X-ray diffraction data sets were collected at the Advanced Light Source on beamlines 8.2.1 and 5.0.2. Diffraction sets were indexed, integrated, and scaled by XDS57 or HKL200058 together with the microdiffraction assembly method59 (link). The best diffraction data for Data set 1 were derived from merging data from three crystals60 (link). A single crystal was used for Data set 2. Structure 1 was determined by molecular replacement with Phaser61 (link) using the isolated Xenopus/rat GluN1/GluN2B ATD domains in complex with Ro25-6981 (PDB code 3QEM)24 (link) and the isolated rat/rat GluN1/GluN2A LBD (PDB code 2A5T)32 (link) structures as search probes. The molecular replacement solutions were robust with the highest best log likelihood gain and translation function Z-score of 3071.7 and 31.9, respectively. Initial maps were improved by density modification62 (link). A partial model of the transmembrane domain was manually built into ‘omit’ style electron density maps. Cycles of manual model building and crystallographic refinement were carried out using the computer graphic program Coot63 and the crystallographic refinement software package Phenix64 (link). During the course of model building and refinement, the amino acid sequence and corresponding structure within the ATDs and LBDs were adjusted to the Xenopus amino acid sequences. The model was refined to a nominal resolution of 3.7 Å with reasonable R-factors. Structure 2 derived from Data set 2 was solved by molecular replacement using Structure 1 as a search probe. Upon inspection of electron density maps, density for the pore loops was visible, along with additional residues in the other TM segments. The final Structure 2 was obtained by cycles of manual model building and crystallographic refinement, as described above. Stereochemistry of the model was evaluated by MolProbity 65 (link), pore dimensions were estimated using HOLE66 (link) and figures were created using Pymol67 . Important information on the qualities of the structures is provided in Supplementary Information .
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Structural Determination of NMDA Receptor Complexes
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- X-ray diffraction data sets were collected at the Advanced Light Source on beamlines 8.2.1 and 5.0.2.
- Diffraction sets were indexed, integrated, and scaled by XDS or HKL2000 together with the microdiffraction assembly method.
- The best diffraction data for Data set 1 were derived from merging data from three crystals.
- A single crystal was used for Data set 2.
- Structure 1 was determined by molecular replacement with Phaser using the isolated Xenopus/rat GluN1/GluN2B ATD domains in complex with Ro25-6981 (PDB code 3QEM) and the isolated rat/rat GluN1/GluN2A LBD (PDB code 2A5T) structures as search probes.
- Structure 2 derived from Data set 2 was solved by molecular replacement using Structure 1 as a search probe.
- The molecular replacement solutions were robust with the highest best log likelihood gain and translation function Z-score of 3071.7 and 31.9, respectively.
- The model was refined to a nominal resolution of 3.7 Å with reasonable R-factors.
- Upon inspection of electron density maps, density for the pore loops was visible, along with additional residues in the other TM segments.
- Initial maps were improved by density modification.
- Cycles of manual model building and crystallographic refinement were carried out using the computer graphic program Coot and the crystallographic refinement software package Phenix.
- During the course of model building and refinement, the amino acid sequence and corresponding structure within the ATDs and LBDs were adjusted to the Xenopus amino acid sequences.
- Stereochemistry of the model was evaluated by MolProbity, pore dimensions were estimated using HOLE, and figures were created using Pymol.
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