Skip to main content


2019 METRIC NMR and X-ray Crystallography Workshop

May 22, 2019 Talley Student Union – Room 3285

8:15 AM     Registration

8:30 AM     Welcome.

8:45 AM     Dr. Andrew N. Lane, Center for Environmental and Systems Biochemistry, University of Kentucky

Stable Isotope Resolved Metabolomics for Cancer Biochemistry- the NMR View

Stable Isotope Resolved Metabolomics (SIRM) is an approach that determines isotope and istopologue distributions downstream of an enriched precursor administered to the cells, tissue or organism. This enables reconstruction of pathway utilization and response to external perturbations, and provides the raw material for flux analyses. Despite its modest sensitivity, NMR has several desirable features including the ability to determine structures, atomic level details of labeling, isotope editing capabilities and accurate quantification in unfractionated mixtures. New developments in multiple labeling and isotope editing schemes will be presented, in the context of cancer biochemistry.

9:45 AM     Dr. Robert London, NMR Group Leader, Genome Integrity and Structural Biology Laboratory. National Institute of Environmental Health Sciences

Structural Maturation of HIV-1 Reverse Transcriptase—A Metamorphic Solution to Genomic Instability

There has been a steadily increasing appreciation of the fact that the relationship between protein sequence and structure is often sufficiently ambiguous to allow a single sequence to adopt alternative, stable folds. Living organisms have been able to utilize such metamorphic proteins in remarkable and unanticipated ways. HIV-1 reverse transcriptase is among the earliest such proteins identified and remains a unique example in which a functional heterodimer contains two, alternatively folded polymerase domains. Structural characterization of the p66 precursor protein combined with NMR spectroscopic and molecular modeling studies have provided insights into the factors underlying the metamorphic transition and the subunit-specific programmed unfolding step required to expose the protease cleavage site within the ribonuclease H domain, supporting the conversion of the p66 precursor into the mature p66/p51 heterodimer. In the human immunodeficiency virus, the use of a metamorphic reverse transcriptase provides an effective strategy for minimizing the size of its relatively unstable RNA genome while maintaining functionality.

10:30 AM  Break

10:45 AM  Dr. Flora Meilleur, Molecular & Structural Biochemistry, North Carolina State University & Neutron Sciences Directorate, Oak Ridge National Laboratory

Toward Elucidating The Mechanism Of Lytic Polysaccharide Monooxygenases: Chemical Insights from X-ray and Neutron Crystallography

Fungal lytic polysaccharide monooxygenases (LPMOs) have been intensely studied since their first characterization in 2010 as a unique class of copper enzymes capable of oxidizing carbohydrates. Academic and insdutrial research efforts are conducted worldwide to understand how LPMOs accelerate the breakdown of recalcitrant cellulose. The Meilleur lab combines insights from X-ray and neutron cystallographic structures and computational analyses to elucidate the enzymatic mechanism of LPMOs. LPMOs require the input of two electrons and of one oxygen molecule (O2 or H2O2) to achieve hydroxylation of one carbon in the glycosidic bond. Our lab focuses on three poorly understood aspects of the LPMO’s reaction mechanism: 1) What are the structural determinants of O2 and H2O2 binding? 2) How do conserved second shell residues contribute to activity? 3) Does the O2 based mechanism follow a superoxyl, hydroperoxyl or oxyl catalytic pathway? The ability to pinpoint hydrogen atoms to determine protonation states at and around the active site through the catalytic pathway is key to decipher the chemistry catalyzed by LPMOs. To achieve this, we combine high resolution X-ray and neutron protein crystallography to deliver precise, all atom structures of key reaction intermediates that can reveal i) the positions and interactions of all hydrogen atoms in the enzyme, ii) atomistic details of the active site without perturbing the metal oxidation state, and iii) the chemical nature of the activated dioxygen species coordinated to the active site copper. In this talk, we will discuss our recent X-ray and neutron crystallographic studies that provide new insights into the LPMO mechanism.

11:30 AM   Dr. Mark Wilson, Redox Biology Center & Department of Biochemistry, University of Nebraska

Combining old and new crystallographic approaches to characterize enzyme dynamics during turnover

A vast number of enzymes rely on cysteine modification for function. Transient covalent modification of enzyme active sites during catalysis can be coupled to catalytically important conformational changes. In this talk, I will describe our work using conventional and serial mix-and-inject X-ray free electron laser (XFEL) crystallography, computer simulations, and enzyme kinetics to show that the modification of the active site cysteine residue in isocyanide hydratase (ICH) triggers a cascade of conformational changes that are important enzyme turnover. XFEL crystallography allows the direct visualization of the proposed thioimidate intermediate and the resulting enzyme conformational variabilty, validating the ICH mechanism. As most enzymes that form catalytic intermediates will experience similar transient changes in active site interactions, modification-gated conformational dynamics may be a widespread and underreported means for regulating protein conformational dynamics.

12:30 PM  LUNCH

1:30 PM     Break Out Workshops

NMR Metabolomics: Nuclear magnetic resonance (NMR) spectroscopy are and mass spectrometry (MS) the two leading analytical approaches to examine metabolomics. While each approach has its strengths and limitations, NMR-based methods are generally less well-known. This session will focus on the hands-on aspects of NMR-based metabolomics. Using pre-made samples, you will learn how to collect a 1D 1H spectrum on a Bruker spectrometer and analyze the data using Chenomx (Polk 50).

Cambridge Structural Database: Cambridge Structural Database (CSD) is a world-wide repository for small-molecule organic and metal-organic crystal structures hosted by Cambridge Crystallographic Data Center (CCDC).  NC State has a campus-wide site license for CSD-Enterprise, which provides access to the CSD and all CCDC application software. This hands-on workshop will provide participants the opportunity to explore this powerful software suite (Dabney 120).

Introduction to Macromolecular Crystallography: Participants will be provided an overview of macromolecular crystallography and then focus on CrysAlisPro, a data collection and data processing software for macromolecular and small molecule crystallography. Designed around an easy-to-use graphical user interface, CrysAlisPro can be operated under fully automatic, semi-automatic or manual control. This workshop will demonstrate data collection, assessment of crystal quality, data reduction and processing. As time permits, advanced strategies for challenging samples will be discussed, The workshop will primarily be focused on protein samples (Polk 1).

Click Here to Register (Deadline Passed: Friday, May 17, 2019)