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Search in content. Search in excerpt. Search in posts. See more. Video player. Very fast, high quality and professional. Although the uptake rate of aspartate by ASCT2 CR was slower than that of glutamine by ASCT2 wt , the results confirm that a single point mutation in the substrate binding site was sufficient to switch the substrate specificity of ASCT2 from neutral to acidic amino acids.

Data points and error bars represent means and s. Small schemes depict proteoliposomes with internal and external compound compositions used in respective experiments. Source data are provided as a Source Data file. The quality of the cryo-EM map was sufficient to unambiguously model residues 43— and — Notably, despite different strategies employed during image processing, including 3D classifications with and without symmetry imposed on the trimer or single protomers, only a single conformational state was captured for all protomers, indicating a preferred state of the transporter in the given conditions Supplementary Fig.

Compared to the previously determined substrate-bound inward-occluded state of ASCT2 2 , the most prominent difference is the conformation of the loop connecting both helices of hairpin 2 HP2 loop. Similar movements of the HP2 loop have been observed on the extracellular side between the outward-occluded and outward-open states of EAAT1 and Glt Ph structures 15 , Although the remainder of the transport domain has virtually the same conformation as in the inward-occluded state Fig.

The scaffold domains are coloured in yellow, the extracellular antenna in red, the transport domains in blue, the HP2 loop in purple, and non-protein densities in grey see also Supplementary Fig. The membrane boundary is indicated and the location of the substrate binding site as shown in Fig. Substrate-binding site and movement of the HP2 loop. The lipid head group is positioned between the HP1 and HP2 loops and its negative charge appears to be stabilised by the helix dipole of hairpin helix HP2b.

Accessibility of the substrate-binding site and lipid densities. The open HP2 gate provides access to the binding site from the cytoplasm indicated by arrows. In the substrate-binding site we observe a patch of non-protein density close to R Although the density is too weak to allow for an unambiguous assignment, it most likely represents a TBOA molecule, although the occupancy of the site appears low Supplementary Fig.

This is surprising, as the binding of bulky inhibitors was required to obtain all of the reported outward-open states of EAAT1 and Glt Ph 15 , 19 , To exclude that the CR mutation itself induced the observed opening of HP2, we also investigated the conformational state of ASCT2 wt in absence of its substrate glutamine.

Albeit of lower resolution, the cryo-EM map of the substrate-free ASCT2 wt shows that the protein accommodates the same inward-open state, where the HP2 loop has moved Supplementary Figs. Thus, neither the presence of the inhibitor nor the point mutation itself is responsible for gate opening on the intracellular side, allowing the use of the higher resolved ASCT2 CR cryo-EM map for a detailed description of the inward-open state.

The characteristic shape of this density indicates that it most likely corresponds to a lipid molecule. The exact lipid identity is ambiguous at the given resolution, but the tentatively modelled diundecyl phosphatidylcholine fits well in the density Fig. Notably, the head group occupies the position where the HP2 loop is located in the inward-occluded state, indicating that the lipid would need to relocate for gate closure Fig.

Consistently, no lipid density was observed in the substrate-bound inward-occluded ASCT2 wt structure 2. Further unassigned densities were found between the transport and scaffold domains Figs. The density at the interface between HP2 and TM2 of the scaffold domain is located near the putatively bound cholesterols found in the inward-occluded ASCT2 structure 2 Fig. However, as the position of HP2 in the inward-open structure partly overlaps with the potential lipid densities found in the inward-occluded structure, the previously assigned lipids must relocate upon HP2 opening.

This putative lipid is adjacent to the phosphatidylcholine found at the tip of both hairpins described above Figs.

It might be responsible for the observed helix-rearrangement of TM1 of the scaffold domain, which is not directly interacting with HP2, when compared to the inward-occluded ASCT2 structure 2 Supplementary Fig. A large group of well defined, unassigned densities, which likely represent lipids or cholesterol molecules, is found in the cavity formed by TM5 of one protomer, and TM and the back of HP2 of the adjacent protomer Fig. Interestingly, one of these densities is located at a similar region as where the allosteric inhibitor UCPH binds EAAT1 19 , hinting at a potential regulatory role.

