In transmission electron microscopy (TEM), a high-energy electron beam is used to examine the structures of molecules, down to the level of atomic details. The electron beam passing through a very thin specimen projects a two-dimensional image of the sample onto the detector (a charge-coupled device; CCD or a direct electron detector; DED). Computational algorithms are used to align the two-dimensional image projections to generate a three-dimensional reconstruction which in turn is used for atomic model building. Cryogenic Electron Microscopy (Cryo-EM) is an approach that allows the observation of hydrated biological specimens in their native environment at cryogenic temperatures in TEM. Cryo-EM broadly encompasses three different approaches: electron crystallography, single-particle cryo-EM, and electron cryo tomography. Recent technological advancements in cryo-EM have ushered in a new era of structural biology enabling exclusive views of the complex biological molecules. Atomic details of biomolecules allows us to better understand physiological phenomena that govern life and aid in drug design and development.
CWRU cryo-EM facility houses state-of-the-art electron microscopes in a newly renovated space including FEI Titan Krios G3i 300 kV X-FEG TEM with BioQuantum K3, Phase Plate; an FEI Tecnai TF20 (200kV, FEG) transmission electron microscope with a 4k x 4k CMOS-based Tietz TemCam-F416 and a DE-20 direct electron detector, and FEI Tecnai G² Spirit (120kV) transmission electron microscope with a 4k x 4k Gatan US4000 CCD camera; The facility is set up to prepare flash-frozen samples for single particle, cryo-ET and crystallography analysis.
The Facility also offers complete conventional TEM services including sample processing, ultramicrotomy, film developing, and photographic printing, as well as immunocytochemistry. Intramural collaboration is available. We welcome trained EM users as well as researchers who wish to be trained.
Nuclear Magnetic Resonance (NMR) Spectroscopy is a very versatile technology for the characterization of structure and dynamics of small molecules as well as biological macromolecules in solution, but also in membrane mimicking environments, in solid powders or microcrystals.
The CCMSB building houses state of the art equipment primarily for solution NMR (Bruker 600, 800 and 900 MHz spectrometers), and a solid state capacity has been added to the Bruker 800 MHz spectrometer. A typical aqueous sample consists of a 200-500 μL solution with 10μM to > 1mM of the relatively pure molecule of interest. In optimal cases the technique can be used to obtain structural information up to 60-200 kDa molecular weight. Structure determination, characterization of molecular dynamics and protein-ligand or protein-protein interactions are more routine in the sub-30 kDa range. For most applications, it is necessary to obtain isotope-labeled forms of the molecule to facilitate the high- resolution spectral analysis (15N and/or 13C labelling). Thus most proteins are expressed in bacteria or other cells which can be grown in isotope enriched media.
Electron Paramagnetic Resonance (EPR) spectroscopy is an approach used for measuring long-range distances between paramagnetic centers (either naturally occurring or engineered) and/or coupled to nuclei within clinically relevant proteins, peptides, and nucleic acids at cryogenic temperatures. EPR is ideally suited for structural dynamic studies of large bio-macromolecular assemblies that include soluble proteins, membrane proteins, and protein-nucleic acid complexes. EPR spectroscopic applications span a wide range of areas, from nucleic acids and biomembranes, to protein research. This technology provides valuable information about biological processes critical for identification of therapeutic targets in human diseases.
In the last decade, pulsed-EPR methods have emerged as an exceptionally sensitive and versatile for quantifying conformational changes in large protein complexes in their native environment. Double electron-electron resonance (DEER) methods are sensitive to spin-spin distance from 15 to ~80 Å, which is suitable for studying conformational changes in large proteins and nucleic acid complexes. The macromolecule dynamics data obtained from pulsed-EPR are complementary to the high-resolution, yet static, information gained from X-ray crystallography and cryo-electron microscopy. Additionally, this approach is applicable for systems too large for NMR measurements.
