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@ARTICLE{Niebling:604257,
author = {Niebling, Stephan and Veith, Katharina and Vollmer,
Benjamin and Lizarrondo, Javier and Burastero, Osvaldo and
Schiller, Janina and Struve Garcia, Angelica and Lewe,
Philipp and Seuring, Carolin and Witt, Susanne and
García-Alai, María},
title = {{B}iophysical {S}creening {P}ipeline for {C}ryo-{EM} {G}rid
{P}reparation of {M}embrane {P}roteins},
journal = {Frontiers in molecular biosciences},
volume = {9},
issn = {2296-889X},
address = {Lausanne},
publisher = {Frontiers},
reportid = {PUBDB-2024-01055},
pages = {882288},
year = {2022},
note = {Part of this work was performed at the Cryo-EMFacility at
CSSB, supported by the UHH and DFG grantnumbers (INST
152/772-1|152/774-1|152/775-1|152/776-1|152/777-1 FUGG)},
abstract = {Successful sample preparation is the foundation to any
structural biology technique. Membrane proteins are of
particular interest as these are important targets for drug
design, but also notoriously difficult to work with. For
electron cryo-microscopy (cryo-EM), the biophysical
characterization of sample purity, homogeneity, and
integrity as well as biochemical activity is the
prerequisite for the preparation of good quality cryo-EM
grids as these factors impact the result of the
computational reconstruction. Here, we present a quality
control pipeline prior to single particle cryo-EM grid
preparation using a combination of biophysical techniques to
address the integrity, purity, and oligomeric states of
membrane proteins and its complexes to enable reproducible
conditions for sample vitrification. Differential scanning
fluorimetry following the intrinsic protein fluorescence
(nDSF) is used for optimizing buffer and detergent
conditions, whereas mass photometry and dynamic light
scattering are used to assess aggregation behavior,
reconstitution efficiency, and oligomerization. The data
collected on nDSF and mass photometry instruments can be
analyzed with web servers publicly available at
spc.embl-hamburg.de. Case studies to optimize conditions
prior to cryo-EM sample preparation of membrane proteins
present an example quality assessment to corroborate the
usefulness of our pipeline.},
cin = {CSSB-CF-PP / CSSB-LIV-KG / CSSB-CF-SPC / CSSB-CF-CRYO},
ddc = {570},
cid = {I:(DE-H253)CSSB-CF-PP-20210530 /
I:(DE-H253)CSSB-LIV-KG-20220525 /
I:(DE-H253)CSSB-CF-SPC-20210520 /
I:(DE-H253)CSSB-CF-CRYO-20210520},
pnm = {899 - ohne Topic (POF4-899)},
pid = {G:(DE-HGF)POF4-899},
experiment = {EXP:(DE-MLZ)NOSPEC-20140101},
typ = {PUB:(DE-HGF)16},
pubmed = {pmid:35813810},
UT = {WOS:000861787900001},
doi = {10.3389/fmolb.2022.882288},
url = {https://bib-pubdb1.desy.de/record/604257},
}