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Enabling membrane protein structural analysis: Tools for capillary diffusion crystallization and remote in situ diffraction experiments

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 1R41GM139420-01
Agency Tracking Number: R41GM139420
Amount: $239,206.00
Phase: Phase I
Program: STTR
Solicitation Topic Code: 400
Solicitation Number: PA19-270
Timeline
Solicitation Year: 2019
Award Year: 2020
Award Start Date (Proposal Award Date): 2020-09-07
Award End Date (Contract End Date): 2021-03-06
Small Business Information
7400 VAN CAMP RD
Girard, PA 16417-8312
United States
DUNS: 831189571
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 RICHARD HOWELLS
 (716) 483-3276
 richard@crystalpositioningsystems.com
Business Contact
 RICHARD HOWELLS
Phone: (716) 483-3276
Email: richard@crystalpositioningsystems.com
Research Institution
 UNIVERSITY OF ALABAMA IN HUNTSVILLE
 
301 SPARKMAN DRIVE, SKH 229
HUNTSVILLE, AL 35899-0001
United States

 Nonprofit College or University
Abstract

Project SummaryThis proposal is to develop an integrated capillary counter diffusion-based approach to in situ collection
of macromolecule diffraction data. The method to be developed will enable both cryogenic and ambient
temperature data collection, with the latter being critical to structure-based drug design. The approach is
based on a capillary that is pre-mounted in a holder that facilitates its being moved from loading to crystal
growth to diffraction analysis without ever having to physically touch or directly manipulate the crystal. The
capillary is closed at one end and is filled by centrifugation, which method enables filling with very high
viscosity solutions. Crystallization is by capillary counter diffusion (CCD), a method that often results in the
growth of crystals, often multiple crystals, that fill the internal diameter of the capillary.The immediate research goals of this effort are the development of and improvements to the mounted
capillary design. These are more specifically focused on the capillary holder design, how the capillary is mated
to the holder, the lower size limits for the capillary internal diameter that can be employed, the capillary materials
that can be used, and the methodology by which the capillary is secured to the holder. Different solution holder
designs, by which the mounted capillary is introduced to the precipitant solution for crystallization, will be
investigated, as well as the holder used for centrifugally filling the capillary. As the designs are implemented
they will then be tested by the growth and subsequent diffraction of soluble and integral membrane protein
crystals.This proposal is a collaborative effort between the PI, Mr. Richard Howells of Crystal Positioning Systems,
Dr. Marc Pusey of the University of Alabama in Huntsville, and Dr. Aina Cohen of Stanford Synchrotron Radiation
Laboratory. RH will be responsible for all machine shop work, fabricating parts to designs as suggested by MP
and AC. MP will be responsible for assembly of the mounted capillaries, preparation of the proteins to be used
as test materials, setting up the crystallization experiments, and all functional testing of the hardware developed.
AC will be responsible for all beamline-specific aspects of this effort; to test the mounted capillaries on the
beamline and to ensure their compatibility with sample exchange robots employed at synchrotron sources.
Diffraction data collection and data analysis will use the SSRL beamlines and computing infrastructure. Data and
structure analysis will be overseen by RH with input from MP and AC. AC and AH, together with MP, will provide
input to RH for improvements to the designs, and will then test those improvements as they are fabricated.Project NarrativeThis research directly addresses the ability to carry out structure-based drug design targeting
macromolecules, through development of a means to grow protein crystals and carry them through to X-ray
diffraction analysis without ever having to physically touch them. The methods to be developed are proposed
to be applicable to both soluble and integral membrane proteins, with diffraction data being collectable either in
situ and remotely at either room or cryogenic temperatures and with full rotational access.

* Information listed above is at the time of submission. *

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