Isolation and Characterization of Endogenous Nuclear Pore Complexes

Описание: Join us to discuss cutting-edge technologies and advances in the biomedical and life sciences!

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🔬 The characterization of native protein assemblies requires the production of a relatively pure sample that maintains the full complement, native organization, and function of that complex. This can be particularly challenging to achieve for large, multi-component, membrane embedded complexes using the traditional recombinant expression and reconstitution methodologies. However, using affinity capture from native cells, suitable whole endogenous protein complexes can be isolated.

In this webinar, I will present a method for the affinity isolation and characterization of baker's yeast (S. cerevisiae) nuclear pore complexes, which are ~50 MDa assemblies made up of 552 distinct proteins and embedded in a double-membraned nuclear envelope. Producing this sample allowed us to perform analyses to characterize the mass, stoichiometry, morphology, and connectivity of this complex and to obtain its integrative structure with subnanometer precision. We believe this methodology can be applied to other challenging protein complexes to produce similar results.

⏰ Timestamps
0:00 Introduction
3:35 About nuclear pore complexes (NPCs)
10:08 Structural determination of NPCs
13:08 Step 1: Isolating NPCs
30:53 Step 2: Verifying sample quality
34:00 Step 3: Gathering structural data
42:27 Step 4: Final integrative structure
43:47 Connecting structure to function
58:14 Summary and acknowledgements
Q&A
1:01:18 Choosing protease inhibitor mixtures
1:03:11 Protease cleavage
1:04:45 Cell number of stocking material for mass spec analysis
1:06:19 Do you have to tag the protein at the yeast genomic level?
1:08:22 Ideal protein concentration for cryo-EM analysis
1:10:20 Glycosylation in NPCs
1:11:15 Chemical crosslinkers
1:13:34 Focusing on more specific parts of the NPC
1:15:13 NPCs of cancer cells vs. normal cells
1:17:07 Is crosslinking done in vivo or in vitro?
1:18:39 Using other affinity peptides + reusing Dynabeads
1:20:57 Potential phosphorylation impacts on NPC structure
1:22:31 Genetically modifying haploid yeast
1:24:15 Regulating NPC pathways via drugs
1:25:50 How to co-express two interrupting proteins
1:26:36 Applications in mammalian cell lines/tissues
1:28:14 Getting an in situ model of the NPC
1:29:10 If you have any questions... + Closing remarks

🔧 Helpful resources:
National Center for Dynamic Interactome Research
Website: https://www.ncdir.org/
Twitter:   / intactome  
Webinar Q&A
https://bio-protocol.org/webinar/deta...

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The Bio-protocol webinar series highlights technical advances in biomedical and life science, focusing on a detailed presentation of cutting-edge technologies and techniques. We aim to make this program a go-to place for sharing, learning, discussing, and uncovering how to apply new technologies and tools in the life science community.

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