X-ray Crystallography
The X-ray crystallography is a widely used technique for determining the atomic and molecular structure of biological macromolecules like proteins, nucleic acids, and their complexes.
Shared Facility
https://www.med.upenn.edu/jf/bsbcore/centers/crystallography.html
Researchers
The Marmorstein laboratory studies the molecular mechanisms of (1) epigenetic regulation (2) protein post- and co-translational modification with a particular focus on protein acetylation, and (3) enzyme signaling in cancer and metabolism. The laboratory uses a broad range of biochemical, biophysical and structural research tools (X-ray crystallography and cryo-EM) to determine macromolecular structure and mechanism of action. The laboratory also uses high-throughput small molecule screening and structure-based design strategies to develop protein-specific small-molecule probes to interrogate protein function and for preclinical studies.
Chemical Biology cryoEM X-ray Crystallography
We are interested in understanding on a structural and biochemical level how DNA and RNA molecules are maintained and processed by living cells. In addition to the mechanisms and regulation of genome replication and transcription, this includes processes such as DNA repair, site-specific and homologous recombination, transposition, condensation of chromosomes, chromosome pairing and segregation, and RNA trafficking and splicing. Our approach is to establish three-dimensional models of macromolecular assemblies relating to a particular biological question using X-ray diffraction methods and to then develop mechanistic and functional models that can be tested experimentally.
X-ray Crystallography cryoEM
The actin cytoskeleton plays an essential role in multiple cellular functions, including cytokinesis, vesicular trafficking, and the maintenance of cell shape and polarity. To accomplish these functions, the cytoskeleton undergoes constant remodeling into various forms of structural and functional networks, such as lamellipodia, filopodia, stress fibers and focal adhesions. Remodeling of the cytoskeleton of eukaryotic cells is a tightly regulated process, involving hundreds of actin-binding and signaling proteins. Additionally, many bacterial pathogens highjack the eukaryotic actin cytoskeleton during invasion. The main focus of the research in our lab is to understand the molecular basis for how protein-protein interaction networks bring together cytoskeleton scaffolding, nucleation, elongation, and signaling proteins to accomplish specific cellular functions. We are also interested in understanding the function of BAR domain-containing proteins, which are emerging as a critical linkage between signaling, the cytoskeleton and cellular membranes. Our lab is finally deeply invested in understanding the role of cytoskeleton dynamics in muscle cells, particularly the differentiated vascular smooth muscle cell. Our primary research tool is structural biology, including cryo-EM and X-ray crystallography. The atomic snapshots resulting from these structural methods provide a wealth of knowledge, but lack information about the function, dynamics and energetic aspects of protein-protein recognition. To obtain this kind of information we use a host of other approaches, including bio-informatics, biophysical and biochemical methods and collaborative cellular studies.
X-ray Crystallography cryoEM
Dr. Krishnaswamy studies molecular mechanisms underlying the reactions of blood coagulation. His laboratory investigates how the proteins of blood coagulation interact with each other and with membranes to yield a regulated clotting response to vascular injury or an unregulated response in thrombotic or bleeding disease.
X-ray Crystallography cryoEM
Kushol Gupta is a Research Assistant Professor in the Department of Biochemistry & Biophysics at the Perelman School of Medicine of The University of Pennsylvania, a member of the BMB graduate group, and directs the Johnson Foundation Structural Biology and Biophysics Core, a departmental resource that serves Penn and the greater region.
He is a structural biologist with expertise in both X-ray crystallography and solution biophysical methods, including small-angle X-ray and neutron scattering, light scattering, and analytical ultracentrifugation. His ongoing research focuses on retroviral integrases, their interaction with host factors, and a new class of drugs known as allosteric inhibitors of integrase (ALLINIs), which are potent antivirals against HIV. His research also includes other projects in the areas of phenylketonuria, RNA splicing, and site-specific recombination, highlighting the collaborative nature of research at Penn.
