Structural Biology Facility
The Structural Biology Core Facility provides state-of-the art equipment for structural modeling in crystals and in solution using X-ray diffraction and scattering as well as magnetic resonance.
Structural Biology Facility
The Structural Biology Core Facility provides state-of-the art equipment for structural modeling in crystals and in solution using X-ray diffraction and scattering as well as magnetic resonance.
The Structural Biology Core Facility at Brown University provides state-of-the-art equipment for researchers at Brown University and the surrounding area. Our instruments can be used for structure determination in crystals and in solution, using X-ray diffraction as well as magnetic resonance.
The core facility is equipped with instruments for creating high-resolution biomolecular structural models. We have NMR spectrometers suitable for acquiring high-resolution structural and dynamics data on proteins in the solution state. Additionally, our X-ray source can be used for collecting diffraction data and is equipped to automatically screen up to 80 crystals in a run.
Find us at CoresRI!
Services and Instruments
Service Request and Reservations
Designated established users have priority to schedule instrument time in the respective instrument calendar for the upcoming week before Thursday 9:00 am in the preceding week. A week in instrument schedule starts on Monday at 9:00 am and ends next Monday at 8:59 am. After Thursday 9:00 am, unreserved time is open for all current users. While making the reservation, users should provide their name and details of the proposed experiment and the time they plan to use the instrument. A day in the NMR schedule starts at 9 am.
If you plan something for a week where your lab does not have a priority, please mark the reservation in the calendar with the word “(tentative)”. Tentative on our calendars means you plan to use the time if not reserved by the users in the priority group for that week. Do not schedule instrument time more than three weeks in the future without prior permission.
If you are a new user, please contact the facility manager for gaining access to the instrument reservation.
Requests for instrument time should be directed to the facility manager via the contact page.
The Structural Biology Core Facility instrument schedules are available as Google calendars.
|Service||Units||Internal Rate*||External Academic||Commercial|
|ASCEND 850 MHz NMR||Hours||$12||$18||$79|
|AVANCE III HDTM 600 MHz NMR||Hours||$9||$15||$80|
|Avance II Ultrashield 11.7 T 500 MHz NMR||Hours||$2||$3||$45|
|Rigaku X-ray Source||Hours||$27||$43||$103|
*Rates for Brown and Rhode Island Academic and Hospital Affiliates
Effective as of 11/01/2022
The BMRB is a repository of NMR data and also aggregates this data for purposes of comparison and study.
C6 at CSIRO is a tool for comparing crystallization conditions across many different kinds of screens. This may help develop a screening strategy.
The PDB is a repository for biomolecular structures.
Resources for Grants
Structural Biology Facility. The facility, located at the Laboratories for Molecular Medicine, is directed and managed by two PhD-level scientists. The facility houses instruments for NMR spectroscopy and X-ray crystallography. The facility instrumentation includes Bruker AVANCE III HD 850 MHz, Bruker NEO 600MHz NEO, and Bruker AVANCE II 500 MHz spectrometers. The 850 and 600 MHz spectrometers are equipped with TCI cryoprobes and have nitrogen liquefiers. The 500 is currently equipped with a TXI HCN room-temperature probe. Room temperature probes are also available for the 850 and 600 MHz spectrometers. All spectrometers are operated by Linux workstations running Bruker TopSpin software. For protein crystallographic research, the facility instrumentation includes a Rigaku MicroMax-003 microfocus sealed tube X-ray generator and Saturn 944HG detector. The setup is equipped with ACTOR crystal mounting robot that can be operated using the J Director software from a Linux workstation.
Scientific reproducibility is enhanced through scientific rigor and transparency. Scientific rigor is the strict application of the scientific method to ensure unbiased and well-controlled experimental design, methodology, analysis, interpretation, and reporting of results. The Structural Biology Facility is committed to supporting research excellence by adopting the following practices of scientific rigor.
- Purchase and maintain a variety of high-quality instruments from established vendors such that the best instrument is available for any given research analysis.
