Michael H. B. Stowell, PhD - US grants

This proposal to acquire a nanoindentor system from MTS corporation to support exciting and diverse
research and training at the intersection of nano and microsystems technology. In its
base configuration, the load resolution is 50 nN and the displacement resolution is 0.01 nm. These can be
significantly lowered to 1 nN and 0.0002 nm, respectively with an additional indentation head. The
nanoindentor is typically used to indent (push the tip into) a material while simultaneously measuring the
load and displacement. The concept is common to many techniques in materials characterization, except
that the mode of deformation is extremely complicated. However, with suitable analysis, typically via the
finite element method, the measured load vs. displacement curve can be inverted to infer a host of
important material properties. Because the measurement area is so small (on the order of 100s of nms),
the instrument is ideally suited to obtain quantitative information regarding mechanical properties as a
function of position in complex material systems and structures.
The nanoindentor will be the heart of a unique micro/nanomechanical characterization facility at the
University of Colorado (CU). It will greatly impact numerous diverse research efforts that are currently
supported including applications in the characterization of advanced materials, experimental mechanics of
micromechanical structures and devices, studies of biological materials/systems ranging from protein/cell
structure up to full arteries, and as a nanomanufacturing tool for the development of nanoscale circuits
using biomolecular templates.
Initially the nanoindentor will be used by the groups of 12 faculty members, spanning six
Departments and three Colleges at CU. It will be used to support research projects and instructional
activities at the graduate and undergraduate levels. A number of activities are planned to increase our
user base and the overall level of diverse expertise with the instrument. These include: i) the development
of a Micro/nanosystems Forum in which two types speakers will be solicited: those with expertise in
nanoindentation and related fields, and those with interesting research activities that may be able to make
use of the nanoindentor; ii) the development of a website devoted to activities using the nanoindentor; iii)
dissemination of capabilities and results, along with the solicitation of potential collaborative users at
semiannual meetings of our NSF Industry/University Cooperative Research Centers (CAMPmode and
MAST); iv) a user-fee structure that requires users to disseminate their results to our user community; and
v) a plan to recruit new users, particularly underrepresented groups and women pursuing advanced
degrees.

DESCRIPTION (provided by applicant): This proposal aims to minimize the three bottlenecks of membrane proteins structure determination with a novel approach that combines high throughput electron microscopy single particle analysis with a novel self-assembling method. Validation of this approach will be realized by application to the structural analysis of several proteins which represent major categories of eukaryotic membrane proteins of biomedical importance. The overall objective of this proposal will be determining their structures and to simultaneously fully develop this approach to tackle further membrane protein targets in our own and other laboratories. The majority of known human pharmaceutical targets are membrane proteins and the future success of structure based drug design efforts will rely heavily on membrane protein structural information. This proposal will minimize the three bottlenecks of membrane proteins structure determination with a novel approach that combines high throughput electron microscopy single particle analysis with a novel self-assembling method. The success of this project will lower the barrier for obtaining the needed information for structure based drug design to proceed for a wide range of diseases.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Several ion channels are being studied with the plan of determining the molecular mechanism of their action. These include voltage gated channels responsible for propagation and termination of action potentials, calcium channels involved in signal amplification and ligand gated ion channels involved in signal detection and modulation. Using rapid affinity purification methods, along with x-ray crystallography and electron microscopy, our goal is to elucidate the structural elements of these channels in various state. Systems of current interest include the prokaryotic mechanosensitive channels (Msc), including those of large (MscL) and small (MscS) conductance that couple channel gating with membrane tension. We have begun to collect cryo-tomograms of membrane preparations of prokaryotes that contain mechanosensitive channels and are applying 3D volume averaging using out PEET software program.