X-ray Crystallography X-ray Crystallography and Biophysics cryoEM
Our work focuses on the structural and chemical biology of metal-requiring enzymes in human disease as well as biosynthetic enzymes that generate complex terpenoid natural products. Our research incorporates modern techniques of structural biology, such as X-ray crystallography, small-angle X-ray scattering, and cryo-EM, as well as techniques rooted in bioinorganic, bioorganic, and biophysical chemistry.
Our group, along with the entire Penn Chemistry community, is committed to providing an open and engaging environment in which all persons are welcome and respected. We are a diverse group of faculty, staff, and students, and our strength derives from our diversity. We value the highest ideals of respect and dignity in our daily interactions as we maintain an environment that is free from all forms of discrimination, harassment, and intimidation. Whoever you are and wherever you are on your journey of scholarship in the molecular sciences, you are welcome here.
X-ray Crystallography cryoEM
The Liu Lab is affiliated with the Department of Biochemistry and Biophysics at the University of Pennsylvania Perelman School of Medicine. We utilized a battery of biochemical, cellular, and high-throughput sequencing approaches to study the function of RNA modifying enzymes and sex chromosome-encoded protein homologs in human physiology and disease.
X-ray Crystallography Mass Spectrometry
The goal of our research is to understand the development of antibodies against viruses. We study both signaling complexes inside immune cells, as well as complexes between antibodies and viral glycoproteins. To do this, we employ the structural biology approaches X-ray crystallography and cryo-EM, along with other biochemical techniques.
cryoEM cryoET X-ray Crystallography Faculty CryoEM and CryoET X-ray Crystallography and Biophysics Research Areas
At Muzykantov's Lab, our mission is to revolutionize drug delivery for a healthier future. Since our establishment in the mid-nineties, we've been at the forefront of tackling a critical problem: drugs often lack the natural ability to reach their intended targets in the body, leading to the need for high doses and causing unwanted side effects. Additionally, some drugs simply don't work unless they are precisely localized in specific microenvironments within the body.
CryoEM and CryoET X-ray Crystallography and Biophysics X-ray Crystallography Chemical Biology
At the Feradical (Aimin Liu) Lab, we study how enzymes harness metals and radical chemistry to carry out vital reactions in living systems. Our research focuses on metalloenzymes, protein radicals, and the unique catalytic intermediates that shape biological pathways. By integrating spectroscopy, structural biology, and mechanistic analysis, we seek to understand how these remarkable molecular machines work — and how their chemistry influences health, metabolism, and cellular regulation.
Chemical Biology CryoEM and CryoET X-ray Crystallography Biophysics cryoEM Mass Spectrometry
The Fuglestad lab is interested in expanding molecular study of peripheral membrane proteins and enabling new strategies in drug development for this difficult category of protein. Their approach is rooted in detailed structural and biophysical investigation of fundamental protein function, information that is used to better understand peripheral membrane proteins as drug targets and to guide inhibitor design efforts. In addition, the Fuglestad group develops new tools and methods to enhance the ability to better study peripheral membrane proteins and initiate drug discovery for these elusive targets. The Fuglestad lab has developed a unique membrane model, membrane-mimicking reverse micelles, which allow access to experiments that are otherwise difficult or impossible for this class of protein. Protein targets of interest to the Fuglestad lab, among others, include glutathione peroxidase 4, an important cancer target, p47phox, which is implicated in a variety of inflammatory and other diseases, and a range of lipid transport proteins which are fundamental to lipid organization in the cell, many of which are important therapeutic targets. The unique tools and approaches that the Fuglestad lab utilizes has answered important functional questions surrounding these proteins. Their work has demonstrated unique inhibition strategies that may help peripheral membrane proteins become more routine targets for drug discovery.