- The equipment is overseen by highly trained Structural Biology experts and well maintained under service contracts or funds budgeted for annual preventive maintenance and repairs.
- The Structural Biology experts are available for experimental design consults or troubleshooting.
- All Structural Biology Facility users are thoroughly trained by the expert staff, one on one, and not granted access to the instruments until they are deemed to be suitably trained.
- All newly trained users are also required to use the Structural Biology instruments during daytime hours to increase interaction with the expert staff members.
Read about the science being performed at Brown University's Structural Biology Core Facility.
The high-field magnets housed in the Structural Biology Core Facility were used to collect all of the experimental data presented in these publications. In these reports, NMR spectroscopy uncovered mechanistic signatures, both structural and dynamic, that led to new insight regarding the propagation of biomolecular signals in a diverse set of enzymes.
Chen, E.; Reiss, K.; Shah, D.; Manjula, R.; Allen, B.; Murphy, E.L.; Murphy, J.W.; Batista, V.S.; Bhandari, V.; Lolis, E.J.*; Lisi, G.P. "A Structurally Preserved Allosteric Site in the MIF Superfamily Affects Enzymatic Activity and CD74 Activation in D-dopachrome Tautomerase" J. Biol. Chem. 2021. 297. 101061-101073. DOI: https://doi.org/10.1016/j.jbc.2021.101061
The macrophage migration inhibitory factor (MIF) family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism(s). Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. Despite structural and functional similarity, the MIF homolog D-dopachrome tautomerase (also called MIF-2) has low sequence identity (35%), prompting the question of whether this dynamic regulatory network is conserved. Here, we establish the structural basis of an allosteric site in MIF-2, showing with solution NMR that dynamic communication is preserved in MIF-2 despite differences in the primary sequence. X-ray crystallography and NMR detail the structural consequences of perturbing residues in this pathway, which include conformational changes surrounding the allosteric site, despite global preservation of the MIF-2 fold. Molecular simulations reveal MIF-2 to contain a comparable hydrogen bond network to that of MIF, which was previously hypothesized to influence catalytic activity by modulating the strength of allosteric coupling. Disruption of the allosteric relay by mutagenesis also attenuates MIF-2 enzymatic activity in vitro and the activation of the cluster of differentiation 74 receptor in vivo, highlighting a conserved point of control for nonoverlapping functions in the MIF superfamily.
East, K.W.; Delaglio, F.; Lisi, G.P. “A Simple Approach for Reconstruction of Non-uniformly Sampled Pseudo-3D NMR Data for Accurate Measurement of Spin Relaxation Parameters" J. Biomol. NMR. 2021. 75. 213-219. DOI: https://doi.org/10.1007/s10858-021-00369-7
We explain how to conduct a pseudo-3D relaxation series NUS measurement so that it can be reconstructed by existing 3D NUS reconstruction methods to give accurate relaxation values. We demonstrate using reconstruction algorithms IST and SMILE that this 3D approach allows lower sampling densities than for independent 2D reconstructions. This is in keeping with the common finding that higher dimensionality increases signal sparsity, enabling lower sampling density. The approach treats the relaxation series as ordinary 3D time-domain data whose imaginary part in the pseudo-dimension is zero, and applies any suitably linear 3D NUS reconstruction method accordingly. Best results on measured and simulated data were achieved using acquisitions with 9 to 12 planes and exponential spacing in the pseudo-dimension out to ~ 2 times the inverse decay time. Given these criteria, in typical cases where 2D reconstructions require 50% sampling, the new 3D approach generates spectra reliably at sampling densities of 25%.
