The Harvard Center for Cryo-electron microscopy (HC2EM) is a joint effort by Harvard Medical School, Dana-Farber Cancer Institute, Boston Children’s Hospital, and Massachusetts General Hospital to provide state-of-the-art cryo-EM instrumentation and expertise for the Harvard structural biology community. This user facility offers consultation and training by staff in specimen preparation, microscope operation, image acquisition, and data analysis. 

Additionally, the Molecular Electron Microscopy Suite (MEMS) at Harvard Medical School is a separate user resource currently available to qualified researchers. This user facility offers training and supervision in negative-stain and cryo-transmission electron microscopy. Equipment includes three transmission electron microscopes, two cryo plungers, and sample preparation areas.

Recent Publications

Jaron A. M. Mercer, Stephan J. DeCarlo, Shourya S. Roy Burman, Vedagopuram Sreekanth, Andrew T. Nelson, Moritz Hunkeler, Peter J. Chen, Katherine A. Donovan, Praveen Kokkonda, Praveen K. Tiwari, Veronika M. Shoba, Arghya Deb, Amit Choudhary, Eric S. Fischer, and David R. Liu. 3/14/2024. “Continuous evolution of compact protein degradation tags regulated by selective molecular glues.” Science, 383, 6688, Pp. eadk4422. Publisher's VersionAbstract
Conditional protein degradation tags (degrons) are usually >100 amino acids long or are triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous protein levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved a 36–amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex with PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame with SD40 using prime editing are degraded by otherwise inert PT-179. Cryo–electron microscopy structures of SD40 in complex with ligand-bound cereblon revealed mechanistic insights into the molecular basis of SD40’s activity and specificity. Our efforts establish a system for continuous evolution of molecular glue complexes and provide ZF tags that overcome shortcomings associated with existing degrons. Degron tags enable rapid and tunable control of target protein levels using small molecules. The ability to develop tags with desirable properties could expand their use in research and biotechnology. Mercer et al. report a continuous evolution platform for generating high-affinity molecular glue complexes. Using this approach, the authors evolved a compact zinc-finger degron that engages the protein cereblon in the presence of thalidomide derivatives that avoid endogenous proteins, unlike the immunomodulatory drugs commonly used to trigger protein degradation. This work provides a compact orthogonal degron tag and a powerful system with which to engineer molecular glue interactions using diverse small molecules. —Di Jiang Rapid evolution of molecular glue interfaces yields a compact small molecule–triggered degron with high target protein specificity.
May S. Freag, Mostafa T. Mohammed, Arpita Kulkarni, Hagar E. Emam, Krishna P. Maremanda, and Ahmed O. Elzoghby. 2/28/2024. “Modulating tumoral exosomes and fibroblast phenotype using nanoliposomes augments cancer immunotherapy.” Science Advances, 10, 9, Pp. eadk3074. Publisher's VersionAbstract
Cancer cells program fibroblasts into cancer associated fibroblasts (CAFs) in a two-step manner. First, cancer cells secrete exosomes to program quiescent fibroblasts into activated CAFs. Second, cancer cells maintain the CAF phenotype via activation of signal transduction pathways. We rationalized that inhibiting this two-step process can normalize CAFs into quiescent fibroblasts and augment the efficacy of immunotherapy. We show that cancer cell–targeted nanoliposomes that inhibit sequential steps of exosome biogenesis and release from lung cancer cells block the differentiation of lung fibroblasts into CAFs. In parallel, we demonstrate that CAF-targeted nanoliposomes that block two distinct nodes in fibroblast growth factor receptor (FGFR)–Wnt/β-catenin signaling pathway can reverse activate CAFs into quiescent fibroblasts. Co-administration of both nanoliposomes significantly improves the infiltration of cytotoxic T cells and enhances the antitumor efficacy of αPD-L1 in immunocompetent lung cancer–bearing mice. Simultaneously blocking the tumoral exosome-mediated activation of fibroblasts and FGFR-Wnt/β-catenin signaling constitutes a promising approach to augment immunotherapy. Blocking exosome-mediated activation of fibroblasts and FGFR/β-catenin axis using nanoliposomes augments αPD-L1 immunotherapy.
Friederike MC Benning, Simon Jenni, Coby Y Garcia, Tran H Nguyen, Xuewu Zhang, and Luke H Chao. 1/4/2024. “Helical reconstruction of VP39 reveals principles for baculovirus nucleocapsid assembly.” Nature Communications, 15, 1, Pp. 250.Abstract
Baculoviruses are insect-infecting pathogens with wide applications as biological pesticides, in vitro protein production vehicles and gene therapy tools. Its cylindrical nucleocapsid, which encapsulates and protects the circular double-stranded viral DNA encoding proteins for viral replication and entry, is formed by the highly conserved major capsid protein VP39. The mechanism for VP39 assembly remains unknown. We use electron cryomicroscopy to determine a 3.2 \AA helical reconstruction of an infectious nucleocapsid of Autographa californica multiple nucleopolyhedrovirus, revealing how dimers of VP39 assemble into a 14-stranded helical tube. We show that VP39 comprises a distinct protein fold conserved across baculoviruses, which includes a Zinc finger domain and a stabilizing intra-dimer sling. Analysis of sample polymorphism shows that VP39 assembles in several closely-related helical geometries. This VP39 reconstruction reveals general principles for baculoviral nucleocapsid assembly.
