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Research Highlights 2014

Electrostatic interactions help an enzyme do its job

A catalytic enzyme facilitates a reaction by bringing one or more molecules into its active site and there providing an environment conducive to the reaction. Electrostatic interactions are important for both phases, and conformational changes occurring in an enzyme during its catalytic cycle modify these interactions. For complete understanding of the catalytic process, we require knowledge of the contribution of electrostatic effects to each step in the process, as well as an understanding of how conformational changes affect the electrostatic environment in the active site.

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent conversion of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF), a step in the synthesis of some amino acids. During the catalytic process, the NADP+/NADPH cofactor is bound, altered, and released, as is the DHF/THF substrate, and the enzyme conformation switches between “closed” and “occluded” forms (Figure 1C). Using a combination of techniques, the Benkovic group (Penn. State) has followed the electrostatic microenvironment in the active site of E. coli DHFR (ecDHFR) throughout its catalytic cycle. As aids to monitoring the state of the active site, two mutant DHFRs were generated, each introducing a new Cys residue which could be modified by addition of a -CN (sometimes -13CN) reporter group (Figure 1A).


Figure 1. A, ecDHFR in the closed conformation with folate (FOL) and NADP+ bound. Modified residues in L45C-CN and T46C-CN mutants are shown. B, Closed (red) and occluded (blue) conformations of ecDHFR. C, Major complexes in the catalytic cycle of ecDHFR, color-coded according to ecDHFR conformation at each stage.

The CN vibrational stretching frequency, measured by FTIR (Fourier transform infrared) spectroscopy, and the 13C chemical shift, measured using NMR (nuclear magnetic resonance), were sensitive to the electrostatic environment of the modified residue. To determine the exact location of the reporter groups, crystal structures were determined for the two mutants, to about 2 Å resolution, using data taken at CHESS A1 station. Structures were determined using molecular replacement, with the wild-type structure as a model: the backbones for the mutants were nearly the same as for the wild-type, with an RMSD of the heavy atoms of just 0.5 Å. Kinetics measurements verified that the activity of the mutant enzymes was comparable to that of the wild-type.

FTIR and NMR measurements, along with QM/MM (quantum mechanics/molecular dynamics) simulations, provided information about the electrostatics, as well as the degree of hydration, of the environment of the CN probes. Moreover, it was possible to calculate the contributions of the ligands, surrounding residues, and solvent molecules. Significant changes occurred as the enzyme progressed along the catalytic cycle, particularly near the hydride transfer site, with smaller changes in the folate-binding pocket. The interpretation of these changes is that electrostatic interactions between protein and ligands act to orient the reactants in such a way as to create an electric field favoring the hydride transfer reaction; active site residues also contribute to this field. Future work with more probes and additional states of DHFR will expand knowledge of electrostatics in DHFR to other parts of the molecule and other conformational states.


[1] C.T. Liu, J.P. Layfield, R.J. Stewart III, J.B. French, P. Hanoian, J.B. Asbury, S. Hammes-Schiffer, S.J. Benkovic, "Probing the electrostatics of active site microenvironments along the catalytic cycle for Escherichia coli dihydrofolate reductase," J. Am. Chem. Soc. 136, 10349-10360 (2014).

Submitted by: Marian Szebenyi, MacCHESS, Cornell University 2014

Professor Eddy Arnold elected as 2014 American Crystallographic Association Fellow


Eddy Arnold, Board of Governors Professor of Chemistry and Chemical Biology at Rutgers University, and Resident Faculty Member at the Center for Advanced Biotechnology and Medicine (CABM), was recently elected as a Fellow of the American Crystallographic Association (ACA). Professor Arnold, a long-time user of CHESS and MacCHESS facilities, was honored for his research in macromolecular crystallography and drug design targeting infectious disease agents. Also cited were his contributions to the field through scholarly and organizational activities, including serving on advisory boards for macromolecular crystallography and synchrotron radiation. Arnold was among eight ACA Fellows named this year, bringing the total to 35. ACA Fellows serve as scientific ambassadors to the broader scientific community and the general public to advance science education, research, knowledge, interaction, and collaboration.

Professor Eddy Arnold is an elected Fellow of the American Association for the Advancement of Science (2001), and of the American Academy of Microbiology (2006). Since its inception in 1987, Professor Arnold’s laboratory has been continuously funded by the National Institutes of Health (NIH), and he is the recipient of two consecutive NIH MERIT Awards (1998-2008, 2009-2019), which extend five-year grants to ten years and are awarded to less than 5% of NIH investigators. In 2013 Dr. Arnold received the Hyacinth Award “Honoring outstanding achievements in the struggle against HIV/AIDS,” recognizing work that he and his group have done to understand the structure and function of the AIDS virus reverse transcriptase enzyme, and to develop drugs that can overcome resistance. HIV reverse transcriptase is responsible for copying the viral genetic material in infected cells and is the target of many of the most widely used anti-AIDS drugs.