This Major Research Instrumentation (MRI) award is awarded to the University of Colorado for the development of a novel ultrastable atomic force microscope (US-AFM) designed to address the pressing need in structural biology for an atomically precise tool to study protein structure and conformational dynamics. Atomic force microscopy (AFM) is limited by unwanted mechanical drift between the tip and sample. This limitation is exacerbated by environmental perturbations, which occur in biologically relevant conditions (room temperature in liquid). To be broadly applicable, operation and control of US-AFM needs to be user-friendly, robust, and reliable for room temperature operation in liquid. The resulting instrumentation allows the atomic sensitivity of AFM to be fully exploited for a range of disciplines, including biology, physics, chemistry, and nanotechnology. Student participation in this project is an outstanding research and educational experience because of its multi-disciplinary nature. Results from these studies will be presented by students and faculty at regional or national meetings, and published in peer-reviewed journals.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our research is focused on molecular and supramolecular structures that facilitate communication between neurons at the chemical synapse and how such structures are perturbed in neurological disease. We are particularly interested in the architectural arrangement of signaling molecules and enzymes, and characterizing the ways in which such molecular assemblies are formed and undergo changes during synaptic transmission and modulation. Our approach is to investigate individual proteins using x-ray and electron crystallographic methods and to combine this information with EM images obtained via 3-D reconstruction of supramolecular assemblies and tomographic analysis of the intact chemical synapse. Our long-term goal is to construct a dynamic molecular and architectural map for the chemical synapse that will help to understand synaptic formation, transmission and plasticity. Using electron tomographic methods we have begun to study the architecture of the chemical synapse in cultured neurons. Our first goal is to establish the common architectural elements present at the synapse and to identify the molecules involved using specific antibody labeling or genetic tagging. Subsequently, we will perform field potential stimulations coupled with cryogenic trapping to investigate the dynamic processes involved in synaptic transmission. Ultimately we plan to study long-term, stimulation dependent, synaptic changes in the hopes of gaining insight into the architectural elements underlying synaptic plasticity. FUNDING

DESCRIPTION (provided by applicant): This proposal aims to study the structure and function of membrane proteins using a novel approach that combines a microfluidic membrane self-assembling method with high throughput electron microscopy single particle analysis. We will leverage our previous work to realize a systematic approach to structural analysis of membrane proteins in a biological membrane. The overall objective of this proposal will be determining the structures of several membrane proteins both in the absence and presence of ligands and transmembrane gradients that alter the activity of the membrane protein.

Summary: We will establish a satellite service center within a larger network according to RFAs RM-19-009 and -010, named the CU-Boulder Center for Cryo-Electron Tomography (CCET). Our satellite will focus on cryo- specimen preparation and cryo-electron tomography (cryo-ET) applied to cells, organelles and large supramolecular assemblies. The applicants combine the expertise of three P.I.s, Michael Stowell (high- resolution cryo-electron microscopy (cryo-EM)), Karolin Luger (biochemistry of large complex assemblies, especially protein-nucleic acid interactions), and Andreas Hoenger (former P41 director, cryo-EM and cryo-ET from macromolecules to cells and tissues). With our current staff, we combine extensive expertise with cryo-ET and relevant software. We will offer a broad range of cryo-electron microscopy (cryo-EM) and cryo-ET preparation procedures for cellular structures and intracellular macromolecular assemblies, as well as training opportunities for scientists who would like to establish these technologies, or at least part of them, for their home labs. Here in Boulder, we have several high-end microscopes available, including a newly installed Titan-Krios. Three of our microscopes are equipped with direct electron detectors. In addition, we have a comprehensive periphery for cryo-specimen preparation, storage, and handling resources, including computational tools. We will add cryo-Focused Ion Beam (cryo-FIB) milling to produce vitrified lamellae of cells and tissues. We propose the following organization of the facility: Specific Aim 1: Cryo-ET specimen preparation methods offered for service: Our technology and expertise will be made accessible for our users and for further collaborations with the data collection hub proposed in a parallel RFA (RM-19-010). Specific Aim 2: Cross-training opportunities for service users: We will offer comprehensive training for users, including one-on-one instruction on microscopes and related equipment. As appropriate, we will pool users into small groups and run a training week as a hands-on workshop on equipment as well as related software developed by our facility such as Serial-EM, IMOD and PEET. While the emphasis with training will be on practical aspects of specimen preparation and data collection, we often encounter novice users in strong need for a thorough introduction into image analysis software as well. Specific Aim 3: Administration and organization of the facility: Users, assigned to us via the network-wide application process will have the opportunity to participate in the specimen refinement process, and receive training on the cryo-ET processes involved. Projects with high potential will be referred for high-throughput data collection to the central hub facility of the network. Boulder, Colorado is centrally located and well-connected nationwide through Denver International Airport.