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Application of the SPREAD method to XFEL serial crystallography for the spatial and temporal resolution of metalloenzyme electronic structures

Award Number: F32GM161078
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 02/15/2027
PERIOD OF PERFORMANCE END DATE: 02/14/2028

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Application of the SPREAD method to XFEL serial crystallography for the spatial and temporal resolution of metalloenzyme electronic structures - Project Summary Metalloenzymes are crucial in mediating biological processes. Catalysis is controlled by precise movement of protons and electrons at active sites, which are often coordinated with ligand binding, side-chain movement, or larger protein conformational changes. To fully characterize enzyme mechanisms, simultaneous interrogation of protein structure and the metal oxidation state of each atom in their metallocofactors is required. A key motivation of this proposal is to understand the mechanism of Photosystem II (PSII), where the coordinated movement of substrate and electrons within a Mn4CaO5 cofactor catalyzes biological water oxidation. These research questions can be addressed by X-ray crystallography by analyzing the anomalous scattering of each metal atom, even for metallocofactors which contain multiple copies of the same element. The spatially resolved anomalous dispersion (SPREAD) method depends on the wavelength dependent behavior of scatterers around their absorption K-edge. With a tunable and monochromatic X-ray source with a resolution of <1 eV, scattering factors can be assessed by obtaining partial datasets in energy increments along the absorption edge, which yields the oxidation state of each atom. However, these measurements are obtained for static crystal structures at cryogenic temperatures, and thus, this method lacks the ability to follow metal oxidation states through catalysis. Time-resolved crystallographic methods using X-ray free electron lasers (XFELs) could provide spatial and temporal resolution of oxidation states, but adapting SPREAD for use with XFELs is inherently challenging due to the shot-to-shot variability and the ~30 eV bandwidth of an XFEL pulse. This proposal will translate SPREAD to XFELs by using the intrinsic pulse energy distribution to collect full datasets without monochromatic X-rays. Then, energy contributions to each pixel contributing to Bragg spots are modeled, allowing structure factors to be extracted. The resulting software will enable time-resolved SPREAD serial crystallography experiments using reaction triggers. This technology will allow the isolation of transient catalytic intermediates, including the four metastable intermediates in the catalytic cycle of PSII, and the elusive S4 state where O-O bond formation occurs. With SPREAD, we will be able to definitively assign oxidation states to each Mn in the Mn4CaO5 cluster to determine whether the catalytic cycle proceeds through a high-valent or low-valent paradigm, and potentially if a high-valent Mn(V) or Mn(IV)-oxyl is utilized during the O-O bond formation step. We will generalize the method for different types of metalloenzymes relevant to human health, starting with class I di-iron ribonucleotide reductases which generate the pool of dNTPs for DNA synthesis and repair. Precise electron flow is critical in these enzymes, and SPREAD would give direct evidence of the oxidation states of the iron atoms in the active site during catalysis. This proposal will enable temporal and spatial resolution of metal electronic structures in metalloenzymes, advancing our understanding of their function in biology, health, and the environment.


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 = $76,300 )
2026	2026	THE REGENTS OF THE UNIVERSITY OF CALIFORNIA	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	000	1	8/12/2026	NEW	$76,300
														Subtotal = $76,300

Grand Total All Awards = $76,300

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Application of the SPREAD method to XFEL serial crystallography for the spatial and temporal resolution of metalloenzyme electronic structures

Award Number: F32GM161078

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 02/15/2027

PERIOD OF PERFORMANCE END DATE: 02/14/2028

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