Schwartz J, Son J, Brugger C, Deaconescu AM. “Phospho-dependent signaling during the general stress response by the atypical response regulator and ClpXP adaptor RssB.” Protein Sci. 2021 Apr;30(4):899-907. doi: 10.1002/pro.4047. Epub 2021 Mar 1.PMID: 33599047
This study sheds light on the regulation of the general stress response by post-translational modification of RssB, a key protein factor that regulates the intracellular levels of the master transcription factor RpoS. We report and compare two structures of the receiver domain of RssB, in the apo form and bound to the phosphomimetic beryllofluoride. These structures put decades of research into a structural framework and cement the role of phosphorylation in increasing the efficiency of RpoS proteolysis. Preliminary X-ray diffraction experiments analyses were carried out using core facility instrumentation.
Cui, J.Y.; Zhang, F.; Nierzwicki, L.; Palermo, G.; Linhardt, R.J.; Lisi, G.P. “Mapping the Structural and Dynamic Determinants of pH-sensitive Heparin Binding to Granulocyte Macrophage-colony Stimulating Factor" Biochemistry. 2020. 59. 3541-3553. DOI: https://doi.org/10.1021/acs.biochem.0c00538
Granulocyte macrophage colony stimulating factor (GMCSF) is an immunomodulatory cytokine that is harnessed as a therapeutic. GMCSF is known to interact with other clinically important molecules, such as heparin, suggesting that endogenous and administered GMCSF has the potential to modulate orthogonal treatment outcomes. Thus, molecular level characterization of GMCSF and its interactions with biologically active compounds is critical to understanding these mechanisms and predicting clinical consequences. Here, we dissect the biophysical factors that facilitate the GMCSF–heparin interaction, previously shown to be pH-dependent, using nuclear magnetic resonance spectroscopy, surface plasmon resonance, and molecular dynamics simulations. We find that the affinity of GMCSF for heparin increases not only with a transition to acidic pH but also with an increase in heparin chain length. Changes in local flexibility, including a disruption of the N-terminal helix at acidic pH, also accompany the binding of heparin to GMCSF. We use molecular dynamics simulations to propose a mechanism in which a positive binding pocket that is not fully solvent accessible at neutral pH becomes more accessible at acidic pH, facilitating the binding of heparin to the protein.
Pantouris, G.; Khurana, L.; Ma, A.; Skeens, E.; Reiss, K.; Batista, V.S.; Lisi, G.P.*; Lolis, E.J.* “Regulation of MIF Enzymatic Activity by an Allosteric Site at the Central Solvent Channel" Cell Chem. Biol. 2020. 27. 740-750. DOI: https://doi.org/10.1016/j.chembiol.2020.05.001 (*corresponding authors)
In proteins with multiple functions, such as macrophage migration inhibitory factor (MIF), the study of its intramolecular dynamic network can offer a unique opportunity to understand how a single protein is able to carry out several nonoverlapping functions. A dynamic mechanism that controls the MIF-induced activation of CD74 was recently discovered. In this study, the regulation of tautomerase activity was explored. The catalytic base Pro1 is found to form dynamic communications with the same allosteric node that regulates CD74 activation. Signal transmission between the allosteric and catalytic sites take place through intramolecular aromatic interactions and a hydrogen bond network that involves residues and water molecules of the MIF solvent channel. Once thought to be a consequence of trimerization, a regulatory function for the solvent channel is now defined. These results provide mechanistic insights into the regulation of catalytic activity and the role of solvent channel water molecules in MIF catalysis.
East, K.W.; Newton, J.C.; Morzan, U.N.; Narkhede, Y.; Acharya, A.; Skeens, E.; Jogl, G.; Batista, V.S.; Palermo, G.*; Lisi, G.P.* “Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics” J. Am. Chem. Soc. 2020. 142. 1348-1358. DOI: https://doi.org/10.1021/jacs.9b10521 (*corresponding authors)
CRISPR–Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian-accelerated molecular dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond time scale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in full-length CRISPR–Cas9. These findings reveal a potential route of signal transduction within the CRISPR–Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR–Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome-editing capability.