Finally, a density was found on the extracellular side of the scaffold domain. Zenger News. Live News. Besides a fun script and never-a-dull-moment directing, Jason Statham really deserves praise for h My Movies. Transporter 2. Confirm current pricing with applicable retailer. All transactions subject to applicable license terms and conditions. Depending on your client machine and network setup, editing and managing files stored on a remote DAV server can be almost as easy as editing and managing the files in your local directory.

Using Transporter you can specify success and failure directories to help you determine which packages succeeded and which packages failed.

In this example, when Transporter finishes executing the command, it returns eXtreme log information to the console. Transporter includes an embedded integration with the Aspera delivery software to allow fast delivery of your Store Packages to the iTunes Store, Apple Books, or App Store. To avoid the issue, you can create separate firewall rules to allow UDP activities for a longer period of time for Aspera users. For more detailed examples, including Transporter output and logging, see Aspera and Aspera on Windows.

When setting up a Signiant integration, if you have outbound and inbound firewall rules, you must open a direct outbound and inbound connection to Transmission Control Protocol TCP port and User Datagram Protocol UDP ports through To avoid the issue, you can create separate firewall rules to allow UDP activities for a longer period of time for Signiant users.

For more information, see Solving Problems. For more detailed examples, including Transporter output and logging, see Signiant , Signiant on Windows , and Signiant failing over to Aspera. For more detailed examples, including Transporter output and logging information, see Validate assets mode.

When you send a package to Apple for verification, Transporter parses the metadata file and verifies that the file is well formed and the data is correct, including dates, products, and more.

Transporter also validates all assets for example, audio files, video files, book files, an apps you send to Apple. For a more detailed example, including Transporter output and logging information, see Verify mode.

If an artist exists, instead of creating the new music artist, Transporter returns information about the existing iTunes Store music artist. Transporter also returns a force-creation token "1d82c6fef-3eac7cda" you can use to force Transporter to create the new music artist.

To force Transporter to create the new music artist, add -forceCreateToken "1d82c6fef-3eac7cda" to the command:. Transporter returns information indicating the metadata ticket was successfully created, and displays eXtreme logging information. In the following example, the user runs Transporter in diagnostic mode without specifying a specific delivery method. In this example, when Transporter finishes executing the command, it displays diagnostic information indicating a successful connection between your computer and the Apple web service and successful connections between your computer and the Apple DAV, Aspera, and Signiant servers.

Able to connect to the host s. In the following example, the user runs Transporter in diagnostic mode using the DAV delivery method. Transporter runs a diagnostic test to check the web service and the status and availability of the Apple DAV servers. In this example, when Transporter finishes executing the command, it displays diagnostic information indicating a successful connection between your computer and the Apple web service and a successful connection between your computer and the Apple DAV server.

In the following example, the user runs Transporter in diagnostic mode using the Aspera delivery method, and specifies that Transporter display eXtreme logging information.

Transporter runs a diagnostic test to check the web service and the status and availability of the Aspera servers and displays eXtreme logging information. In this example, when Transporter finishes executing the command, it displays eXtreme logging information, as well as diagnostic information indicating a successful connection between your computer and the Apple web service and a successful connection between your computer and the Aspera servers. In the following example, the user runs Transporter in diagnostic mode using the Signiant delivery method, and specifies that Transporter display eXtreme logging information.

Transporter runs a diagnostic test to check the web service and the status and availability of the Signiant servers and displays eXtreme logging information. In this example, when Transporter finishes executing the command, it displays eXtreme logging information, as well as diagnostic information indicating a successful connection between your computer and the Apple web service and a successful connection between your computer and the Signiant servers.

In the following example, the user runs Transporter in diagnostic mode using without specifying a specific delivery method, username, or password. In this example, when Transporter finishes executing the command, it displays diagnostic information indicating successful TCP connectivity. In the following example, the user, bob, runs Transporter in list report types mode and specifies that Transporter display the list of his available report content types for example, Apps or Music.

