Computational protocol: Structure of mammalian endolysosomal TRPML1 channel in nanodiscs

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Protocol publication

[…] Motion correction was performed using MotionCorr2 program, and the CTF parameters of the micrographs were estimated using the GCTF program. All other steps of image processing were performed using RELION. Initially, ∼ 1,000 particles were manually picked from a few micrographs. Class averages representing projections of MmTRPML1 in different orientations were selected from the 2D classification of the manually picked particles, and used as templates for automatic particle picking from the full data set of 2,974 micrographs. The extracted particles were binned 3 times and subjected to two rounds of 2D classification, and a total of 519,463 particles were finally selected for 3D classification and 3D refinement. For the first round of 3D classification, the TRPV1 structure (EMDB code 5778) was used as the reference. One of the 3D classes showed good secondary structural features and is significantly different from TRPV1. It was then selected as the reference for the second round of 3D classification. One of the resulting 3D reconstructions from about 71,000 particles showed improved cryo-EM density and a clear 4-fold symmetry with a resolution of ∼ 4 Å after 3D refinement using the un-binned particles (C4 symmetry imposed) and particle polishing. However, part of the transmembrane domain showed poor density, indicative of local structural heterogeneity. Therefore, we performed a focused 3D classification with density subtraction in order to improve the density of the transmembrane domain. In this approach, the density corresponding to the soluble domain of the channel as well as the belt-like density from the nanodisc was subtracted from the original particles. The subsequent 3D classification on the modified particles was carried out by applying a mask around the transmembrane domain and having all of the orientations fixed at the value determined in the 3D refinement. This round of classification yielded 6 classes of particles, among which one major class (∼50,000 particles) showed poor density in the transmembrane region and was discarded. After a new 3D refinement using the particles from the 5 classes (∼20,000 total particles), the resolution of the map was improved from 4 Å to 3.59 Å. In addition, these five classes of particles can be partitioned into two groups with 9,000 and 11,000 particles, respectively. 3-D refinement of each group yielded structures at 3.64 Å (closed I state) or 3.75 Å (closed II state) resolution, respectively, each showing distinct conformations at the S4-S5 linker. All resolutions were estimated by applying a soft mask around the protein density and the gold-standard Fourier shell correlation (FSC) = 0.143 criterion. ResMap was used to calculate the local resolution map. [...] For the soluble domain, the structure of HsTRPML1 I-II liner (PDB code: 5TJC) was docked into the cryo-EM map of MmTRPML1 followed by manual adjustment and mutation of residues that differed between MmTRPML1 and HsTRPML1. For the transmembrane domain, de novo atomic model building was conducted in Coot. Amino acid assignment was achieved based mainly on the clearly defined densities for bulky residues (Phe, Trp, Tyr, and Arg). Models were refined against summed maps using phenix.real_space_refine implemented in PHENIX. The model was validated using previously described methods to avoid overfitting. The final structure models include residues 40–198, 215-285, and 296–527. About 40 residues at the amino-terminus and 50 residues at the carboxyl-terminus are disordered and not modeled. The statistic of the models' geometries was generated using MolProbity. Pore radii were calculated using the HOLE program. All the figures were prepared in PyMol or Chimera. […]

Pipeline specifications

Software tools MotionCorr, Gctf, RELION, ResMap, Coot, PHENIX, MolProbity, PyMOL
Applications cryo-EM, Protein structure analysis
Organisms Homo sapiens, Mus musculus
Diseases Mucolipidoses, Lysosomal Storage Diseases
Chemicals Phenobarbital