Cryo-EM single-particle structure refinement and map calculation using Servalcat.

In 2020, cryo-EM single-particle analysis achieved true atomic resolution thanks to technological developments in hardware and software. The number of high-resolution reconstructions continues to grow, increasing the importance of the accurate determination of atomic coordinates. Here, a new Python package and program called Servalcat is presented that is designed to facilitate atomic model refinement. Servalcat implements a refinement pipeline using the program REFMAC5 from the CCP4 package. After the refinement, Servalcat calculates a weighted Fo - Fc difference map, which is derived from Bayesian statistics. This map helps manual and automatic model building in real space, as is common practice in crystallography. The Fo - Fc map helps in the visualization of weak features including hydrogen densities. Although hydrogen densities are weak, they are stronger than in the electron-density maps produced by X-ray crystallography, and some H atoms are even visible at ∼1.8 Å resolution. Servalcat also facilitates atomic model refinement under symmetry constraints. If point-group symmetry has been applied to the map during reconstruction, the asymmetric unit model is refined with the appropriate symmetry constraints

ALS mutations in the TIA-1 prion-like domain trigger highly condensed pathogenic structures

筋萎縮性側索硬化症（ALS）の発症機構の一端を解明 --タンパク質の高密度な凝縮構造が鍵--. 京都大学プレスリリース. 2022-09-13.T cell intracellular antigen-1 (TIA-1) plays a central role in stress granule (SG) formation by self-assembly via the prion-like domain (PLD). In the TIA-1 PLD, amino acid mutations associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or Welander distal myopathy (WDM), have been identified. However, how these mutations affect PLD self-assembly properties has remained elusive. In this study, we uncovered the implicit pathogenic structures caused by the mutations. NMR analysis indicated that the dynamic structures of the PLD are synergistically determined by the physicochemical properties of amino acids in units of five residues. Molecular dynamics simulations and three-dimensional electron crystallography, together with biochemical assays, revealed that the WDM mutation E384K attenuated the sticky properties, whereas the ALS mutations P362L and A381T enhanced the self-assembly by inducing β-sheet interactions and highly condensed assembly, respectively. These results suggest that the P362L and A381T mutations increase the likelihood of irreversible amyloid fibrillization after phase-separated droplet formation, and this process may lead to pathogenicity

An isomorphous replacement method for efficient de novo phasing for serial femtosecond crystallography.

SACLAのX線自由電子レーザーを用いた新規タンパク質立体構造決定に世界で初めて成功. 京都大学プレスリリース. 2015-09-14.Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) holds great potential for structure determination of challenging proteins that are not amenable to producing large well diffracting crystals. Efficient de novo phasing methods are highly demanding and as such most SFX structures have been determined by molecular replacement methods. Here we employed single isomorphous replacement with anomalous scattering (SIRAS) for phasing and demonstrate successful application to SFX de novo phasing. Only about 20,000 patterns in total were needed for SIRAS phasing while single wavelength anomalous dispersion (SAD) phasing was unsuccessful with more than 80,000 patterns of derivative crystals. We employed high energy X-rays from SACLA (12.6 keV) to take advantage of the large anomalous enhancement near the LIII absorption edge of Hg, which is one of the most widely used heavy atoms for phasing in conventional protein crystallography. Hard XFEL is of benefit for de novo phasing in the use of routinely used heavy atoms and high resolution data collection

An oxyl/oxo mechanism for dioxygen bond formation in PSII revealed by X-ray free electron lasers

Photosynthetic water oxidation is catalyzed by the Mn4CaO5 cluster of photosystem II (PSII) with linear progression through five S-state intermediates (S0 to S4). To reveal the mechanism of water oxidation, we analyzed structures of PSII in the S1, S2, and S3 states by x-ray free-electron laser serial crystallography. No insertion of water was found in S2, but flipping of D1 Glu189 upon transition to S3 leads to the opening of a water channel and provides a space for incorporation of an additional oxygen ligand, resulting in an open cubane Mn4CaO6 cluster with an oxyl/oxo bridge. Structural changes of PSII between the different S states reveal cooperative action of substrate water access, proton release, and dioxygen formation in photosynthetic water oxidation

Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine

新規光駆動型イオンチャネルの構造解明と高性能分子ツールの創出 --神経科学に光を当てる--. 京都大学プレスリリース. 2022-02-03.ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology

The FOXE1 and NKX2-1 loci are associated with susceptibility to papillary thyroid carcinoma in the Japanese population.

Background FOXE1 and NKX2-1 are two known genetic risk factors for the predisposition to sporadic papillary thyroid carcinoma (PTC) in Europeans, but their association in other ethnicities is still unknown. Objective We aim to examine the association of the two genes with Japanese sporadic PTC, which exhibits high BRAF(V600E) mutation rate. Methods 507 Japanese sporadic PTC cases and 2766 controls were genotyped for rs965513 (FOXE1) and rs944289 (NKX2-1). PTC cases were also examined for their BRAF(V600E) mutational status. Results The association of both rs965513 (p=1.27×10(-4), OR=1.69, 95% CI 1.29 to 2.21) and rs944289 (p=0.0121, OR=1.21, 95% CI 1.04 to 1.39) with the risk of sporadic PTC was confirmed. Subgroup analysis based on the BRAF mutational status showed strong association of rs965513 with BRAF(V600E)-positive cases (p=2.26×10(-4), OR=1.72, 95% CI 1.29 to 2.29), but not with BRAF(V600E)-negative cases (p=0.143, OR=1.52, 95% CI 0.87 to 2.65). However, there was no difference in the observed effect size between both subgroups. For rs944289, both subgroups showed marginal association (p=0.0585, OR=1.17, 95% CI 0.99 to 1.37 for BRAF(V600E)-positive cases; p=0.0492, OR=1.35, 95% CI 1.00 to 1.81 for BRAF(V600E)-negative cases). Conclusions Both FOXE1 and NKX2-1 were associated with the increased risk of sporadic Japanese PTC. No clear associations were observed for either SNP with BRAF(V600E) status

Bipartite binding and partial inhibition links DEPTOR and mTOR in a mutually antagonistic embrace.

The mTORC1 kinase complex regulates cell growth, proliferation, and survival. Because mis-regulation of DEPTOR, an endogenous mTORC1 inhibitor, is associated with some cancers, we reconstituted mTORC1 with DEPTOR to understand its function. We find that DEPTOR is a unique partial mTORC1 inhibitor that may have evolved to preserve feedback inhibition of PI3K. Counterintuitively, mTORC1 activated by RHEB or oncogenic mutation is much more potently inhibited by DEPTOR. Although DEPTOR partially inhibits mTORC1, mTORC1 prevents this inhibition by phosphorylating DEPTOR, a mutual antagonism that requires no exogenous factors. Structural analyses of the mTORC1/DEPTOR complex showed DEPTOR's PDZ domain interacting with the mTOR FAT region, and the unstructured linker preceding the PDZ binding to the mTOR FRB domain. The linker and PDZ form the minimal inhibitory unit, but the N-terminal tandem DEP domains also significantly contribute to inhibition

Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin

X線自由電子レーザーを用いて、光照射によるチャネルロドプシンの構造変化の過程を捉えることに成功. 京都大学プレスリリース. 2021-03-26.Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore