Skip to main content
News   |   Events   |   Safety   |   CHESS-U>   |   InSitμ   |   MacCHESS   |   CLASSE

X-RAY RUNS: Apply for Beamtime

2017  Nov 1 - Dec 21

2018  Feb 7 - Apr 3
2018  Proposal/BTR deadline: 12/1/17

2018  Apr 11 - Jun 4
2018  Proposal/BTR deadline: 2/1/18



Tuesday, June 4th

"Structural basis of rifampicin resistance by bacterial RNA polymerase"

Katsuhiko Murakami
Pennsylvania State University

Presentation: PDF

Abstract:  Tuberculosis (TB) is one of the most significant global challenges to human health. For over four decades, Rifampicin (Rif, aka Rifampin), a semi-synthetic derivative of Rifamycin, has been used as a first line antibiotic treatment of TB and is the cornerstone of current short-term TB treatment. The mode of action involves tight Rif binding to a beta subunit of bacterial RNA polymerase (RNAP) (Kd is sub nanomolar) to inhibit RNA transcription. Although many Rif resistant (RifR) strains with mutations in the Rif-binding pocket can be isolated in bacterial culture, only three specific RifR mutations account for over 80 % of Mycobacterium tuberculosis (MTB) RifR strains in clinical isolates due to RifR associated fitness costs. Recently, we have shown that the Escherichia coli RNAP can be prepared from a convenient overexpression system and its X-ray crystal structure can be determined (1). We have also shown that the E. coli RNAP crystal can be used for finding the binding site of ligand such as bacterial alarmone (p)ppGpp (2) and for investigating the interaction between RNAP and benzoxazinorifamycins, rifampicin-derivatives having superior affinity toward RifR RNAP mutants (3). In this study, we have determined the crystal structures of the E. coli RNAP RifR mutants each having one of three major RifR mutations found in clinical isolates. Each RifR RNAP structure shows a unique conformation of the Rif binding pocket and their structural deviations from the wild-type Rif binding pocket are consistent with their Rif resistances suggesting that the RifR results from alternating the shape complementary between the Rif binding pocket and Rif in addition to disrupting hydrophilic and hydrophobic interactions. This study provides an important step toward developing superior Rif analogues for the RifR MTB.

1. K.S. Murakami. The X-ray crystal structure of Escherichia coli RNA polymerase sigma70 holoenzyme. J. Biol. Chem. 2013, 288, 9126-9134.

2. U. Mechold, K. Potrykus, H. Murphy, K.S. Murakami and M. Cashel. Differential regulation by ppGpp versus pppGpp in Escherichia coli. Nucleic Acids Research 2013, in press.

3. V. Molodtsov, I.N. Nawarathne, N.T. Scharf, P.D. Kirchhoff, H.D.H. Showalter, G.A. Garcia and K.S. Murakami. X-ray crystal structures of the Escherichia coli RNA polymerase in complex with Benzoxazinorifamycins. J. Medicinal Chem. in press.


"Introducing cryo-SAXS: solution scattering from nanoliter volumes"

Steve P. Meisburger, Matthew Warkentin, Andrea Katz, Jesse B. Hopkins, Huimin Chen, Richard E. Gillilan, Robert E. Thorne and Lois Pollack
Cornell University

Abstract: Small angle X-ray scattering (SAXS) is an increasingly popular technique for obtaining low resolution solution structures of dynamic macromolecules and complexes. Because SAXS does not require special sample preparation beyond that needed to ensure monodispersity, it is commonly used to probe the dependence of macromolecular conformation or association on different solution conditions (such as temperature, pH, and ionic strength). Such combinatorial experiments are often limited by consumption of both sample and synchrotron time. Therefore, for high throughput applications of SAXS, it is desirable to find ways to economize sample and to accelerate data collection. Although modern X-ray sources can deliver sufficient flux for millisecond data acquisition in micron-scale samples, the high sensitivity to radiation damage of macromolecules in solution demands that the total dose be distributed over a large volume (typically > 10 microliters), either by defocusing the beam or flowing the sample through it. Furthermore, frequent, aggressive cleaning of X-ray windows of the sample cell is often required due, for example to protein adsorption. Experience from electron microscopy (EM) and X-ray crystallography (MX) suggests that the maximum allowable radiation dose may be significantly larger at low temperatures than at ambient conditions. In addition to reducing radiation damage, cryocooling prevents evaporation of the sample droplet, and therefore “window-free” sample holders may be used.