Set up, in part, with NIH S10 instrumentation grant support, the EPR facility features a ELEXSYS E580 Q-Band pulsed-EPR spectrometer equipped with spinJet Arbitrary Waveform Generator, E580-AmpQ300 Q-band pulsed-300W TWT, ER5106QT-II resonator, Xepr software package for system control, acquisition control and data acquisition. Continuous wave-EPR spectrometer (Bruker EMX) is equipped with resonators (dielectric, super high Q, and loop-gap resonators), Bruker N2 temperature controller
Proteomics entails the in depth structural analysis of individual proteins in human and animal cells. In studying proteins and their changes, bioinformatics enables researchers to take an integrated -omics approach for discovering networks involved in human disease. The School of Medicine has established the Center for Proteomics and Bioinformatics to perform research to better understand the genetic and environmental bases of disease as well as provide new technologies to diagnose diseases such as cancer, heart disease, and diabetes.
New technologies in mass spectrometry are also allowing protein expression, localization, structure, post-translational modifications, and interactions to be studied in increasing detail and on a genome wide scale. The Center is also developing and applying state-of-the-art-structural proteomics technologies to understand the function and interactions of macromolecular complexes.
Macromolecular crystallography (MX), available at the FMX, AMX, and NYX beamlines, is the most fundamental technique of all approaches providing high-resolution structure and both local and global structural details. Footprinting (at XFP) and solution scattering (at LiX) are complementary solution biophysics methods well suited to studying structure and dynamics of large macromolecular complexes. In particular, MX can provide static high-resolution structural information, while footprinting gives structural insight into local conformational dynamics at the binding interfaces and solution scattering can provide structural details on the overall global conformation of the full complex. In addition, Cryo-EM is emerging as a very competitive technique for structural insight, with rapidly improving resolution capabilities. These structural probes, connected to laboratory-based methods and computational approaches, can provide a comprehensive picture of biological structure-function relationships relevant to a wide range of biomedical research problems.
Researchers - Focco van den Akker, Walter Boron, Sudha Chakrapani, Mark Chance, Chris Dealwis, Marcin Golczak, David Lodowski, Mike Maguure, Rajesh Ramachandran, Nami Tajima, Tsan Sam Xiao, Vivien Yee, Edward Yu
The Protein Expression Purification Crystallization and Molecular Biophysics Core (PEPCMBC) is a state-of-art laboratory that provides access to protein expression purification crystallization and molecular biophysical instrumentation. The Molecular Biophysical Core covers most current technologies for protein characterization and binding assay, and major instrumentation includes the following: 1) Biacore T200 (Cytiva, formerly GE healthcare) is suitable for affinity and kinetics analysis for biomacromolecules, including protein, DNA, RNA, whole cell, interacting with other biomacromolecules or ligands or small molecules. It is also suitable for high throughput screening from a kinetic perspective. 2) Tecan microplate reader M1000 (Tecan Inc.) is good for multi-channel absorbance, fluorescence, time resolved fluorescence (TRF), Fluorescence resonance energy transfer (FRET), fluorescence polarization (FP) and luminescence measurement. It also has an injector module that allows users to dispense reagents. The main purposes of this equipment are for primary and secondary screening, cell-based assays, receptor-ligand binding studies and other interaction assays etc. 3) MicroCal ITC-200 (Malvern Inc.) is an isothermal titration calorimeter that allows direct, label-free in solution measurement of binding affinity, stoichiometry and thermodynamics in a single experiment. 4) Microscale Thermophoresis (Nanotemper Technologies) provides the broadest application range detecting interactions between any type of biomolecules and for pM to mM binding affinities. 5) SEC-MALS (size-exclusion chromatography with multi-angle light scattering, Wyatt Inc.) allows the determination of absolute molecular weight and the amount of aggregation through gel filtration and miniDawn TREOS and Optilab rEX. 6) DynoPro NanoStar (Wyatt Inc.) measures the hydrodynamic radii and monodispersity of a biomolecular sample. Melting temperature of a sample can also be measured under temperature scan mode.
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