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

Jiazhi Li, Longfei Wang, Quentin Hahn, Radosław P. Nowak, Thibault Viennet, Esteban A. Orellana, Shourya S. Roy Burman, Hong Yue, Moritz Hunkeler, Pietro Fontana, Hao Wu, Haribabu Arthanari, Eric S. Fischer, and Richard I. Gregory. 1/4/2023. “Structural basis of regulated m7G tRNA modification by METTL1–WDR4.” Nature, 613, 7943, Pp. 391-397. Publisher's VersionAbstract
Chemical modifications of RNA have key roles in many biological processes1–3. N7-methylguanosine (m7G) is required for integrity and stability of a large subset of tRNAs4–7. The methyltransferase 1–WD repeat-containing protein 4 (METTL1–WDR4) complex is the methyltransferase that modifies G46 in the variable loop of certain tRNAs, and its dysregulation drives tumorigenesis in numerous cancer types8–14. Mutations in WDR4 cause human developmental phenotypes including microcephaly15–17. How METTL1–WDR4 modifies tRNA substrates and is regulated remains elusive18. Here we show,  through structural, biochemical and cellular studies of human METTL1–WDR4, that WDR4 serves as a scaffold for METTL1 and the tRNA T-arm. Upon tRNA binding, the $\alpha$C region of METTL1 transforms into a helix, which together with the $\alpha$6 helix secures both ends of the tRNA variable loop. Unexpectedly, we find that the predicted disordered N-terminal region of METTL1 is part of the catalytic pocket and essential for methyltransferase activity. Furthermore, we reveal that S27 phosphorylation in the METTL1 N-terminal region inhibits methyltransferase activity by locally disrupting the catalytic centre. Our results provide a molecular understanding of tRNA substrate recognition and phosphorylation-mediated regulation of METTL1–WDR4, and reveal the presumed disordered N-terminal region of METTL1 as a nexus of methyltransferase activity.
Le Xiao, Venkat Giri Magupalli, and Hao Wu. 11/28/2022. “Cryo-EM structures of the active NLRP3 inflammasome disk.” Nature. Publisher's VersionAbstract
Inflammasomes are cytosolic innate immune complexes that activate caspase-1 upon detection of pathogenic and endogenous dangers1-5, and NLRP3 is an inflammasome sensor of membrane damage highly important in inducing inflammation2,6,7. Here we report cryo-EM structures of disk-shaped active NLRP3 oligomers in complex with ATP&\#x1D6FE;S, the centrosomal kinase NEK7, and the adaptor protein ASC which recruits caspase-1. In these NLRP3-NEK7-ASC complexes, the central NACHT domain of NLRP3 assumes an ATP-bound conformation in which two of its subdomains rotate by \textasciitilde85 ° relative to the ADP-bound inactive conformation8-12. The FISNA domain conserved in NLRP3 but absent in most NLRPs13 becomes ordered in its key regions to stabilize the active NACHT conformation and mediate most interactions in the disk. Mutations on these interactions compromise NLRP3-mediated caspase-1 activation. The N-terminal PYDs from all the NLRP3 subunits gather together to form a PYD filament that recruits ASC PYD to elicit downstream signalling. Surprisingly, the C-terminal LRR domain and the LRR-bound NEK7 do not participate in disk interfaces. Together with previous structures of inactive NLRP3 cage in which LRR-LRR interactions play an important role8-11, we propose that the role of NEK7 is to break the inactive cage to transform NLRP3 into the active NLRP3 inflammasome disk.
José A. Velilla, Matthew R. Volpe, Grace E. Kenney, Richard M. Walsh, Emily P. Balskus, and Rachelle Gaudet. 10/17/2022. “Structural basis of colibactin activation by the ClbP peptidase.” Nature Chemical Biology. Publisher's VersionAbstract
Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-d-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads.