Professor Arnold has served on the MacCHESS Advisory Committee since 1992, and was Chair from 2006-2009. Much of the work that led to the ACA Fellowship honor relied on using CHESS facilities for structural studies of complex and biologically important viruses and components of viruses. Using crystallography at synchrotron radiation sources, primarily at CHESS, BNLS, and APS, the Arnold group has been able to obtain comprehensive pictures of how a key part of the AIDS virus, the enzyme HIV-1 reverse transcriptase, carries out its functions. Our studies at CHESS enabled the discovery and development of two approved drugs used to treat HIV infection, Edurant/rilpivirine and Intelence/etravirine, that are especially resilient to drug resistance. Professor Eddy Arnold has been a faculty member at Rutgers University and CABM since 1987. He pursued undergraduate and graduate study in organic chemistry and crystallography at Cornell University with Professor Jon Clardy. Eddy then performed postdoctoral research at Purdue University, where he worked with Professor Michael G. Rossmann (ACA Fellow 2011) to obtain a picture of a human common cold virus in atomic detail, the first animal virus structure. The spectacular common cold virus structure determination was made possible by use of CHESS. Arnold is author of more than 250 publications in prominent peer-reviewed scientific journals. With Professor Rossmann, Arnold co-edited the first International Tables for Crystallography volume devoted to crystallography of biological macromolecules (Volume F, editions published in 1999 and 2012). Eddy Arnold also has served on several national and international advisory committees, including for synchrotron X-ray facilities, and served as Member (1999-2011) of and Chair (2005-2011) of the International Union of Crystallography Commission on Biological Macromolecules.

The year 2014 has been designated by UNESCO as the International Year of Crystallography (IYCr) to celebrate the 100th anniversary of the discovery of X-ray diffraction, the phenomenon that enables determination of the structures of molecules ranging in complexity from table salt to entire viruses and other complex biological machines. Professor Arnold was Director of an International School of Crystallography course on structure-based drug design in Erice, Sicily, Italy in June 2014, attended by 160 scientists from around the world. Dr. Arnold also presented a Keynote Lecture at the IUCr Congress in Montreal in August describing his laboratory’s structural studies of HIV reverse transcriptase and how that information has contributed to the discovery and development of two drugs used for treating HIV infection.

Professor Arnold said: “Being named an ACA Fellow is particularly gratifying because of my passion for and long-term involvement in crystallography and the pivotal contributions of this field to fundamental chemical and biological knowledge and ongoing biomedical discovery. Crystallographic studies at synchrotrons such as CHESS continually contribute some of the most exciting experimental science that has translated into so many fundamental discoveries across the chemical, physical, and life sciences.”

Submitted by: Eddy Arnold, Rutgers University 11/14/2014

Fine details of transcribing DNA to RNA

RNA polymerase (RNAP) assembles an RNA strand corresponding to the DNA sequence of a gene, in a precisely choreographed series of molecular motions. To understand this critical cellular process, biologists are studying the fine details of the structural changes involved, and how they are regulated. RNAP complexes vary from one species to another, but a core subset of proteins is found throughout archaeal and eukaryotic life forms. Comparison of archaeal and eukaryotic proteins reveals how structural motifs have been modified during evolution, so that function is maintained while regulation has become more complex in eukaryotic species.

The Murakami group (Penn. State) determined the crystal structure of RNAP from the archaeal species Thermococcus kodakarensis, which is believed to be similar to the common ancestor of Archaea and Eukaryota. Diffraction data were collected at CHESS F1 station, and the structure was solved, to 3.5 Å resolution, with the help of known RNAP and RNAP subunit structures. The final refinement gave Rwork/Rfree of 27.7/31.6%.

EuryarchaealRNAP archaeal

Structures of archaeal and eukaryotic forms of RNAP. Numbers identify individual proteins in the complex. Proteins 4 and 5 are hidden on the far side of the complex; no equivalents to proteins 8 and 9 exist in the T. kodakarensis structure.

The overall T. kodakarensis RNAP structure is similar to that of the yeast RNAP Pol II, but has the “clamp” (protein 1 in the figure) in an opened position and the “stalk” (proteins 4 and 7) rotated 12° relative to the Pol II position. The new structure leads to a model for the domain motions involved as the RNAP “clamp” closes about a DNA strand. Concerted motion of the clamp and stalk resolve the problem of steric clashes observed in open models constructed from the Pol II crystal structure by moving only the clamp domain. Alignment of the two structures also helps to identify the surface regions involved in regulation by eukaryotic-specific transcription factors.


