![130319-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-04/130319-Thumbnail%20Image.png?versionId=vGJCgNNle7WqEw_Y.qHeSBSG9Zi2UIo9&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240530/us-west-2/s3/aws4_request&X-Amz-Date=20240530T153726Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=eaaa8d753fbf71d15e04639d798ecf41b58f5bd0ac8cca3e8a70877e439fe5ea&itok=hfN9x8Il)
Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices.
![130322-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-04/130322-Thumbnail%20Image.png?versionId=YCHSKaVcygXd3HHenvL2xzS.G.kixC32&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240530/us-west-2/s3/aws4_request&X-Amz-Date=20240530T153726Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=84121a565f274f48d6dad85b0da158b4de106a8b80e67f52cd88a28592f99586&itok=I8O649eW)
Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.
![130311-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-04/130311-Thumbnail%20Image.png?versionId=qHMde1a9_azrWBw6LMhLxblmGpj1T5VL&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240530/us-west-2/s3/aws4_request&X-Amz-Date=20240530T153726Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=41adb2a374858bed3f69bffdc20ae60b6118ac9b4a692ad1a10cfcb435e6fa33&itok=WBhJHD7-)
![128362-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-04/128362-Thumbnail%20Image.png?versionId=2z1DUohMEvRSZyQKO5hxakzJuuMjAYYa&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240612/us-west-2/s3/aws4_request&X-Amz-Date=20240612T200524Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=96121de368c02fff0e4d8d27ddefd4e4f967fd9d39eb53d91b2dbe1da5659a93&itok=GZ5jXgvg)
The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.
![130274-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-04/130274-Thumbnail%20Image.png?versionId=y2otTFmNxVfGdNlboOFY5tZyqTmTOPne&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240605/us-west-2/s3/aws4_request&X-Amz-Date=20240605T154426Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=3a8830c70f28afb2b8ae5b8804cf05fb2eb9ae8030624b569a5ff1321faffbf4&itok=3GiCMrZE)
![130284-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-04/130284-Thumbnail%20Image.png?versionId=RL18_PeDsi0QZ7ifKxgMiJEVL3i4mc.x&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240606/us-west-2/s3/aws4_request&X-Amz-Date=20240606T054759Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=f3d6b15d8c73c0cf7d6967a573a9bcefad0d52398c294984b06db431689cb0d5&itok=MkSjOHVT)
![130301-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-04/130301-Thumbnail%20Image.png?versionId=xqj1fyeOr9I9fWJmok_xApUZSi.vLJWr&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240612/us-west-2/s3/aws4_request&X-Amz-Date=20240612T055045Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=3af1580345077680de92545cffca2847e8ddb7c9e7a997cee287918d1b3ade0f&itok=_EM0ee44)
![130302-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-04/130302-Thumbnail%20Image.png?versionId=bRVeSoMNtjtOc8xFDxSUq67rlfgCYFH1&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240605/us-west-2/s3/aws4_request&X-Amz-Date=20240605T205427Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=5397aef26823e23c5d1b8a9b8546cb83c5d889ac489d568fdbe3d29d6b0d301f&itok=yeSP2o7w)
![130303-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-04/130303-Thumbnail%20Image.png?versionId=49yWpTidtPKMv58NC2TOxiCt7hU1PUbz&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240612/us-west-2/s3/aws4_request&X-Amz-Date=20240612T062125Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=c896761c215e9840bc8d062542fbfe120adf19505e31c6a9378da7f4b459bbc0&itok=8-_QFPiz)
![130306-Thumbnail Image.png](https://d1rbsgppyrdqq4.cloudfront.net/s3fs-public/styles/width_400/public/2021-04/130306-Thumbnail%20Image.png?versionId=v631S8PiiTl8DPeW3_O4fQhoei2bVdDu&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIASBVQ3ZQ42ZLA5CUJ/20240613/us-west-2/s3/aws4_request&X-Amz-Date=20240613T040041Z&X-Amz-SignedHeaders=host&X-Amz-Expires=120&X-Amz-Signature=ebc3223d6ed5bacb625e6fbacdc59a6c5529a4a8adba3578d7781c4e6628a532&itok=oFxZCiJK)