polymeric Ig-receptor ectodomain, called secretory component (SC). Here, we report the cryo-electron microscopy structures of murine SIgA and dIgA. Structures reveal two IgAs conjoined through four heavy-chain tailpieces and the JC that together form a β-sandwich-like fold. The two IgAs are bent and tilted with respect to each other, forming distinct concave and convex surfaces. In SIgA, SC is bound to one face, asymmetrically contacting both IgAs and JC. The bent and tilted arrangement of complex components limits the possible positions of both sets of antigen-binding fragments (Fabs) and preserves steric accessibility to receptor-binding sites, likely influencing antigen binding and effector functions

The structures of secretory and dimeric immunoglobulin A

Secretory (S) Immunoglobulin (Ig) A is the predominant mucosal antibody, which binds pathogens and commensal microbes. SIgA is a polymeric antibody, typically containing two copies of IgA that assemble with one joining-chain (JC) to form dimeric (d) IgA that is bound by the polymeric Ig-receptor ectodomain, called secretory component (SC). Here, we report the cryo-electron microscopy structures of murine SIgA and dIgA. Structures reveal two IgAs conjoined through four heavy-chain tailpieces and the JC that together form a β-sandwich-like fold. The two IgAs are bent and tilted with respect to each other, forming distinct concave and convex surfaces. In SIgA, SC is bound to one face, asymmetrically contacting both IgAs and JC. The bent and tilted arrangement of complex components limits the possible positions of both sets of antigen-binding fragments (Fabs) and preserves steric accessibility to receptor-binding sites, likely influencing antigen binding and effector functions

Characterization of Reactive Organometallic Species via MicroED

Here we apply microcrystal electron diffraction (MicroED) to the structural determination of transition-metal complexes. We find that the simultaneous use of 300 keV electrons, very low electron doses, and an ultrasensitive camera allows for the collection of data without cryogenic cooling of the stage. This technique reveals the first crystal structures of the classic zirconocene hydride, colloquially known as “Schwartz’s reagent”, a novel Pd(II) complex not amenable to solution-state NMR or X-ray crystallography, and five other paramagnetic and diamagnetic transition-metal complexes

Characterization of Reactive Organometallic Species via MicroED

Here we apply microcrystal electron diffraction (MicroED) to the structural determination of transition-metal complexes. We find that the simultaneous use of 300 keV electrons, very low electron doses, and an ultrasensitive camera allows for the collection of data without cryogenic cooling of the stage. This technique reveals the first crystal structures of the classic zirconocene hydride, colloquially known as “Schwartz’s reagent”, a novel Pd(II) complex not amenable to solution-state NMR or X-ray crystallography, and five other paramagnetic and diamagnetic transition-metal complexes

The structure of the teleost Immunoglobulin M core provides insights on polymeric antibody evolution, assembly, and function

Abstract Polymeric (p) immunoglobulins (Igs) serve broad functions during vertebrate immune responses. Typically, pIgs contain between two and six Ig monomers, each with two antigen binding fragments and one fragment crystallization (Fc). In addition, many pIgs assemble with a joining-chain (JC); however, the number of monomers and potential to include JC vary with species and heavy chain class. Here, we report the cryo-electron microscopy structure of IgM from a teleost (t) species, which does not encode JC. The structure reveals four tIgM Fcs linked through eight C-terminal tailpieces (Tps), which adopt a single β-sandwich-like domain (Tp assembly) located between two Fcs. Specifically, two of eight heavy chains fold uniquely, resulting in a structure distinct from mammalian IgM, which typically contains five IgM monomers, one JC and a centrally-located Tp assembly. Together with mutational analysis, structural data indicate that pIgs have evolved a range of assembly mechanisms and structures, each likely to support unique antibody effector functions

Budding Pathway in the Templated Assembly of Viruslike Particles

A new pathway for the assembly of viral capsid protein around inorganic nanoparticle cores was observed by time-course light scattering and cryo-electron tomography. Gold nanoparticles with an average diameter of 11.3 nm have been used as a template for the assembly of Brome mosaic virus (BMV) capsid protein at different concentrations. At least at low protein concentrations the kinetic features of the scattering and extinction measurements are consistent with the initial rapid formation of large nanoparticle–protein clusters, which subsequently separate into individual viruslike particles (VLPs). The occurrence of multiparticle clusters at short times after mixing nanoparticles and proteins was confirmed by cryo-EM. Cryo-electron tomography of the multiparticle clusters yielded an average surface-to-surface interparticle distance of ∼7.5 nm, equivalent to ∼1.5 times the thickness of a protein shell. We propose a scenario in which VLP generation may take place through monomer exchange between aggregated particles with defect-ridden or incomplete shells, leading to the formation of stable icosahedral shells, which eventually bud off the aggregate. Together with results from previous works, the findings highlight the astonishing versatility of plant virus capsid protein assembly. This previously unknown mechanism for VLP formation has features that may have relevance for the crowded environment characterizing virus factories in the cell

Allele specificity between <i>RPL10</i> and <i>TIF6</i>.