Dorich V, Brugger C, Tripathi A, Hoskins JR, Tong S, Suhanovsky MM, Sastry A, Wickner S, Gottesman S, Deaconescu AM. “Structural basis for inhibition of a response regulator of σS stability by a ClpXP antiadaptor.” Genes Dev. 2019 Jun 1;33(11-12):718-732. doi: 10.1101/gad.320168.118. Epub 2019 Apr 11. PMID: 30975721
This manuscript reports on the first structural information on the degradation pathway of RpoS, a major transcriptional regulator in E. coli and related species, which controls a large regulon of up to 24% of the E. coli genes and orchestrates the general stress response. My group solved the only existing crystal structure of full-length RssB (an RpoS adaptor that delivers it to ClpXP for degradation) bound to one of its many anti-adaptors, the IraD protein, and carried out in vitro functional studies of RssB and IraD. Our data points to RssB using distinct mechanisms for recognition of its many, sequence-dissimilar anti-adaptors, which is enabled by the remarkable plasticity of an interdomain segmented helical linker. Since anti-adaptors represent the main strategy for stabilizing RpoS for stress adaptation, our analysis suggests a strategy for antimicrobial development via targeting of the RssB linker region or anti-adaptor/RssB interfaces for inhibition. Preliminary X-ray diffraction experiments analyses were carried out using core facility instrumentation.
Burke KA, Janke AM, Rhine CL, and Fawzi NL. "Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Polymerase II" Mol Cell. 2015, (online ahead of print).
The N-terminal domain of the RNA-binding protein Fused in Sarcoma (FUS) plays a role in amyotrophic lateral sclerosis and frontotemporal dementia. Brown University postdoctoral fellow Kathleen Burke, graduate student Christy Rine, and undergraduate Abigail Jahnke from the lab of Dr. Nicolas Fawzi investigated the behavior of this domain using the SBCF 850 MHz NMR and demonstrated that it was disordered in solution. They also observed that as the protein concentration increased, FUS formed phase-separated droplets. Both RNA and RNA polymerase II facilitated the formation of these droplets. Unlike solid inclusions such as amyloid plaques, the droplets are dynamic, and FUS diffuses freely within them. Thanks to the high sensitivity of the 850, Burke and colleagues were able to demonstrate that FUS is still disordered and flexible even inside the droplets. This dynamic character is unique and may have important physiological implications.
Krishnan N, Krishnan K, Connors CR, Choy MS, Page R, Peti W, Van Aelst L, Shea SD, and Tonks NK. "PTP1B inhibition suggests a therapeutic strategy for Rett syndrome" J Clin Invest. 2015, 125(8):3163-77.
Choy MS, Yusoff P, Lee IC, Newton JC, Goh CW, Page R, Shenolikar S, and Peti W. "Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase" Cell Rep. 2015, 11(12):1885-91.
Conicella A, and Fawzi NL. "The C-Terminal Threonine of Aβ43 Nucleates Toxic Aggregation via Structural and Dynamical Changes in Monomers and Protofibrils" Biochemistry 2014, 53 (19): 3095-3195.
The aggregation of amyloid-β (Aβ) peptides into plaques or soluble oligomers is believed to cause Alzheimer's disease. However, many different Aβ peptides are present in the brain. The most common are Aβ40 and Aβ42, but some studies have suggested that other forms of the peptide may be the trigger for aggregation and disease. Aβ43 is one of these suspected culprits, because it is significantly enriched in plaques and is more toxic than Aβ42. Using the 850 MHz NMR spectrometer of the Structural Biology Core Facility, Brown University graduate student Alex Conicella and Professor Nick Fawzi studied the dynamics of Aβ43 in order to understand the cause of this enhanced toxicity. They found that the Aβ43 peptide formed protofibrils faster than Aβ42, and at lower concentrations. They also determined that Aβ43 is more rigid at its C-terminus than either of the other two peptides, as well as being very similar to the aggregation-prone Aβ42 when in protofibrils. These data support the hypothesis that Aβ43 serves as a trigger for the formation of toxic assemblies of Aβ and thus plays a significant role in the pathology of Alzheimer's.