Karanbir S Pahil, Morgan SA Gilman, Vadim Baidin, Thomas Clairfeuille, Patrizio Mattei, Christoph Bieniossek, Fabian Dey, Dieter Muri, Remo Baettig, Michael Lobritz, Kenneth Bradley, Andrew C Kruse, and Daniel Kahne. 1/3/2024. “A new antibiotic traps lipopolysaccharide in its intermembrane transporter.” Nature.Abstract
Gram-negative bacteria are extraordinarily difficult to kill because their cytoplasmic membrane is surrounded by an outer membrane that blocks the entry of most antibiotics. The impenetrable nature of the outer membrane is due to the presence of a large, amphipathic glycolipid called lipopolysaccharide (LPS) in its outer leaflet1. Assembly of the outer membrane requires transport of LPS across a protein bridge that spans from the cytoplasmic membrane to the cell surface. Maintaining outer membrane integrity is essential for bacterial cell viability, and its disruption can increase susceptibility to other antibiotics2–6. Thus, inhibitors of the seven lipopolysaccharide transport (Lpt) proteins that form this transenvelope transporter have long been sought. A new class of antibiotics that targets the LPS transport machine in Acinetobacter was recently identified. Here, using structural, biochemical and genetic approaches, we show that these antibiotics trap a substrate-bound conformation of the LPS transporter that stalls this machine. The inhibitors accomplish this by recognizing a composite binding site made up of both the Lpt transporter and its LPS substrate. Collectively, our findings identify an unusual mechanism of lipid transport inhibition, reveal a druggable conformation of the Lpt transporter and provide the foundation for extending this class of antibiotics to other Gram-negative pathogens.
João PL Coelho, Matthew CJ Yip, Keely Oltion, Jack Taunton, and Sichen Shao. 1/3/2024. “The eRF1 degrader SRI-41315 acts as a molecular glue at the ribosomal decoding center.” Nature Chemical Biology.Abstract
Translation termination is an essential cellular process, which is also of therapeutic interest for diseases that manifest from premature stop codons. In eukaryotes, translation termination requires eRF1, which recognizes stop codons, catalyzes the release of nascent proteins from ribosomes and facilitates ribosome recycling. The small molecule SRI-41315 triggers eRF1 degradation and enhances translational readthrough of premature stop codons. However, the mechanism of action of SRI-41315 on eRF1 and translation is not known. Here we report cryo-EM structures showing that SRI-41315 acts as a metal-dependent molecular glue between the N domain of eRF1 responsible for stop codon recognition and the ribosomal subunit interface near the decoding center. Retention of eRF1 on ribosomes by SRI-41315 leads to ribosome collisions, eRF1 ubiquitylation and a higher frequency of translation termination at near-cognate stop codons. Our findings reveal a new mechanism of release factor inhibition and additional implications for pharmacologically targeting eRF1.
Carol Cho, Christian Ganser, Takayuki Uchihashi, Koichi Kato, and Ji-Joon Song. 9/28/2023. “Structure of the human ATAD2 AAA+ histone chaperone reveals mechanism of regulation and inter-subunit communication.” Communications Biology, 6, 1, Pp. 993.Abstract
ATAD2 is a non-canonical ATP-dependent histone chaperone and a major cancer target. Despite widespread efforts to design drugs targeting the ATAD2 bromodomain, little is known about the overall structural organization and regulation of ATAD2. Here, we present the 3.1 \AA cryo-EM structure of human ATAD2 in the ATP state, showing a shallow hexameric spiral that binds a peptide substrate at the central pore. The spiral conformation is locked by an N-terminal linker domain (LD) that wedges between the seam subunits, thus limiting ATP-dependent symmetry breaking of the AAA+ ring. In contrast, structures of the ATAD2-histone H3/H4 complex show the LD undocked from the seam, suggesting that H3/H4 binding unlocks the AAA+ spiral by allosterically releasing the LD. These findings, together with the discovery of an inter-subunit signaling mechanism, reveal a unique regulatory mechanism for ATAD2 and lay the foundation for developing new ATAD2 inhibitors.
Pascal Devant, Ying Dong, Julian Mintseris, Weiyi Ma, Steven P Gygi, Hao Wu, and Jonathan C Kagan. 11/22/2023. “Structural insights into cytokine cleavage by inflammatory caspase-4.” Nature, 624, 7991, Pp. 451–459.Abstract
Inflammatory caspases are key enzymes in mammalian innate immunity that control the processing and release of interleukin-1 (IL-1)-family cytokines1,2. Despite the biological importance, the structural basis for inflammatory caspase-mediated cytokine processing has remained unclear. To date, catalytic cleavage of IL-1-family members, including pro-IL-1$\beta$ and pro-IL-18, has been attributed primarily to caspase-1 activities within canonical inflammasomes3. Here we demonstrate that the lipopolysaccharide receptor caspase-4 from humans and other mammalian species (except rodents) can cleave pro-IL-18 with an efficiency similar to pro-IL-1$\beta$ and pro-IL-18 cleavage by the prototypical IL-1-converting enzyme caspase-1. This ability of caspase-4 to cleave pro-IL-18, combined with its previously defined ability to cleave and activate the lytic pore-forming protein gasdermin D (GSDMD)4,5, enables human cells to bypass the need for canonical inflammasomes and caspase-1 for IL-18 release. The structure of the caspase-4–pro-IL-18 complex determined using cryogenic electron microscopy reveals that pro-lL-18 interacts with caspase-4 through two distinct interfaces: a protease exosite and an interface at the caspase-4 active site involving residues in the pro-domain of pro-IL-18, including the tetrapeptide caspase-recognition sequence6. The mechanisms revealed for cytokine substrate capture and cleavage differ from those observed for the caspase substrate GSDMD7,8. These findings provide a structural framework for the discussion of caspase activities in health and disease.
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