CryoEM and CryoET NMR X-ray Crystallography and Biophysics X-ray Crystallography Biophysics Chemical Biology NMR
Related people
Publications
Padilla MS, Li JJ, Zhang Q, Patwari K, Shi S, Yamagata HM, Joseph RA, Hymms BN, Teerdhala SV, Fitzgerald E, Chalom OZ, Gupta K, Alameh MG, Weissman D, Le AD, Mitchell MJ. Lipid Nanoparticle Co-Delivery of mRNA and a Small Molecule Drug for Oral Cancer Chemoimmunotherapy. Adv Mater. 2026 Aug;38(47):e73721. doi: 10.1002/adma.73721. Epub 2026 Jul 14. PMID: 42446053; PMCID: PMC13496076.
Abstract
Oral squamous cell carcinoma (OSCC) represents 90% of all head and neck cancers. Despite decades of research, the 5-year survival rate is 50%. Strikingly, the overall incidence rate is projected to increase by 30% in the next 10 years, which may result in a sharp increase in mortality. Two fundamental aspects of OSCC are that it progresses via the inactivation and mutation of tumor suppressor genes (TSGs) and has a "cold" tumor microenvironment (TME). A major barrier in the treatment of OSCC is the lack of novel therapies clinicians have at their disposal that are designed to disrupt tumor progression by reshaping the cold tumors into inflammatory "hot" tumors. To overcome these obstacles, we employed a lipid nanoparticle (LNP) that co-encapsulates p53 mRNA and the small molecule ciclopirox (CPX). We demonstrate that both drugs have innate chemotherapeutic properties by facilitating caspase activation. Moreover, these therapies can create a less immunosuppressive TME in part by repolarizing tumor-associated macrophages (TAMs) to M1-like phenotypes. When formulated together, our platform provides an all-in-one approach for OSCC, effective in both p53-therapy-susceptible and p53-therapy-resistant models. Additionally, this work offers a template for a delivery platform capable of tackling multiple mechanisms of OSCC progression and survival.
McShan AC, Flores-Solis D, Sun Y, Garfinkle SE, Toor JS, Young MC, Sgourakis NG. Nat Commun. 2023 Dec 11. doi: 10.1038/s41467-023-43654-9.
Abstract
The conformational landscapes of peptide/human leucocyte antigen (pHLA) protein complexes encompassing tumor neoantigens provide a rationale for target selection towards autologous T cell, vaccine, and antibody-based therapeutic modalities. Here, using complementary biophysical and computational methods, we characterize recurrent RAS55-64 Q61 neoepitopes presented by the common HLA-A*01:01 allotype. We integrate sparse NMR restraints with Rosetta docking to determine the solution structure of NRASQ61K/HLA-A*01:01, which enables modeling of other common RAS55-64 neoepitopes. Hydrogen/deuterium exchange mass spectrometry experiments alongside molecular dynamics simulations reveal differences in solvent accessibility and conformational plasticity across a panel of common Q61 neoepitopes that are relevant for recognition by immunoreceptors. Finally, we predict binding and provide structural models of NRASQ61K antigens spanning the entire HLA allelic landscape, together with in vitro validation for HLA-A*01:191, HLA-B*15:01, and HLA-C*08:02. Our work provides a basis to delineate the solution surface features and immunogenicity of clinically relevant neoepitope/HLA targets for cancer therapy.
Sun Y, Florio TJ, Gupta S, Young MC, Marshall QF, Garfinkle SE, Papadaki GF, Truong HV, Mycek E, Li P, Farrel A, Church NL, Jabar S, Beasley MD, Kiefel BR, Yarmarkovich M, Mallik L, Maris JM, Sgourakis NG. Sci Immunol. 2023 Dec 1. doi: 10.1126/sciimmunol.adj5792.
Abstract
Peptide-centric chimeric antigen receptors (PC-CARs) recognize oncoprotein epitopes displayed by cell-surface human leukocyte antigens (HLAs) and offer a promising strategy for targeted cancer therapy. We have previously developed a PC-CAR targeting a neuroblastoma-associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes. Here, we determine the 2.1-angstrom crystal structure of the PC-CAR–PHOX2B–HLA-A*24:02–β2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). This PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactive group, covering a combined global population frequency of up to 46.7%. Biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation, and CAR T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.
