Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins

Niebling, Stephan; Veith, Katharina; Vollmer, Benjamin; Lizarrondo, Javier; Burastero, Osvaldo; Schiller, Janina; Garcia, Angelica Struve; Lewe, Philipp; Seuring, Carolin; Witt, Susanne; García-Alai, María

doi:10.3389/fmolb.2022.882288

Cited by 19 publications

(13 citation statements)

References 57 publications

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“…The workflow was published previously. 74 NP was measured in 50 mM Tris (HCl), 150 mM NaCl, 5% glycerol, and a concentration of 25 nM. For the NP + RNA condition, NP at 500 nM was mixed with a single-stranded 9mer (Table S3) at a 4 molar-excess to the NP monomer.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

RNA to Rule Them All: Critical Steps in Lassa Virus Ribonucleoparticle Assembly and Recruitment

Sänger,

Williams,

et al. 2023

J. Am. Chem. Soc.

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Section: ■ Materials and Methodsmentioning

confidence: 99%

RNA to Rule Them All: Critical Steps in Lassa Virus Ribonucleoparticle Assembly and Recruitment

Sänger,

Williams,

et al. 2023

J. Am. Chem. Soc.

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“…Data were analyzed using the provided Discover analysis software. Masses were estimated by multi-Gaussian fitting using the online PhotoMol software (https://spc.emblhamburg.de/app/photoMol) 39 .…”

Section: Mass Photometrymentioning

confidence: 99%

BRCA2-HSF2BP Oligomeric Ring Disassembly by BRME1 Promotes Homologous Recombination

Ghouil

Miron

Sato

et al. 2023

Preprint

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In meiotic homologous recombination (HR), BRCA2 facilitates loading of the recombinases 2 RAD51 and DMC1 at the sites of double-strand breaks. The HSF2BP-BRME1 complex interacts 3 with BRCA2 to support its function in meiotic HR. In somatic cancer cells ectopically producing 4 HSF2BP, DNA damage can trigger HSF2BP-dependent degradation of BRCA2, which prevents 5 HR. Here we show that, upon binding to BRCA2, HSF2BP assembles into a large ring-shaped 6 24-mer consisting of three interlocked octameric rings. Addition of BRME1 leads to 7 dissociation of this ring structure, and cancels the disruptive effect of HSF2BP on cancer cell 8 resistance to DNA damage. It also prevents BRCA2 degradation during inter-strand DNA 9 crosslink repair in Xenopus egg extracts. We propose that the control of HSF2BP-BRCA2 10 oligomerization by BRME1 ensures timely assembly of the ring complex that concentrates 11 BRCA2 and controls its turnover, thus promoting meiotic HR.

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“…Similarly, combining native MS directly with cryo-EM via selective deposition using electrospray ionisation offers additional benefits of increasing the scope and throughput of structural determination [24][25][26]. Native MS can serve as a preliminary tool to screen for the best protein homologues and optimal purification conditions for subsequent interrogation by other structural techniques [27,28]. Additionally, native MS enables the detection of lipids bound to membrane proteins [29,30] -offering valuable insights into the critical role of lipids in membrane protein structures and functions.…”

Section: Introductionmentioning

confidence: 99%

Mass spectrometry of intact membrane proteins: shifting towards a more native-like context

Oluwole

Shutin

Bolla

2023

Essays in Biochemistry

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Integral membrane proteins are involved in a plethora of biological processes including cellular signalling, molecular transport, and catalysis. Many of these functions are mediated by non-covalent interactions with other proteins, substrates, metabolites, and surrounding lipids. Uncovering such interactions and deciphering their effect on protein activity is essential for understanding the regulatory mechanisms underlying integral membrane protein function. However, the detection of such dynamic complexes has proven to be challenging using traditional approaches in structural biology. Native mass spectrometry has emerged as a powerful technique for the structural characterisation of membrane proteins and their complexes, enabling the detection and identification of protein-binding partners. In this review, we discuss recent native mass spectrometry-based studies that have characterised non-covalent interactions of membrane proteins in the presence of detergents or membrane mimetics. We additionally highlight recent progress towards the study of membrane proteins within native membranes and provide our perspective on how these could be combined with recent developments in instrumentation to investigate increasingly complex biomolecular systems.

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Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins

Cited by 19 publications

References 57 publications

RNA to Rule Them All: Critical Steps in Lassa Virus Ribonucleoparticle Assembly and Recruitment

RNA to Rule Them All: Critical Steps in Lassa Virus Ribonucleoparticle Assembly and Recruitment

BRCA2-HSF2BP Oligomeric Ring Disassembly by BRME1 Promotes Homologous Recombination

Mass spectrometry of intact membrane proteins: shifting towards a more native-like context

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