[1] S.-H. Jun, A. Hirata, T. Kanai, T. J. Santangelo, T. Imanaka, and K. S. Murakami, "The X-ray crystal structure of the euryarchaeal RNA polymerase in an open-clamp configuration," Nature Commun. DOI: 10.1038/ncomms5132 (2014).

Submitted by: Marian Szebenyi, MacCHESS, Cornell University 11/17/2014

Users show off innovative work at BioSAXS Essentials V workshop

The Macromolecular Diffraction Facility at the Cornell High Energy Synchrotron Source (MacCHESS) held its fifth annual BioSAXS Essentials workshop on October 30 to November 1, 2014. The workshop convened 6 speakers, all expert practitioners in various topics related to BioSAXS, who in a full first day of lectures, provided a solid foundation of the theory and application of solution X-ray scattering to an eager class with various industrial and academic appointments.

Richard Gillilan (MacCHESS) kicked off the morning session with scattering principles and captivating images during his BioSAXS introductory talk, saving a more in-depth lecture of scattering theory for a later session. Kushol Gupta (UPenn Med) followed with an overview of best practices for sample preparation and data collection, focusing on critical details often overlooked by newcomers. Part 2 of Richard’s BioSAXS introductory talk followed with an overview of BioSAXS data reduction and elucidation of the more basic structural parameters encoded in scattering profiles.


The early afternoon session started with Thomas Grant (Hauptman-Woodward Institute) providing a basic but complete case for interpreting BioSAXS scattering profiles to model structural envelopes. Thomas keenly pointed out the possibilities but also limitations of this approach, and appropriate uses. Next, guest speaker Nozomi Ando (Princeton) gave her data processing tutorial using the ATSAS package while the class followed her step-by-step on their personal laptops. She covered a range of topics, not limited to data quality assessment, Guinier and Kratky analysis, Pair-distance distribution analysis and bead modeling through simulated annealing. The subsequent lecture by Alvin Acerbo (MacCHESS) covered the fundamentals and benefits of incorporating size exclusion chromatography, multi-angle and dynamic light scattering in a BioSAXS experiment, in an in-line configuration.

The final lectures of the day started with Steve Meisburger’s (Princeton) discussion of his pioneering work on time-resolved BioSAXS performed right here at CHESS. Steve also gave examples of how sucrose can be used as a nifty trick to turn off the scattering contribution from protein in protein-DNA complexes, effectively making only the DNA visible. Kushol gave his second lecture afterwards on atomistic modeling using scattering data from either x-ray or neutron based experiments with X-ray crystallographic information. He highlighted the computational tool SASSIE and its application to ensemble analysis. The final talk was by Richard, who concluded with clear pointers and guidelines for publishing scattering data.

The second day of the workshop began with a hands-on tutorial by Alvin Acerbo on processing of scattering data from inline HPLC-SAXS experiments using the US-SOMO software package and sample scattering data taken at G1. The tutorial covered several advanced topics, including data quality assessment and global fitting strategies. During the remainder of that and the subsequent day, workshop participants were given 6-hour slots on the BioSAXS stations at beamlines G1 and F1 to become familiar with basic BioSAXS data collection procedures. Besides performing BioSAXS experiments on protein standards supplied by the workshop, several participants brought their own sample for analysis.

Submitted by: Alvin Acerbo, MacCHESS, Cornell University 12/04/2014

Crystallography at A1: new detector for a new beam

A Pilatus3 6M pixel array detector has replaced the Q-210 CCD detector in the A1 hutch. This is the preferred detector worldwide for macromolecular crystallography, because of its large dynamic range, zero background, very small point spread function, and millisecond readout time.

The fast readout allows data to be collected in “shutterless” mode, meaning that the shutter remains open during a large-angle rotation, e.g. 180°, of the crystal, while the detector records a series of images. This avoids the overhead, mechanical wear, and small inaccuracies in starting and ending points of the many short, small-rotation exposures that must be used with a detector that takes longer to read out.


The X-ray energy at A1 is currently 19.6 KeV (corresponding to a wavelength of 0.63 Å), due to the use of a diamond monochromator and constraints on where the beam must be positioned in the hutch. At this energy, diffraction spots produced by crystals with unit cell dimensions of a few hundred Ångstroms are only a few pixels apart on the detector, even when it is as far back as possible, and the small point spread function of the Pilatus is critically important in preventing strong spots from “blooming” and contaminating neighboring spots.

The first undulator feeding A1 for the beginning of the Fall 2014 run is a 30-cm prototype, which produces much less flux than the 1.5 m device to be installed later in the run, and the X-ray beam coming off the monochromator is currently unfocused. Hence the beam through the A1 collimator is weaker than it was in the past and will be in the future, and the zero background due to the detector is advantageous in detecting the weak diffraction from small and/or poorly ordered crystals.