Yarmarkovich M, Marshall QF, Warrington JM, Premaratne R, Farrel A, Groff D, Li W, di Marco M, Runbeck E, Truong H, Toor JS, Tripathi S, Nguyen S, Shen H, Noel T, Church NL, Weiner A, Kendsersky N, Martinez D, Weisberg R, Christie M, Eisenlohr L, Bosse KR, Dimitrov DS, Stevanovic S, Sgourakis NG, Kiefel BR, Maris JM. Nature. 2023 Nov 8. doi: 10.1038/s41586-023-06706-0
Abstract
The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.
Papadaki GF, Woodward CH, Young MC, Winters TJ, Burslem GM, Sgourakis NG. J. Biol. Chem. 2023 Oct 1. doi: 10.1016/j.jbc.2023.105136.
Human Tapasin (hTapasin) is the main chaperone of MHC-I molecules, enabling peptide loading and antigen repertoire optimization across HLA allotypes. However, it is restricted to the endoplasmic reticulum (ER) lumen as part of the protein loading complex (PLC), and therefore is highly unstable when expressed in recombinant form. Additional stabilizing co-factors such as ERp57 are required to catalyze peptide exchange in vitro, limiting uses for the generation of pMHC-I molecules of desired antigen specificities. Here, we show that the chicken Tapasin (chTapasin) ortholog can be expressed recombinantly at high yields in a stable form, independent of co-chaperones. chTapasin can bind the human HLA-B∗37:01 with low micromolar-range affinity to form a stable tertiary complex. Biophysical characterization by methyl-based NMR methods reveals that chTapasin recognizes a conserved β2m epitope on HLA-B∗37:01, consistent with previously solved X-ray structures of hTapasin. Finally, we provide evidence that the B∗37:01/chTapasin complex is peptide-receptive and can be dissociated upon binding of high-affinity peptides. Our results highlight the use of chTapasin as a stable scaffold for protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.
Gupta S, Nerli S, Kutti Kandy S, Mersky GL, Sgourakis NG. Nat. Commun. 2023 Oct 10. doi: 10.1038/s41467-023-42163-z.
Abstract
The class I proteins of the major histocompatibility complex (MHC-I) display epitopic peptides derived from endogenous proteins on the cell surface for immune surveillance. Accurate modeling of peptides bound to the human MHC, HLA, has been mired by conformational diversity of the central peptide residues, which are critical for recognition by T cell receptors. Here, analysis of X-ray crystal structures within our curated database (HLA3DB) shows that pHLA complexes encompassing multiple HLA allotypes present a discrete set of peptide backbone conformations. Leveraging these backbones, we employ a regression model trained on terms of a physically relevant energy function to develop a comparative modeling approach for nonamer pHLA structures named RepPred. Our method outperforms the top pHLA modeling approach by up to 19% in structural accuracy, and consistently predicts blind targets not included in our training set. Insights from our work may be applied towards predicting antigen immunogenicity, and receptor cross-reactivity.
Sun Y, Young MC, Woodward CH, Danon JN, Truong HV, Gupta S, Winters TJ, Font-Burgada J, Burslem GM, Sgourakis NG. Proc. Natl. Acad. Sci. U.S.A. 2023 Jun 20. doi: 10.1073/pnas.2304055120.
