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This is because these systems are easy to prepare and are often of small size, which is favorable for solution NMR spectroscopy. Usually detergents are employed for membrane protein preparation and detergent micelles are the most common media for structural investigations 1. A major part of this problem is the selection of an appropriate membrane-mimicking environment supporting both function and stability of a particular membrane protein. This is primarily due to difficulties of producing integral membrane proteins in folded and active forms as well as in sufficient yields. Membrane proteins only represent about 2% of all structures deposited in the protein data bank. While the focus of this protocol is on protein NMR, these nanodiscs can also be used for (cryo-) EM and small-angle X-ray and neutron scattering studies.ĭespite the high relevance of membrane proteins in health and disease, as well as drug design, little is known about the structures of this protein class. Depending on the target protein of interest, nanodisc assembly and purification can be achieved within 12 – 24h. We discuss critical points and pitfalls relating to optimizing nanodiscs for NMR spectroscopy and outline a strategy for the high-resolution structure determination and positioning of isotope-labeled membrane proteins in nanodiscs using NOESY spectroscopy, residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs). We describe specific isotope labeling schemes required for working with large membrane protein systems in nanodiscs and provide guidelines on the setup of NMR non-uniform sampling (NUS) data acquisition and high-resolution NMR spectra reconstruction.
Here, we present a protocol on the assembly of phospholipid nanodiscs of various sizes for structural studies of membrane proteins with solution-state NMR spectroscopy. We have recently constructed truncated versions of the membrane scaffold protein allowing the preparation of a range of stepwise smaller nanodiscs (6-8 nm diameter) to overcome this limitation. These have been used for a variety of functional studies, but structural studies by high-resolution solution-state NMR spectroscopy of membrane proteins in commonly-used nanodiscs of 10 nm diameter were limited by the high molecular weight of these particles causing unfavorably large NMR line widths. Phospholipid nanodiscs (formed when a membrane scaffold protein encircles a small portion of a lipid bilayer) have native-like membrane properties.
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These include fast and accurate phase-detection autofocus with both Face Detection and real-time Eye autofocus, both auto and manual ND filter settings for quick exposure compensation, and a 3.5" LCD monitor that boasts time saving touch screen access to key menu settings and versatile positioning for comfortable operating.Suitable membrane mimetics are crucial for performing structural and functional studies of membrane proteins. While the FX6 fits seamlessly into a pro-cine on-set workflow, it also offers several features that are a boon for a single-operator user.
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Weighing just under 2 lb, the FX6 body is a full 2 lb lighter than the FX9 and measuring 6 x 4.6", it packs a full-frame sensor into a body comparable to the S35-sensor Sony FS5.
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The FX6 camera combines a 4K full-frame Exmor R sensor with a BIONX XR engine for extremely sensitive capture in a range of settings using a base ISO of 800 as well as an ISO 12800 high-sensitivity mode for nuanced low-light scenes.
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With the ability to capture up to 15+ stops of dynamic range, Sony's S-Cinetone gamma for film like skin tones, and up to 10-bit, 4:2:2 XAVC-I recording, the FX6 is poised to both supplement your FX9 or VENICE capture and to nimbly take on documentary, gimbal, and drone shoots on its own. The FX6 Full-Frame Camera from Sony was developed to offer versatile, cine-style imaging in a truly compact form. Inside a compact body that's ready for grab-and-shoot action, the FX6 packs a highly sensitive, full-frame image sensor with the power to expand your moviemaking creativity. Sony’s soon-to-be-announced new Alpha camera will also be discussed in depth.Ĭlick DETAILS button at left to register. Band Pro’s Senior Technical Advisor, Randy Wedick, and Sony’s Digital Motion Picture Center Manager, Samuel Fares, will demo the new Sony FX6 in this live interactive Q&A.
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Join us on Zoom Wednesday 3/3 at 11:00 am PST for a free Sony FX6 Workshop.
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