The figure shows diffraction from a crystal of moderate quality, collected by the first outside user of the new A1 setup. An entire 400-image data set was collected in 7 minutes. Processing statistics were as expected for this type of crystal. Once a longer undulator is in use, data collection times will drop even further.

Submitted by: Marian Szebenyi, MacCHESS, Cornell University 10/31/2014

CRISPR Cas3 structure

Within the past decade it was discovered that many cells have an adaptive immune system to fight off foreign RNA or DNA which may have been inserted by a viral, plasmid or transposon attack. In bacteria, this system is known as CRISPR (Clustered Regularly Interspaced Palindromic Repeats, see CHESS news item: Adaptive Immune Systems of Bacteria, 2012) and consists of RNA which recognizes and binds the foreign DNA or RNA through an RNA-mediated interference mechanism and a number of Cas (CRISPR-associated) proteins which help to either form the complex or to degrade the foreign DNA. The complex is known as CASCADE (CRISPR-Associated Complex for Antiviral Defense).

CASCADE complex

Illustration 1: DNA in yellow, iron atoms in orange, proteins domains in various colors

There are a number of variations of the CRISPR system in assorted types of bacteria; the current work concerns CRISPR type I. This type includes the Cas3 protein which serves both as a helicase to unwind DNA and a nuclease to chop it up. A couple of structures of Cas3 have been solved previously, but the current study by the Ailong Ke laboratory of Cornell University and an international team of collaborators is more complete in that the crystallographically resolved portion includes both the nuclease and helicase domains of Cas3, as well as a 12 nucleotide piece of single-stranded DNA which inhabits the substrate site, and a pair of metal atoms (identified as iron in the current structure by the XAFS technique) which may serve to effect the cleavage of the target DNA.

Thus, the current structure provides new information about how the Cas3 protein is recognized by the CASCADE complex, how the nuclease preferentially receives single-stranded DNA from the helicase, and how the degradation of DNA proceeds. The Ke group solved a number of variant structures with different forms of adenosine nucleotide (ATP, ADP, AMP-PNP) to help resolve the reaction cycle of the helicase. They also performed a number of site-directed mutation studies to glean more information about important residues. The Ke group collected diffraction data on their crystals both at CHESS beamline A1 and at APS NECAT beamline 24-ID-C.

CASCADE, nuclease active site

Illustration 2: The two metal atoms in the nuclease active site

Detailed information about bacterial defense systems may have considerable medical and agricultural importance. Bacteriophages and plasmids have potential for use in bacterial control, and overcoming the defense systems bacteria use to fight them off would make the control agents more effective.


Yanwu Huo, Ki Hyun Nam, Fang Ding, Heejin Lee, Lijie Wu, Yibei Xiao, M Daniel Farchione Jr, Sharleen Zhou, Kanagalaghatta Rajashankar, Igor Kurinov, Rongguang Zhang & Ailong Ke. Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation, Nature Structural & Molecular Biology 21, 771–777 (2014). doi:10.1038/nsmb.2875

Protein Data Bank entries:
4QQW CRISPR Cas3 from Thermobifida fusca with bound DNA 12-mer
4QQX -- complex with ATP
4QQY -- complex with ADP
4QQZ -- complex with AMP-PNP

Submitted by: David J. Schuller, MacCHESS, Cornell University 2014-09-09


Key to pathogenic slime uncovered

In the biofilm regulation pathway of Pseudomonas fluorescens and similar bacteria, high phosphate levels lead to the creation of messenger molecule cyclic-di-guanosin monophosphate (CDGMP). cdGMP activates key regulatory protein LapD through its cytoplasmic domains. LapD then, through its periplasmic domains, binds to enzyme LapG, which keeps LapG from breaking down the molecule responsible for the biofilm assembly, the elastin LapA.

protein LapD

A few years ago the Sondermann group used MacCHESS facilities to solve several molecular structures of cytoplasmic domains of the LapD regulatory protein from Pseudomonas fluorescens, alone and in complex with signaling molecule cdGMP. (Navarro, et al., 2011 DOI 10.1371/JOURNAL.PBIO.1000588)

They also used MacCHESS facilites to solve several structures of the LapG enzyme. (Chatterjee, et al. 2012, 10.1128/JB.00640-12)

For the most recent publication, the Sondermann group returned to MacCHESS to solve structures of the periplasmic domain of protein CdgS9 from Legionella pneumophila (an analog of Pseudomonas LapD) both alone and in complex with LapG. These latest structures reveal much detail about the key binding interface between these proteins.

Read the Cornell Chronicle article here:

Submitted by: David Schuller, CHESS, Cornell University 09/09/2014