Academic Journal
Detailed Preparation of Ni-NTA Lipid Monolayer Affinity Grids for Cryo-EM Structural Analysis of Soluble Polyhistidine-Tagged Protein Complexes.
| Τίτλος: | Detailed Preparation of Ni-NTA Lipid Monolayer Affinity Grids for Cryo-EM Structural Analysis of Soluble Polyhistidine-Tagged Protein Complexes. |
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| Συγγραφείς: | Lenger C; Department of Biochemistry and Biophysics, UNC Chapel Hill School of Medicine., Yoon D; Department of Electrical and Computer Engineering, North Carolina State University., Franco IC; Department of Chemistry and Biochemistry, Division of Physical and Biological Sciences, University of California Santa Cruz., Skrajna A; Department of Chemistry and Biochemistry, Division of Physical and Biological Sciences, University of California Santa Cruz., Baker RW; Department of Biochemistry and Biophysics, UNC Chapel Hill School of Medicine; UNC Lineberger Comprehensive Cancer Center, UNC Chapel Hill School of Medicine; baker@med.unc.edu., Strauss JD; Department of Biochemistry and Biophysics, UNC Chapel Hill School of Medicine; Joshua_Strauss@med.unc.edu. |
| Πηγή: | Journal of visualized experiments : JoVE [J Vis Exp] 2026 Aug 04 (234). Date of Electronic Publication: 2026 Aug 04. |
| Τύπος έκδοσης: | Journal Article; Video-Audio Media; Research Support, N.I.H., Extramural |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: MYJoVE Corporation Country of Publication: United States NLM ID: 101313252 Publication Model: Electronic Cited Medium: Internet ISSN: 1940-087X (Electronic) Linking ISSN: 1940087X NLM ISO Abbreviation: J Vis Exp Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: [Boston, Mass. : MYJoVE Corporation, 2006]- |
| Ιατρικοί όροι (MeSH): | Histidine*/chemistry , Lipids*/chemistry , Cryoelectron Microscopy*/methods , Cryoelectron Microscopy*/instrumentation , Proteins*/chemistry , Nitrilotriacetic Acid*/analogs & derivatives , Nitrilotriacetic Acid*/chemistry , Organometallic Compounds*/chemistry, Nickel/chemistry |
| Περίληψη: | Cryo‑specimen preparation remains a major technical challenge for high‑resolution structural determination of protein complexes. Interactions at the air‑water interface can induce protein denaturation or preferred orientation, thereby limiting achievable resolution. In addition, dilute samples or low‑abundance targets often exhibit insufficient particle density, which hinders efficient data collection. Lipid‑based affinity grids provide an effective strategy to address these limitations. This article presents a step‑by‑step protocol for preparing Ni‑NTA lipid monolayer affinity grids for the structural analysis of soluble polyhistidine‑tagged protein complexes. The major stages of the protocol include preparing a lipid mixture, casting the lipid mixture onto the surface of a reservoir buffer, transferring the resulting lipid monolayer onto TEM grids, optionally drying and storing the lipid grids for up to 1 week, and cryo‑plunging the lipid grids with the sample. Successful implementation of this protocol yields cryo‑grids containing a single layer of well‑dispersed particles with improved particle density relative to grids prepared without a lipid monolayer. The procedure can be completed within a few hours in a laboratory equipped with a cryo‑plunger, a chemical fume hood, and standard tools commonly used for cryo‑specimen preparation. By combining detailed written instructions with video demonstrations, this work aims to improve the accessibility, reproducibility, and broader adoption of Ni‑NTA lipid monolayer affinity grids within the cryo‑EM community. |
| Grant Information: | R35 GM150960 United States GM NIGMS NIH HHS |
| Substance Nomenclature: | 4QD397987E (Histidine) 0 (Lipids) 26062-48-6 (polyhistidine) 0 (Proteins) KA90006V9D (Nitrilotriacetic Acid) 0 (Organometallic Compounds) 34831-03-3 (nickel nitrilotriacetic acid) 7OV03QG267 (Nickel) |
| Entry Date(s): | Date Created: 20260818 Date Completed: 20260818 Latest Revision: 20260917 |
| Update Code: | 20260917 |
| DOI: | 10.3791/71598 |
| PMID: | 42611978 |
| Βάση Δεδομένων: | MEDLINE |
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