Computational protocol: Channel opening and gating mechanism in AMPA subtype glutamate receptors

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Protocol publication

[…] C-flat (Protochips, Inc., Morrisville, NC) CF-1.2/1.3-2Au 200 mesh holey carbon grids were coated with gold using an Edwards Auto 306 evaporator. Carbon was removed using Ar/O2 (6 minutes, 50 watts, 35.0 sccm Ar, 11.5 sccm O2) plasma treatment with a Gatan (Pleasanton, CA, USA) Solarus 950 advanced plasma system, making Au 1.2/1.3 holey /on Au mesh grids. In order to make the surface hydrophilic prior to sample application, the grid surface was plasma-treated with H2/O2 (20s, 10 watts, 6.4 sccm H2, 27.5 sccm O2). Frozen-hydrated grids were prepared using a Vitrobot Mark IV (FEI, Hillsboro, OR, USA). 3 μl of sample (5 mg/ml GluA2-GSG1L with or without 0.3 mM antagonist ZK200775) was applied to the plasma-treated grids using 3.0 s blotting time, 3 blot force, 30.0 s wait time and 100% humidity at 22 ˚C. For the open state, 4 mg/ml GluA2-STZ was incubated for 15 minutes with 100 μM CTZ, then 3 μl of this was quickly added and pipetted up-and-down in a mixture of 0.5 μl 700 mM glutamate (final concentration 100 mM). 3 μl of this mixture was added to the grid, blotted as described above.The GluA2-GSG1L data (apo/ZK) were collected on a Tecnai F30 Polara (FEI) microscope operating at 300 kV, using Leginon with a Gatan K2 Summit electron detection (DED) camera (Gatan, Pleasanton, CA, USA) in counting mode with a pixel size of 0.98 Å. Data were collected across 40 frames (0.2 s per frame), with a dose rate of 8 e− pixel−1 s−1 (total dose of ~67 e− Å−2), within defocus range −1.5 μm to −3.5 μm, Cs 2.26 mm. The GluA2-STZ data were collected on a Titan Krios microscope (FEI) operating at 300 kV, using Leginon with a post-GIF quantum energy filter (20 eV slit) and Gatan K2 Summit direct camera DED camera in counting mode operating at a pixel size of 1.08 Å/pixel. A dose rate of 8 e− physical pixel−1 s−1 (total dose of ~55 e− Å2) was used across 40 frames (0.2 ms per frame), within the defocus range −1.0 μm to −3.0 μm. [...] All frame alignment and dose-weighting was carried out in MotionCor2. CTF correction, with gCTF, was performed on non-dose-weighted micrographs, with all further processing on dose-weighted micrographs using RELION 2.0. From 2,938 micrographs for GluA2-GSG1L bound to ZK, 541,261 particles were picked. The data was binned to 3.92 Å/pixel and subject to 2D classification, which resulted in cleaning the particle pool to 513,406 particles. These particles were then subject to 3D classification without symmetry into ten classes and the GluA2-2xGSG1LZK model low-pass filtered to 40 Å as a reference. Three classes, with two showing different ECD conformations (i.e., state 1 and state 2) and two auxiliary subunits bound were chosen for additional 3D sub-classification. This resulted in a group of 234,426 particles, which were unbinned to a pixel size of 1.96, and subject to classification with the GluA2-2xGSG1LZK model (40 Å filter) as the reference. This resulted in two well-structured groups of particles: GluA2-GSG1LZK-1 (26,971 particles) and GluA2-GSG1LZK-2 (41,926 particles). The particles were unbinned to the original 0.98 Å/pixel size, and refined with C2-symmetry. Of note is that we did not observe any stoichiometric heterogeneity, as was observed in cryo-EM performed on the GluA2-GSG1L complex solubilized in DDM.For the GluA2-GSG1L collection in the absence of ligand, 242,078 particles were automatically picked from 2,593 micrographs using six 2D classes as references (generated from manual picking as described above). The particle images were binned to 3.92 Å/pixel, and 235,543 particles remained following 2D classification. Following, 3D classification was carried out without symmetry into ten classes with the 40 Å low-pass filtered GluA2-GSG1LZK-1 map. Six classes, numerating 115,120 particles showed structural details that warranted further image processing. The particles were unbinned to 0.98 Å/pixel and classified in 3D into 10 classes with the GluA2-GSG1LZK-1. Resulting were two prominent classes, GluA2-GSG1Lapo-1 (20,392 particles) and GluA2-GSG1Lapo-2 (18,926 particles), which were refined with C2-symmetry, and resemble the two states observed for GluA2-GSG1L in the presence of ZK ( and ).A total of 4,116 micrographs were collected for GluA2-STZ, and 595,889 particles were automatically picked, with manually-picked particles used to generate reference classes as described above. The particles were binned twice to 2.16 Å/pixel and subject to 2D classification, which resulted in 581,495 particles being subject to further image processing. The original GluA2-2xSTZ antagonist-bound map, low-pass filtered to 40 Å, was used as a reference model for 3D classification into ten classes without symmetry imposed. 