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TitleMicrofluidic protein isolation and sample preparation for high-resolution cryo-EM.
Journal, issue, pagesProc Natl Acad Sci U S A, Vol. 116, Issue 30, Page 15007-15012, Year 2019
Publish dateJul 23, 2019
AuthorsClaudio Schmidli / Stefan Albiez / Luca Rima / Ricardo Righetto / Inayatulla Mohammed / Paolo Oliva / Lubomir Kovacik / Henning Stahlberg / Thomas Braun /
PubMed AbstractHigh-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often ...High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 μL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes.
External linksProc Natl Acad Sci U S A / PubMed:31292253 / PubMed Central
MethodsEM (helical sym.) / EM (single particle)
Resolution1.92 - 3.5 Å
Structure data

EMDB-4628: Cryo-EM structure of Tobacco Mossaic Virus from microfluidic grid preparation.
PDB-6r7m: Tobacco Mosaic Virus (TMV)
Method: EM (helical sym.) / Resolution: 1.92 Å

EMDB-4738: Endogeneous native human 20S proteasome with bound Fabs isolated from less than 1 ul cell lysate
PDB-6r70: Endogeneous native human 20S proteasome
Method: EM (single particle) / Resolution: 3.5 Å

Source
  • homo sapiens (human)
  • tobacco mosaic virus
KeywordsHYDROLASE / native / human / 20S proteasome / proteasome / cryo-EM / microfluidic / CryoWriter / protein isolation / endogeneous / VIRUS / tobacco / mosaic / TMV

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