Krishnan N, Koveal D, Miller DH, Xue B, Akshinthala SD, Kragelj J, Jensen MR, Gauss C-M, Page R, Blackledge M, Muthuswamy SK, Peti W and Tonks N. "Targeting the disordered C terminus of PTP1B with an allosteric inhibitor." Nature Chem. Biol. 2014 (ahead of print)
Protein-tyrosine phosphatase 1B (ptp1b) is a signaling protein that plays a role in a variety of pathways associated with diseases including cancer, diabetes, and obesity. As a result there has been a great deal of interest in developing inhibitors of this enzyme. However, the nature of the chemistry it performs has proven a significant barrier to creating effective drugs. Brown University students Dorothy Koveal and Dan Miller, with Professors Wolfgang Peti and Rebecca Page, collaborated with research teams at the University of Toronto and Institut de Biologie Structurale in Grenoble to characterize an inhibitor, MSI-1436, that interferes with ptp1b function without binding to the active site. Like most enzymes, ptp1b performs catalysis with a folded domain, but it also has a large C-terminal region that serves a regulatory function and is predominantly unfolded. Using the NMR spectrometers of the Structural Biology Core Facility, the Page and Peti labs were able to show that this C-terminal region, though disordered, possesses residual secondary structure. They were also able to identify two specific sites where MSI-1436 binds. One of these sites lay within one of the predicted helices, and mutations to disrupt the helical structure significantly reduced the inhibitor's affinity. The second binding location also lies within the unfolded domain, but near another allosteric regulation site. Although the mechanism of inhibition is not yet clear, this result suggests promising new avenues for controlling ptp1b activity.
Choy MS, Hieke M, Kumar GS, Lewis GR, Gonzalez-Dewhitt KR, Kessler RP, Stein BJ, Hessenberger M, Nairn AC, Peti W, and Page R. "Understanding the antagonism of retinoblastoma protein dephosphorylation by PNUTS provides insights into the PP1 regulatory code." Proceedings of the National Academy of Sciences 2014, 111 (11): 4097-4102.
The Brown University labs of Rebecca Page and Wolfgang Peti collaborated on an investigation of serine-threonine protein phosphatase 1 (PP1). PP1 is a catlytic unit involved in hundreds of different dephosphorylation reactions involved in numerous regulatory pathways, including processes as diverse as carbohydrate metabolism and cell-cycle progression. It gains specificity for each pathway by binding to other proteins to form a phosphatase holoenzyme, although it has been difficult to predict how any given protein will bind PP1 due to the vast diversity of regulators. One such partner is PNUTS, which plays a key role in many processes of the cell nucleus. Of special interest, the PP1:PNUTS holoenzyme regulates two tumor suppressors: p53 and Rb. Using NMR, the research team identified the smallest piece of PNUTS that encompassed the whole PP1 binding site. Then they used crystallography to determine the structure of this PP1:PNUTS complex. The new structure demonstrated that PNUTS binds to the same part of PP1 that Rb does, but also to two other sites, generating a high-affinity interaction that only breaks down once PNUTS gets phosphorylated or knocked down in concentration. These results not only illuminate the competition between PNUTS and Rb for PP1 binding, but also revealed additional structural motifs related to PP1 binding. This will allow researchers to predict the binding behavior of up to a quarter of known PP1 regulators.
Kumar GS, Zettl H, Page R, and Peti W. "Structural Basis for the Regulation of the MAP Kinase p38α by the Dual Specificity Phosphatase 16 MAP Kinase Binding Domain in Solution" Journal of Biological Chemistry 2013, 288: 28347-56
Brown University postdoctoral associates Senthil Kumar and Heiko Zettl, from the Page-Peti lab, used NMR spectroscopy to figure out how DUSP16 interacts with p38α. DUal-Specificity Phosphatases (DUSPs) regulate MAP kinases, and so are sometimes called MKPs (MAP Kinase Phosphatases). Like the KIM-PTPs, they primarily bind via a short amino acid sequence known as the KIM, but in DUSPs the KIM is part of a completely folded MAP Kinase Binding Domain (MKBD). Dr. Kumar showed that DUSP16 binds more strongly to p38α than DUSP10, another member of the protein family. Using NMR experiments performed on the Structural Biology Core Facility 500 and 850 MHz spectrometers, he found that this heightened affinity was related to an expanded binding site that inolved not only the KIM and nearby helices (as in DUSP10), but also interactions at an additional helix. These results indicate that there are key differences in binding between different DUSPs, explaining their different physiological roles and suggesting that it is likely possible to specifically disrupt particular MAP-DUSP complexes.