3D classification resulted in 278,454 particles, from four classes, being chosen for further image processing, without observation of stoichiometric heterogeneity as in the DDM-solubilized complex, and the particles were unbinned to 1.08 Å/pixel and classified in 3D to ten classes. Particles from three classes, numerating 69,207 particles and showing structurally-similar features, were chosen for refinement. Initial refinement, with C2-symmetry, resulted in a 4.2 Å map, which showed side chain features in the GluA2 core but was lacking details for STZ. To better align particles according to the GluA2 TMD and STZ, we generated a soft spherical mask around the detergent micelle (which encompassed all of the GluA2 and STZ TMDs and STZ extracellular head). This resulted in an overall 4.0 Å map, with distinct side chain features throughout the TMD for model building of the GluA2 and STZ TMDs. We did not observe a digitonin-bound state of GluA2-STZGlu+CTZ, as we did for GluA2-GSG1L in the resting states (GluA2-GSG1LZK-2, GluA2-GSG1Lapo-2).The resolution for all reconstructions was estimated using the FSC=0.143 criterion between independent half maps on corrected FSC curves in which the influences of the mask were removed (). All maps were postprocessed using a softmask in RELION, and B-factors for map sharpening were automatically estimated (). All visualization of EM densities was done in UCSF Chimera. Local resolution for each map () was calculated with unfiltered half maps using ResMap. [...] To build the state 1 ZK-bound and GluA2-GSG1L models we individually isolated the ATD and LBD dimers, as well as the TMD, from the GluA2-2xGSG1LZK complex structure using rigid-body fitting in COOT. Side chains in the TMD were defined based on local resolution, as was the structure of the M2 helix and pore loop. The resulting model was refined against an un-filtered half map (work) in real space with constraints using PHENIX. The refined model was tested for overfitting () by shifting the coordinates with shake in PHENIX and building a density in EMAN2 from the shaken model. FSC was calculated between the densities from the shaken model, the map used in PHENIX refinement (work), second half map (free) and unfiltered sum map, using EMAN2. The resulting GluA2-GSG1LZK-1 model was used as a basis to build GluA2-GSG1Lapo-1. Correspondingly, the ATD and LBD local dimers were moved as rigid bodies to define the state 2 models (GluA2-GSG1LZK-2 and GluA2-GSG1Lapo-2), and features were adjusted manually according to high resolution features in GluA2-GSG1LZK-2. Secondary structure features of the M3 pore were further defined in the GluA2-GSG1LZK-2 M3 density. The resulting models were also real space refined in PHENIX and tested for overfitting (). State 2 models were refined in the absence of digitonin.For GluA2-STZGlu+CTZ, we rigid-body fit the high-resolution structures of the ATD and glutamate/CTZ-bound LBD. We individually rigid body fit helices from the TMD of our GluA2-GSG1LZK-1 refined structure, and rigid-body fit STZ subunits from the GluA2-2xSTZZK structure in COOT for an initial model. After increasing the resolution in the TMD map, we built STZ and the GluA2 TMD de novo. The resulting models were also real space refined in PHENIX and tested for overfitting (), of which there are no signs of over-fit models. Structures were visualized and figures were prepared in Pymol. […]

Pipeline specifications

Software tools Leginon, MotionCor2, Gctf, RELION, UCSF Chimera, ResMap, Coot, EMAN, PyMOL
Databases DED
Applications cryo-EM, Protein structure analysis
Chemicals alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid, Glutamic Acid