While cryocooling is now standard practice for EM and MX, it has not yet been embraced by the SAXS community. Because of the difficulty in producing homogeneous and reproducibly vitrified solutions, the requirement of accurately measuring and subtracting the solvent background scattering for SAXS was seen as incompatible with cryocooling. From a series of experiments performed at CHESS beamlines F2, C1, and G1, we identified cryoprotectants that produce homogeneously vitrified droplets by rapid cooling in a 100 K gas stream. Scattering profiles from similarly vitrified, macromolecule-containing solutions resemble those acquired at room temperature. The ability to expose for longer periods of time before damaging the sample more than compensates for the reduction in signal to background resulting from the cryoprotectants. As a result of increased dose tolerance, the sample volume can be reduced by orders of magnitude relative to room temperature. We collect cryo-SAXS data of sufficient quality to determine molecular shape reconstructions from illuminated volumes as small as 100 nanoliters. Studies to date show minimal artifacts introduced by cryoprotectant and cooling. Possible applications of cryo-SAXS will be discussed, as well as remaining technical challenges for high throughput data collection.


"Radical allostery in a radical protein"

Nozomi Ando
Massachusetts Institute of Technology

Presentation: PDF



Eaton Lattman
Hauptman Woodward


"Ultrafast x-ray imaging of fuel sprays: fluid dynamics on time scale from microseconds to nanoseconds"

Jin Wang
Advanced Photon Source

Presentation: PDF


Wednesday, June 5th - Workshop I

"Tricks and pitfalls in eukaryotic membrane protein crystallization"

Toshi Kawate
Cornell University

Presentation: PDF

Abstract: Crystallization of eukaryotic membrane proteins is extremely challenging due to a number of technical difficulties, and, in fact, there are only less than 70 reported structures compared to more than 86,000 structures for proteins of other kinds. While the rate of successful crystallization has been drastically increasing, there is no golden standard for eukaryotic membrane protein crystallization. Therefore, crystallographers still rely on methods based on their own experience or published techniques, which typically contain only successful attempts. In this talk, I will present unpublished stories behind the successful P2X receptor crystallization, comprising mostly unsuccessful trials. The seven years worth of my struggle will hopefully help the audience succeed in their challenges.


"Crystallographic study of the transport cycle intermediates of a glutamate transporter homologue"

Olga Boudker
Cornell University & Weill Medical College

Presentation: PDF

Abstract: Ion-coupled membrane transporters undergo cycles of conformational changes associated with binding of ions and substrates from the extracellular space, their translocation across the membrane and release into the cytoplasm, and finally the return of the unbound transporter into the initial state. To visualize the key structural states along this cycle crystallographically, it is necessary to isolate them out of the dynamic conformational ensemble sampled by the transporter solubilized in detergent. To achieve this, we have developed several methodologies, including cross-linking of strategically placed cysteines, site directed mutagenesis aided by spectroscopic methods, manipulation of crystallization conditions and crystal soaking. Using these methods, we have determined the structures of several key states of a glutamate transporter homologue, GltPh, providing structural snapshots along the transport cycle and revealing a striking dynamic nature of these transporters.


"Crystal structure of glycoprotein E2 from bovine viral diarrhea virus"

Simon Li
Yale University

Abstract: Pestivuruses, including bovine viral diarrhea virus (BVDV), are important animal pathogens and are closely related to hepatitis C virus (HCV), which remains a major global health threat. They have an outer lipid envelope bearing two glycoproteins, E1 and E2, required for cell entry. They deliver their genome into the host-cell cytoplasm by fusion of their envelope with a cellular membrane, The crystal structure of BVDV E2 reveals a novel protein architecture consisting of two Ig-like domains followed by an elongated beta-stranded domain with a new fold. E2 forms end-to-end homodimers with a conserved C-terminal motif rich in aromatic residues at the contract. A disulfide bond, across the interface explains the acid resistance of pestiviruses ad their requirement for a redox activation step to initiate fusion. From the structure of E2, we propose alternative possible membrane fusion mechanisms. We expect the pestivirus fusion apparatus to be conserved in HCV.


"Structure of the agonist-bound neurotensin receptor NTS1"

Reinhard Grisshammer
National Institutes of Health

Paper: PDF

Abstract: Neurotensin (NT) is a 13 amino acid peptide that functions as both a neurotransmitter and a hormone through activation of the neurotensin receptor NTS1, a G protein-coupled receptor (GPCR) signaling preferentially through Gq. In the brain, NT modulates activity of dopaminergic systems, opioid-independent analgesia, and the inhibition of food intake, and in the gut NT regulates a range of digestive processes. We have solved the structure at 2.8 Å resolution of NTS1 in an active-like state, bound to NT8-13, the C terminal portion of NT responsible for agonist-induced activation of the receptor. Because wild-type NTS1 is unstable and thus not amenable to crystallization, we used alanine- scanning mutagenesis to stabilize NTS1 and to select for an active-like conformation in the presence of agonist, which combined with the bacteriophage T4 lysozyme fusion protein strategy and the lipidic mesophase crystallization method, resulted in diffracting crystals. The agonist binding pocket is located at the extracellular receptor surface. The peptide agonist binds to NTS1 in an extended conformation nearly perpendicular to the membrane plane with the C-terminus oriented towards the receptor core. The NTS1 structure bears many hallmark features of an active-like receptor conformation such as an outward-tilted transmembrane helix 6 at the cytoplasmic surface and key conserved residues in positions characteristic for active but not for inactive GPCRs. Our findings provided for the first time insight into the binding mode of a peptide agonist to a GPCR.


