@article {70340, title = {Rapidly evolving aphid gall effector proteins exhibit saposin-like folds}, journal = {bioRxiv}, year = {2026}, month = {2026 Mar 28}, abstract = {

Many insects manipulate plants by injecting effector proteins. In one extreme example of this molecular {\textquotedblleft}hijacking{\textquotedblright}, Hormaphis cornu aphids inject bicycle proteins into Hamamelis virginiana (Witch Hazel), contributing to the development of novel organs called galls. Bicycle proteins share no amino acid sequence similarity with proteins of known function. Here, we report the crystal structures of two divergent bicycle proteins. Both proteins contain saposin-like folds: one with multiple disulfide bonds exhibits a helix swap; the other has no disulfide bonds and possesses two tandem domains. To explore the structural evolution of bicycle proteins, we predicted bicycle protein structures with Alphafold2 (AF2). While AF2 did not recover the two experimental structures using existing databases, it succeeded after we provided multiple sequence alignments (MSAs) containing protein sequences encoded in new genome sequences from closely related aphid species. Using this customized approach at scale, we generated 2400 high-confidence predictions for bicycle proteins from seven aphid species. This dataset revealed that bicycle proteins without cysteines are outliers in fold space and appear to have evolved from ancestral proteins with disulfide-bonded saposin-like folds. While all bicycle proteins contain predicted saposin-like folds, they display a vast diversity of structural and physicochemical properties. While this diversity thwarts prediction of conserved functions encoded in structure, it suggests that bicycle proteins have evolved to target diverse plant processes and/or to evade plant immune surveillance.Significance statement Parasites introduce specialized {\textquotedblleft}effector{\textquotedblright} proteins into hosts, both to suppress host immunity and to release nutrients. The molecular functions and structures of most effector proteins are unknown. Effector proteins often evolve rapidly and share no similarity with proteins of known function. Here, we demonstrate that machine learning algorithms can accurately predict the structures of aphid {\textquotedblleft}bicycle{\textquotedblright} effector proteins when supplemented with data from closely related species. We exploit this finding to generate predictions of 2400 bicycle protein structures. These proteins exploit a common motif, yet exhibit diverse structures that form distinct structural clusters. Despite the clustering of these proteins in structure space, they occupy a nearly uniformly physicochemical space, suggesting that they encode a large diversity of molecular functions.

}, doi = {10.64898/2026.03.27.712717}, url = {https://www.biorxiv.org/content/early/2026/03/28/2026.03.27.712717}, author = {Bhinderwala, Fatema and Korgaonkar, Aishwarya and Gopalakrishna, Kota and Mathers, Thomas C. and Shigenobu, Shuji and Bazan, J. Fernando and Hogenhout, Saskia A. and Calero, Guillermo and Gronenborn, Angela M. and Stern, David L.} } @article {48265, title = {Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.}, journal = {Nature Methods}, volume = {14}, year = {2017}, month = {2017 Feb 13}, pages = {399-402}, abstract = {

Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.

}, issn = {1548-7105}, doi = {10.1038/nmeth.4178}, author = {de la Cruz, M Jason and Hattne, Johan and Shi, Dan and Seidler, Paul and Rodriguez, Jose and Reyes, Francis E and Sawaya, Michael R and Cascio, Duilio and Weiss, Simon C and Kim, Sun Kyung and Hinck, Cynthia S and Hinck, Andrew P and Calero, Guillermo and Eisenberg, David and Gonen, Tamir} } @article {48617, title = {MicroED structure of Au146(p-MBA)57 at subatomic resolution reveals a twinned FCC cluster.}, journal = {Journal of Physical Chemistry Letters}, volume = {8}, year = {2017}, month = {2017 Oct 31}, pages = {arXiv:1706.07902 [physics.atm-clus]}, abstract = {

Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. We report the structure of Au146(p-MBA)57\ at subatomic resolution (0.85 {\AA}) using electron diffraction (MicroED) and atomic resolution by X-ray diffraction. The 146 gold atoms may be decomposed into two constituent sets consisting of 119 core and 27 peripheral atoms. The core atoms are organized in a twinned FCC structure whereas the surface gold atoms follow a C2\ rotational symmetry about an axis bisecting the twinning plane. The protective layer of 57 p-MBAs fully encloses the cluster and comprises bridging, monomeric, and dimeric staple motifs. Au146(p-MBA)57\ is the largest cluster observed exhibiting a bulk-like FCC structure as well as the smallest gold particle exhibiting a stacking fault.

}, doi = {10.1021/acs.jpclett.7b02621}, url = {https://128.84.21.199/abs/1706.07902}, author = {Vergara, Sandra and Lukes, Dylan A and Martynowycz, Michael W and Santiago, Ulises and Plascencia-Villa, German and Weiss, Simon C and de la Cruz, Michael Jason and Black, David M and Alvarez, Marcos M and Lopez-Lozano, Xochitl and Barnes, Christopher O and Lin, Guowu and Weissker, Hans-Christian and Whetten, Robert L and Gonen, Tamir and Calero, Guillermo} } @article {47422, title = {The collection of MicroED data for macromolecular crystallography.}, journal = {Nature Protocols}, volume = {11}, year = {2016}, month = {2016 May}, pages = {895-904}, abstract = {

The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is \~{}10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.

}, issn = {1750-2799}, doi = {10.1038/nprot.2016.046}, author = {Shi, Dan and Nannenga, Brent L and de la Cruz, M Jason and Liu, Jinyang and Sawtelle, Steven and Calero, Guillermo and Reyes, Francis E and Hattne, Johan and Gonen, Tamir} }