Structure and activation mechanism of the Makes caterpillars floppy 1 toxin.

The bacterial Makes caterpillars floppy 1 (Mcf1) toxin promotes apoptosis in insects, leading to loss of body turgor and death. The molecular mechanism underlying Mcf1 intoxication is poorly understood. Here, we present the cryo-EM structure of Mcf1 from Photorhabdus luminescens, revealing a seahorse-like shape with a head and tail. While the three head domains contain two effectors, as well as an activator-binding domain (ABD) and an autoprotease, the tail consists of two putative translocation and three putative receptor-binding domains. Rearrangement of the tail moves the C-terminus away from the ABD and allows binding of the host cell ADP-ribosylation factor 3, inducing conformational changes that position the cleavage site closer to the protease. This distinct activation mechanism that is based on a hook-loop interaction results in three autocleavage reactions and the release of two toxic effectors. Unexpectedly, the BH3-like domain containing ABD is not an active effector. Our findings allow us to understand key steps of Mcf1 intoxication at the molecular level.

SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM.

Selecting particles from digital micrographs is an essential step in single-particle electron cryomicroscopy (cryo-EM). As manual selection of complete datasets-typically comprising thousands of particles-is a tedious and time-consuming process, numerous automatic particle pickers have been developed. However, non-ideal datasets pose a challenge to particle picking. Here we present the particle picking software crYOLO which is based on the deep-learning object detection system You Only Look Once (YOLO). After training the network with 200-2500 particles per dataset it automatically recognizes particles with high recall and precision while reaching a speed of up to five micrographs per second. Further, we present a general crYOLO network able to pick from previously unseen datasets, allowing for completely automated on-the-fly cryo-EM data preprocessing during data acquisition. crYOLO is available as a standalone program under http://sphire.mpg.de/ and is distributed as part of the image processing workflow in SPHIRE.