Ying Dong, Xiong Pi, Frauke Bartels-Burgahn, Deniz Saltukoglu, Zhuoyi Liang, Jianying Yang, Frederick W. Alt, Michael Reth, and Hao Wu. 10/14/2022. “Structural principles of B-cell antigen receptor assembly.” Nature. Publisher's VersionAbstract
The B-cell antigen receptor (BCR) is composed of a membrane-bound immunoglobulin (mIg) of class M, D, G, E or A for antigen recognition1–3 and a disulfide-linked Ig$\alpha$ and Ig$\beta$ heterodimer (Ig$\alpha$/$\beta$) that functions as the signalling entity through their intracellular immunoreceptor tyrosine-based activation motifs (ITAMs)4,5. The organizing principle of the BCR remains elusive. Here we report cryogenic electron microscopy structures of mouse full-length IgM BCR at 8.2 \AA resolution and its Fab-deleted form at 3.3 \AA resolution. At the ectodomain (ECD), the Ig$\alpha$/$\beta$ heterodimer mainly uses Ig$\alpha$ to associate with Cµ3-Cµ4 domains of one heavy chain (µHC) while leaving the other heavy chain (µHC') empty. The transmembrane domain (TMD) helices of the two µHCs interact with those of the Ig$\alpha$/$\beta$ heterodimer to form a tight 4-helix bundle. The asymmetry at the TMD prevents the recruitment of two Ig$\alpha$/$\beta$ heterodimers. Surprisingly, the connecting peptide between the ECD and TMD of µHC intervenes in between those of Ig$\alpha$ and Ig$\beta$ to guide the TMD assembly through striking charge complementarity. Weaker but distinct density for the Ig$\beta$ ITAMs nestles next to the TMD, suggesting potential autoinhibition of ITAM phosphorylation. Interfacial analyses suggest that all BCR classes utilize a general organizational architecture. Our studies provide a structural platform for understanding B-cell signalling and designing rational therapies against BCR-mediated diseases.
Miao Gui, Jacob T. Croft, Davide Zabeo, Vajradhar Acharya, Justin M. Kollman, Thomas Burgoyne, Johanna L. Höög, and Alan Brown. 10/3/2022. “SPACA9 is a lumenal protein of human ciliary singlet and doublet microtubules.” Proceedings of the National Academy of Sciences, 119, 41, Pp. e2207605119. Publisher's VersionAbstract
The cilium-centrosome complex contains triplet, doublet, and singlet microtubules. The lumenal surfaces of each microtubule within this diverse array are decorated by microtubule inner proteins (MIPs). Here, we used single-particle cryo-electron microscopy methods to build atomic models of two types of human ciliary microtubule: the doublet microtubules of multiciliated respiratory cells and the distal singlet microtubules of monoflagellated human spermatozoa. We discover that SPACA9 is a polyspecific MIP capable of binding both microtubule types. SPACA9 forms intralumenal striations in the B tubule of respiratory doublet microtubules and noncontinuous spirals in sperm singlet microtubules. By acquiring new and reanalyzing previous cryo-electron tomography data, we show that SPACA9-like intralumenal striations are common features of different microtubule types in animal cilia. Our structures provide detailed references to help rationalize ciliopathy-causing mutations and position cryo-EM as a tool for the analysis of samples obtained directly from ciliopathy patients.
