Many multicellular organisms specify germ cells during early embryogenesis by the inheritance of ribonucleoprotein (RNP) granules known as germplasm. However, the role of complex interactions of RNP granules during germ cell specification remains elusive. This study characterizes the interaction of RNP granules, Buc, and zebrafish Vasa (zfVasa) during germ cell specification. We identify a novel zfVasa-binding motif (Buc-VBM) in Buc and a Buc-binding motif (zfVasa-BBM) in zfVasa. Moreover, we show that Buc and zfVasa directly bind in vitro and that this interaction is independent of the RNA. Our circular dichroism spectroscopy data reveal that the intrinsically disordered Buc-VBM peptide forms alpha-helices in the presence of the solvent trifluoroethanol. Intriguingly, we further demonstrate that Buc-VBM enhances zfVasa ATPase activity, thereby annotating the first biochemical function of Buc as a zfVasa ATPase activator. Collectively, these results propose a model in which the activity of zfVasa is a central regulator of primordial germ cell (PGC) formation and is tightly controlled by the germplasm organizer Buc.

CRM1 is a nuclear export receptor that has been intensively targeted over the last decade for the development of antitumor and antiviral drugs. Structural analysis of several inhibitor compounds bound to CRM1 revealed that their mechanism of action relies on the covalent modification of a critical cysteine residue (Cys528 in the human receptor) located in the nuclear export signal-binding cleft. This study presents the crystal structure of human CRM1, covalently modified by 2-mercaptoethanol on Cys528, in complex with RanGTP at 2.58 Å resolution. The results demonstrate that buffer components can interfere with the characterization of cysteine-dependent inhibitor compounds.

While 3D electron diffraction (3D-ED or microcrystal electron diffraction; MicroED) has emerged as a promising method for protein structure determination, its applicability is hindered by a high susceptibility to radiation damage, leading to a decreasing signal-to-noise ratio in consecutive diffraction patterns that limits the quality (resolution and redundancy) of the data. In addition, data completeness may be restricted due to the geometrical limitations of current sample holders and stages. Although specialized equipment can overcome these challenges, many laboratories do not have access to such instrumentation. In this work, we introduce an approach that addresses these issues using a commonly available 200 keV cryo-electron microscope. The multi-position acquisition technique that we present here combines (i) multiple data acquisitions from a single crystal over several tilt ranges and (ii) merging data from a small number of crystals each tilted about a different axis. The robustness of this approach is demonstrated by the de novo elucidation of a protein–peptide complex structure from only two orthorhombic microcrystals.