Wednesday, June 5th - Workshop II

"Scanning nanocalorimetry combined with time-resolved x-ray diffraction - a new tool for studying transformations in complex materials systems"

Joost Vlassak
Harvard University

Presentation: PDF


"Changing the paradigm for engineering design by merging high energy x-ray data with materials modeling"

Jay Schuren
Air Force Research Laboratory


"In-situ analysis of alkali antimonide cathode growth"

John Smedley
Brookhaven National Lab

Presentation: PDF

Abstract: Alkali antimonide photocathodes are a prime candidate for use in high-brightness photoinjectors of 4th generation light sources. These materials have complex growth kinetics - many methods exist for forming the compounds, each with different grain size, roughness, and crystalline texture. These parameters impact the performance of the cathodes, including efficiency, intrinsic emittance and lifetime. In situ analysis of the growth of these materials has allowed investigation of correlations between cathode structure and growth parameters and the resulting quantum efficiency and intrinsic emittance. The best cathodes have a QE at 532 nm in excess of 6% and are structurally textured K2CsSb with grain sizes in excess of 20 nm. X-ray reflection (XRR) and grazing incidence small angle x-ray scattering (GISAXS) have been used to characterize the roughness evolution of the cathode, while x-ray diffraction (XRD) has been used to characterize the texture, grain size and stoichometry. X-ray photoemission spectroscopy (XPS) has been used to analyze the surface chemical makeup, and in-vacuum atomic force microscopy (AFM) has been used to analyze the roughness of the final films.


"Structure of the SrTiO3 (001) surface during photo-assisted water splitting"

Xin Huang2,3*, Manuel Plaza2,3, Joaquin Rodríguez-López1,6, J.Y. Peter Ko2,3, Nicole L. Ritzert1,2, Mei Shen6, Darrell G. Schlom2,4, Joel D. Brock2,3,5, and Héctor D. Abruña1,2

1Department of Chemistry and Chemical Biology
2Energy Materials Center at Cornell
3School of Applied and Engineering Physics
4Materials Science and Engineering,
5Cornell High Energy Synchrotron Source,Cornell University, Ithaca NY 14850, USA
6Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

Abstract: Since the report in 1976 that UV irradiation of an n-type SrTiO3 electrode in an electrochemical cell catalyzes the conversion of H20 to H2 and O2 [1], researchers have been working to develop efficient catalysts for photo-assisted water splitting. While a great deal is known about the electronic structure of SrTiO3 and the redox levels of hydrogen and oxygen, essentially nothing is known about the atomic structure of the catalytically active surface. Using in situ, high-energy x-ray reflectivity, we have measured the electron density, ρ(z), of the (001) surface of a SrTiO3 substrate doped with oxygen vacancies in NaOH solution during photo-assisted electro-catalysis. The structure of the catalytically active surface is not related to the well-known TiO2 double-layer termination [2]. Our x-ray structural measurements are augmented by electrochemical measurements of the O2 evolution, which we then associate with catalytic activity. “Training” the surface by cycling a positive external bias both irreversibly alters the surface structure and enhances the catalytic activity at zero external bias by 300%. Since this simultaneous change in surface structure and chemical activity does not occur for un-doped samples, we conclude the changes are driven by the catalytic reaction, not by changes in pH, applied bias, or ionic species. The next steps are to determine the atomic structure of the catalytically active surface via standard crystallographic techniques and then use that information to guide JDFT studies and the design of hetero-structures engineered for water splitting.

[1] M. S. Wrighton, A. B. Ellis, P. T. Wolczanski, D. L. Morse, H. B. Abrahamson, and D. S. Ginley, "Strontium-Titanate Photoelectrodes. Efficient Photoassisted Electrolysis of Water at Zero Applied Potential," Journal of the American Chemical Society 98 (10), 2774-2779 (1976).
[2] N. Erdman, K.R. Poeppelmeler, M. Asta, O. Warschkow, D.E. Ellis, and L.D. Marks, "The structure and chemistry of the TiO2-rich surface of SrTiO3(001)," Nature 419 (6902), 55-58 (2002).