<p><b>A)</b> Structure showing Tif6 bound to Rpl23 on the 60S subunit. Tif6 residue V192 (green) is located at the interface between Tif6 and Rpl23, while Tif6 residues mutated in <i>rpl10-R98S</i> suppressors (magenta) are clustered in a nearby region distinct from the interface with the 60S subunit. (Assembled from PBD file 5ANB) <b>B)</b> 10-fold serial dilutions of AJY3373 (P_GAL<i>-RPL10</i>) containing <i>WT RPL10</i>, <i>rpl10-R98S</i>, or <i>rpl10-S104D</i> vectors, and either empty vector or the indicated alleles of <i>NMD3</i> on centromeric vectors or <i>TIF6</i> alleles on high copy vectors. Cells were spotted onto glucose-containing selective media to repress genomic <i>RPL10</i>. <b>C)</b> Serial dilutions of the glucose repressible <i>EFL1</i> strain AJY2981 (P_GAL<i>-EFL1</i>) containing empty vector or the indicated <i>TIF6</i> or <i>NMD3</i> plasmids. Cells were spotted onto glucose-containing selective media to repress Efl1.</p

<i>rpl10-R98S</i> suppressors alter the interaction between Nmd3 and 60S.

<p><b>A)</b> Structure of the yeast 60S subunit in complex with Nmd3 (orange density) and Tif6 (yellow density). The eIF5A-like domain of Nmd3 (green ribbon) occupies the E site and associates with the L1 stalk, while the eL22-like domain (yellow ribbon) occupies the P site and comes into contact with the P site loop of Rpl10 (purple). The N-terminal domain of Nmd3 (orange) contacts Tif6. (From PDB 5T62) <b>B)</b> Zoomed view of Tif6 and Nmd3 N-terminal domain. The Tif6 residues mutated in <i>rpl10-R98S</i> suppressors (blue) cluster at the Tif6-Nmd3 interface. <b>C)</b> Zoomed view of Nmd3 eIF5A- and eL22-like domains. Nmd3 residues mutated in <i>rpl10-R98S</i> suppressors (red) map to the Nmd3-60S interface. <b>D)</b> Detail of a portion of the Nmd3-60S interface showing hydrogen bonds between Nmd3 residues N378 and N390 with eL42 and 25S rRNA, respectively. <b>E)</b> 10-fold serial dilutions of AJY3373 (P_GAL<i>-RPL10</i>) expressing <i>WT RPL10</i> or <i>rpl10-R98S</i>, and either WT <i>NMD3</i> or the indicated <i>NMD3</i> mutant from vectors, as indicated. Cells were spotted onto glucose-containing selective media. <b>F-G)</b> Sucrose gradient sedimentation of Nmd3. Extracts were fractionated by sucrose gradient sedimentation and the position of Nmd3 in gradients was monitored by Western blotting using anti-Nmd3 antibody. Anti-Rpl8 was used to monitor the position of 60S subunits. <b>F)</b> Extracts were prepared from AJY3249 (P_GAL<i>-NMD3</i>) cells expressing WT (pAJ409) or mutant <i>NMD3</i> (pAJ2805 or pAJ3609) plasmid as the sole copy. <b>G)</b> Extracts were prepared from AJY1700 (tif6Δ) cells expressing either wild-type (pAJ2846) or mutant <i>TIF6</i> (pAJ2833 or pAJ3401) as the sole copy.</p

The <i>rpl10-R98S</i> defect is not suppressed by Tif6 release.

<p><b>A)</b> The position of Rpl10 in the crown view of the 60S subunit. The central protuberance (CP), helix 38 (H38) and helix 89 (H89) are indicated. A cartoon of Rpl10 structure showing amino acids mutated in T-ALL (blue) (From PDB files 5ANB, 3U5D and 3U5E). <b>B)</b> 10-fold serial dilutions of the glucose repressible <i>RPL10</i> strain AJY3373 (P_GAL<i>-RPL10</i>) harboring <i>WT RPL10</i>, <i>rpl10-S104D</i>, or <i>rpl10-R98S</i> vector, and either empty vector or the indicated <i>TIF6</i> or <i>EFL1</i> plasmids. Cells were spotted onto glucose-containing selective media to repress genomic <i>RPL10</i>. <b>C)</b> Tif6-GFP and Tif6-V192F-GFP localization monitored by fluorescence microscopy in <i>WT RPL10</i>, <i>rpl10-S104D</i>, and <i>rpl10-R98S</i> cells. AJY2766 (P_GAL<i>-RPL10</i>, <i>TIF6-GFP</i>) and AJY3941 (P_GAL<i>-RPL10</i>, <i>TIF6-V192F-GFP</i>) expressing WT <i>RPL10</i>, <i>rpl10-S104D</i>, or <i>rpl10-R98S</i> from plasmids were grown in the presence of glucose to repress genomic <i>RPL10</i>. DIC, differential interference contrast. Scale bar, 5μm.</p