Paula P. Navarro, Andrea Vettiger, Virly Y. Ananda, Paula Montero Llopis, Christoph Allolio, Thomas G. Bernhardt, and Luke H. Chao. 9/12/2022. “Cell wall synthesis and remodelling dynamics determine division site architecture and cell shape in Escherichia coli.” Nature Microbiology. Publisher's VersionAbstract
The bacterial division apparatus catalyses the synthesis and remodelling of septal peptidoglycan (sPG) to build the cell wall layer that fortifies the daughter cell poles. Understanding of this essential process has been limited by the lack of native three-dimensional views of developing septa. Here, we apply state-of-the-art cryogenic electron tomography (cryo-ET) and fluorescence microscopy to visualize the division site architecture and sPG biogenesis dynamics of the Gram-negative bacterium Escherichia coli. We identify a wedge-like sPG structure that fortifies the ingrowing septum. Experiments with strains defective in sPG biogenesis revealed that the septal architecture and mode of division can be modified to more closely resemble that of other Gram-negative (Caulobacter crescentus) or Gram-positive (Staphylococcus aureus) bacteria, suggesting that a conserved mechanism underlies the formation of different septal morphologies. Finally, analysis of mutants impaired in amidase activation ($Δ$envC $Δ$nlpD) showed that cell wall remodelling affects the placement and stability of the cytokinetic ring. Taken together, our results support a model in which competition between the cell elongation and division machineries determines the shape of cell constrictions and the poles they form. They also highlight how the activity of the division system can be modulated to help generate the diverse array of shapes observed in the bacterial domain.
Sai Luo, Jun Zhang, Alex J.B. Kreutzberger, Amanda Eaton, Robert J. Edwards, Changbin Jing, Hai-Qiang Dai, Gregory D. Sempowski, Kenneth Cronin, Robert Parks, Adam Yongxin Ye, Katayoun Mansouri, Maggie Barr, Novalia Pishesha, Aimee Chapdelaine Williams, Lucas Vieira Francisco, Anand Saminathan, Hanqin Peng, Himanshu Batra, Lorenza Bellusci, Surender Khurana, S. Munir Alam, David C. Montefiori, Kevin O. Saunders, Ming Tian, Hidde Ploegh, Tom Kirchhausen, Bing Chen, Barton F. Haynes, and Frederick W. Alt. 8/11/2022. “An Antibody from Single Human VH-rearranging Mouse Neutralizes All SARS-CoV-2 Variants Through BA.5 by Inhibiting Membrane Fusion.” Science Immunology, Pp. eadd5446. Publisher's VersionAbstract
SARS-CoV-2 Omicron sub-variants have generated a world-wide health crisis due to resistance to most approved SARS-CoV-2 neutralizing antibodies and evasion of vaccination-induced antibodies. To manage Omicron sub-variants and prepare for potential new variants, additional means of isolating broad and potent humanized SARS-CoV-2-neutralizing antibodies are desirable. Here, we describe a mouse model in which the primary B cell receptor (BCR) repertoire is generated solely through V(D)J recombination of a human VH1-2 heavy chain (HC) and, substantially, a human Vκ1-33 light chain (LC). Thus, primary humanized BCR repertoire diversity in these mice derives from immensely diverse HC and LC antigen-contact complementarity-region-3 (CDR3) sequences generated by non-templated junctional modifications during V(D)J recombination. Immunizing the human VH1-2/Vκ1-33-rearranging mouse model with SARS-CoV-2 (Wuhan-Hu-1) spike protein immunogens elicited several VH1-2/Vκ1-33-based neutralizing antibodies that bound RBD in a different mode from each other and from those of many prior human patient-derived VH1-2-based neutralizing antibodies. Of these, SP1-77 potently and broadly neutralized all SARS-CoV-2 variants through BA.5. Cryo-EM studies revealed that SP1-77 bound RBD away from the receptor-binding-motif via a CDR3-dominated recognition mode. Lattice-light-sheet-microscopy-based studies showed that SP1-77 did not block ACE2-mediated viral attachment or endocytosis, but rather blocked viral-host membrane fusion. The broad and potent SP1-77 neutralization activity and non-traditonal mechanism of action suggest this antibody might have therapeutic potential. Likewise, the SP1-77 binding epitope may further inform on vacccine strategies. Finally, the general class of humanized mouse models we have described may contribute to identifying therapeutic antibodies against future SARS-CoV-2 variants and other pathogens. A humanized antibody from a recently-developed mouse model potently neutralizes SARS-CoV-2 variants by inhibiting membrane fusion.
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