Abstract
The polymorphic nature and intrinsic instability of class I major histocompatibility complex (MHC-I) and MHC-like molecules loaded with suboptimal peptides, metabolites, or glycolipids presents a fundamental challenge for identifying disease-relevant antigens and antigen-specific T cell receptors (TCRs), hindering the development of autologous therapeutics. Here, we leverage the positive allosteric coupling between the peptide and light chain (β2 microglobulin, β2m) subunits for binding to the MHC-I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/β2m interface, to generate conformationally stable, peptide-receptive molecules named "open MHC-I." Biophysical characterization shows that open MHC-I molecules are properly folded protein complexes of enhanced thermal stability compared to the wild type when loaded with low- to moderate-affinity peptides. Using solution NMR, we characterize the effects of the disulfide bond on the conformation and dynamics of the MHC-I structure, ranging from local changes in β2m-interacting sites of the peptide-binding groove to long-range effects on the α2-1 helix and α3 domain. The interchain disulfide bond stabilizes MHC-I molecules in an open conformation to promote peptide exchange across multiple human leukocyte antigen (HLA) allotypes, covering representatives from five HLA-A supertypes, six HLA-B supertypes, and oligomorphic HLA-Ib molecules. Our structure-guided design, combined with conditional β-peptide ligands, provides a universal platform to generate ready-to-load MHC-I systems of enhanced stability, enabling a range of approaches to screen antigenic epitope libraries and probe polyclonal TCR repertoires covering highly polymorphic HLA-I allotypes, as well as oligomorphic nonclassical molecules.
Papadaki GF, Ani O, Florio TJ, Young MC, Danon JN, Sun Y, Dersh D, Sgourakis NG. Front. Immunol. 2023 Jan 25. doi: 10.3389/fimmu.2023.1116906.
Major Histocompatibility Complex class I (MHC-I) molecules display self, viral or aberrant epitopic peptides to T cell receptors (TCRs), which employ interactions between complementarity-determining regions with both peptide and MHC-I heavy chain ‘framework’ residues to recognize specific Human Leucocyte Antigens (HLAs). The highly polymorphic nature of the HLA peptide-binding groove suggests a malleability of interactions within a common structural scaffold. Here, using structural data from peptide:MHC-I and pMHC:TCR structures, we first identify residues important for peptide and/or TCR binding. We then outline a fixed-backbone computational design approach for engineering synthetic molecules that combine peptide binding and TCR recognition surfaces from existing HLA allotypes. X-ray crystallography demonstrates that chimeric molecules bridging divergent HLA alleles can bind selected peptide antigens in a specified backbone conformation. Finally, in vitro tetramer staining and biophysical binding experiments using chimeric pMHC-I molecules presenting established antigens further demonstrate the requirement of TCR recognition on interactions with HLA framework residues, as opposed to interactions with peptide-centric Chimeric Antigen Receptors (CARs). Our results underscore a novel, structure-guided platform for developing synthetic HLA molecules with desired properties as screening probes for peptide-centric interactions with TCRs and other therapeutic modalities.
Sun Y, Papadaki GF, Devlin CA, Danon JN, Young MC, Winters TJ, Burslem GM, Procko E, Sgourakis NG, Sci. Adv. 2023 Feb 24. doi: 10.1126/sciadv.ade7151.
Abstract
Immunological chaperones tapasin and TAP binding protein, related (TAPBPR) play key roles in antigenic peptide optimization and quality control of nascent class I major histocompatibility complex (MHC-I) molecules. The polymorphic nature of MHC-I proteins leads to a range of allelic dependencies on chaperones for assembly and cell-surface expression, limiting chaperone-mediated peptide exchange to a restricted set of human leukocyte antigen (HLA) allotypes. Here, we demonstrate and characterize xeno interactions between a chicken TAPBPR ortholog and a complementary repertoire of HLA allotypes, relative to its human counterpart. We find that TAPBPR orthologs recognize empty MHC-I with broader allele specificity and facilitate peptide exchange by maintaining a reservoir of receptive molecules. Deep mutational scanning of human TAPBPR further identifies gain-of-function mutants, resembling the chicken sequence, which can enhance HLA-A*01:01 expression in situ and promote peptide exchange in vitro. These results highlight that polymorphic sites on MHC-I and chaperone surfaces can be engineered to manipulate their interactions, enabling chaperone-mediated peptide exchange on disease-relevant HLA alleles.