Francis DM, Kumar GS, Koveal D, Tortajada A, Page R and Peti W. "The Differential Regulation of p38α by the Neuronal Kinase Interaction Motif Protein Tyrosine Phosphatases, a Detailed Molecular Study." Structure 2013, 21 (9): 1612-1623
Brown University graduate students Dana Francis and Dorothy Koveal, and postdoctoral associate Senthil Kumar, from the Page-Peti lab, used NMR spectroscopy, Small-Angle X-ray Scattering (SAXS), Isothermal Titration Calorimetry (ITC), and HADDOCK modeling to identify the structural differences in the interaction of p38α with the family of KIM protein tyrosine phosphatases (KIM-PTPs), which have a catalytic domain and a disordered region that contains a Kinase Interaction Motif. KIM-PTPs regulate the activity of MAP kinases like p38α, which is implicated in inflammatory responses and autoimmune disease. This study focused on PTPSL and STEP. Using NMR spectroscopy and SAXS, the Page-Peti team found that PTPSL binds to p38α primarily using the KIM and another unfolded sequence known as the KIS, and that the complex formed in solution is extended, similarly to the binding interaction they described previously for a KIM-PTP known as HePTP. They found that the interaction of STEP, a drug target in Alzheimer's disease, was very different. SAXS data clearly show that the STEP-p38α complex is compact, and the NMR data indicated that unlike the other KIM-PTPs studied, STEP's catalytic domain interacts directly with p38α. This difference in binding modes explains the lower catalytic efficiency of STEP, and offers novel approaches for controlling the activity of p38α.
Koveal D, Clarkson MW, Wood TK, Page R, and Peti W. "Ligand Binding Reduces Conformational Flexibility in the Active Site of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from Pseudomonas aeruginosa" Journal of Molecular Biology 2013, 425 (12); 2219-2231.
Brown University graduate student Dorothy Koveal, from the Page-Peti lab, used NMR spectroscopy to determine the structure of a phosphatase that controls biofilm formation in P. aeruginosa in isolation and in complex with phosphate. Using the 500 MHz NMR spectrometer at Brown University, she also obtained information about the dynamics of the protein's backbone amide groups. These experiments showed that the unbound protein had significantly greater flexibility in its active site than the phosphate-bound protein. This restraint of motion may be related to enzymatic activity or substrate specificity.
Koveal D, Schuh-Nuhfer N, Ritt D, Page R, Morrison DK, and Peti W. "A CC-SAM, for Coiled-Coil Sterile-α Motif, Domain Targets the Scaffold KSR-1 to Specific Sites in the Plasma Membrane" Science Signaling 2012, 5 (255): ra94.
Brown University graduate student Dorothy Koveal, from the Page-Peti lab, used NMR spectroscopy to determine the structure of a novel domain fusing a coiled-coil motif to a sterile-α motif. With the assistance of collaborators at the National Cancer Institute, she found that this domain targeted the scaffolding protein KSR-1 to membrane ruffles in vivo. Using the Brown University 500, she determined that CC-SAM underwent a structural rearrangement in the presence of specific kinds of lipid micelles and bicelles that explained the domain's targeting capabilities.
This facility was supported in part by Brown University's Division of Biology and Medicine and Provost's office.
We ask that researchers collecting data at the facility acknowledge its use as follows:
This research is based in part on data obtained at the Brown University Structural Biology Core Facility, which is supported by the Division of Biology and Medicine, Brown University.