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Controlled 3D Condensation of Proteins to Avoid Air-water Interface Challenges for High-resolution Single Particle Cryo-electron Tomography

Award Number: R21GM164827
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 08/01/2026
PERIOD OF PERFORMANCE END DATE: 07/31/2028

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Controlled 3D Condensation of Proteins to Avoid Air-water Interface Challenges for High-resolution Single Particle Cryo-electron Tomography - Project Summary/Abstract Cryo-electron microscopy (cryo-EM) is pivotal for revealing the molecular architecture of life, yet its application to many biological systems is frequently hampered by persistent sample grid preparation challenges, including preferred orientations and compositional/conformational heterogeneity caused by air-water interface (AWI) damage. This project is based on the core hypothesis that by forming 3D protein condensates of 200-300 nm in thickness, these AWI challenges can be overcome, as only the surface layers are exposed while the majority of interior proteins remain protected from AWI. By analyzing these condensates with cryo-electron tomography and sub-tomogram averaging (cryo-ET/STA), high-resolution 3D structures can be obtained, an approach we term NAP3D (No AWI Problems in 3D). This Technology Development R21 project will develop and validate a robust toolkit to achieve the NAP3D goal. Aim 1 will develop and validate 'Chemically Induced Condensate AWI- Shielding' (CICA) technologies suitable for all proteins. This aim explores two distinct chemical/biochemical strategies: (i) inducing protective phase separation via controlled solubility modulation using common laboratory reagents such as PEG and ammonium sulfate, and (ii) constructing affinity-mediated condensates based on multivalent biotin-streptavidin network interactions. Proteins will assume random orientations in the condensate to fully cover all views for isotropic quality reconstructions. Aim 2 will engineer and validate 'Plug-and-Play' Genetically Encoded Platforms for more defined 3D protein network formation. This aim will create condensates using Strep-tagged target proteins and multivalent binding to streptavidin, primarily through a symmetry- dependent, dual Strep-tagging approach where N- and C-termini are engineered with affinity tags. For geometrically challenging proteins where this dual Strep-tagging is incompatible with 3D network formation, this strategy incorporates an innovative solution: replacing one tag with a self-assembling D2-symmetric scaffold (e.g., p53tet) to create defined ‘molecular nodes’, providing a flexible and powerful toolkit for organizing proteins in more defined relative orientations to facilitate subtomogram orientation search and averaging for challenging target proteins. The efficacy of these strategies in preserving protein structure and their potential for high- resolution reconstructions will be rigorously validated by cryo-ET/STA using multiple test samples of various sizes, symmetries, and AWI issues. Successful development of the NAP3D approach will deliver a transformative toolkit to overcome AWI challenges, empowering the scientific community to apply these tools to a broad spectrum of challenging biomedical targets and significantly expanding the frontiers of structural biology.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $432,575 )
2026	2026	THE PENNSYLVANIA STATE UNIVERSITY	201 OLD MAIN	UNIVERSITY PARK	PA	16802	CENTRE	USA	Biomedical Research and Research Training	000	1	7/30/2026	NEW	$432,575
														Subtotal = $432,575

Grand Total All Awards = $432,575

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Controlled 3D Condensation of Proteins to Avoid Air-water Interface Challenges for High-resolution Single Particle Cryo-electron Tomography

Award Number: R21GM164827

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 08/01/2026

PERIOD OF PERFORMANCE END DATE: 07/31/2028

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