In the following example, the user, bob, runs Transporter in list report types mode and specifies that Transporter display the list of his available report content types for example, Apps or Music , and display the returned output information as XML. Transporter returns the list of report types available for bob, an app provider, and returns the output information as XML.

In the following example, the user, bob, runs Transporter in lookup artist mode to search for artists with the name John Appleseed, and specifies that Transporter not display any logging information. When Transporter finishes executing the command it displays information about the artists who matched the specified search term, without any additional logging information.

You can use the Apple identifiers in the album artists and track artists block of a music Store Package to precisely specify the intended artist.

In the following example, the user, bob, runs Transporter in lookup artist mode to search for artists with the name John Appleseed and display the returned output information as XML, and specifies that Transporter not display any logging information. This example shows a lookup artist mode for movie artists. Lookup artist with -outputFormat of XML now includes:. To lookup a movie artist, specify the -artistType Movie.

Note that if you do not supply -artistType , it will default to Music. The following is a sample for looking up "Steven Spielberg" using columnar output. The role and localization information will only appear in the xml output format. When Transporter finishes executing the command it displays information about the artist s who matched the specified search term, without any additional logging information. Transporter retrieves the current metadata for the playlist, creates a new In the following example, the user, bob, runs Transporter in lookup metadata mode and specifies that Transporter create a stub package containing the current metadata for the Apple playlists with Apple identifiers and Transporter retrieves the current metadata for the playlist, creates new In the following example, the user, bob, runs Transporter in provider mode and specifies that Transporter not display any logging information.

When Transporter finishes executing the command it displays a list containing the long and short name of providers for which bob has permission to deliver content, without any additional logging information. When uploading assets for this provider, you use the short name Apple with the -s option.

In the following example, the user, bob, runs Transporter in query tickets mode and specifies that Transporter return information about all open iTunes tickets that still require attention, and display eXtreme logging information.

Transporter returns information on all your open iTunes tickets that still require attention, and displays eXtreme logging information. Album title and artist name do not match. In the following example, the user runs Transporter in repair assets mode and specifies that Transporter repair the unmanifested. Transporter attempts to repair the unmanifested. When Transporter finishes executing the command it displays information indicating whether it could repair the file or not.

In this example, Transporter repaired the unmanifested. In the following example, the user, bob, runs Transporter in schema mode and specifies that Transporter return the transitional schema file for the 4. Transporter returns the transitional schema file for the 4. In the following example, the user, bob, runs Transporter in status mode and specifies that Transporter return status information for the most recently uploaded package for the given Apple identifier.

Transporter returns status information for the most recently uploaded package for the given Apple identifier. In this example, since the package you are requesting status for contains a video, status mode also returns component status information name, locale, status, delivered, rejection reason and note, if applicable for each video component thumbnails, subtitles, audio captions, chapters, dub credit, video, preview in addition to the package status.

In the following example, the user, bob, runs Transporter in status mode and specifies that Transporter return status information for the most recently uploaded packages for the comma-separated list of vendor identifiers, and display the returned output information as XML. Transporter returns status information for the most recently uploaded packages for the comma-separated list of vendor identifiers , , and returns the output information as XML.

In this example, Transporter returns an Upload Failed status for vendor identifier and a final upload status of Imported for vendor identifier In the following example, the user, bob, runs Transporter in statusAll mode and specifies that Transporter return status information for all uploaded packages for the given Apple identifier, and display the returned output information as XML.

Transporter returns status information for all uploaded packages for the given Apple identifier, and returns the output information as XML. In this example, since the package you are requesting status for contains a movie, status mode also returns component status information name, locale, status, delivered for each movie component captions, video, audio, chapters, thumbnails in addition to the package status.

In the following example, the user, bob, runs Transporter in statusAll mode and specifies that Transporter return status information for all uploaded packages for the comma-separated list of vendor identifiers, and display the returned output information as XML.

Transporter returns status information for all uploaded packages for the comma-separated list of vendor identifiers, and returns the output information as XML. In this example, Transporter returns a final upload status of Import Error for vendor identifier and Import Error for vendor identifier Sign In.

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