Sampling the conformational space of the catalytic subunit of human γ-secretase

Bai, Xiao Chen; Rajendra, Eeson; Yang, Guanghui; Shi, Yigong; Scheres, Sjors H.W.

doi:10.7554/elife.11182

Cited by 572 publications

(493 citation statements)

References 54 publications

Supporting

Mentioning

468

Contrasting

Unclassified

Order By: Relevance

“…3D classification and refinement were carried out in RELION. The masked 3D classification focusing on the TMDs with residual signal subtraction was done following a previously described procedure 57 . The orientation parameters of the homogenous set of particle images in the selected 3D classes were iteratively refined to yield higher resolution maps using the ‘auto-refine’ procedure.…”

Section: Methodsmentioning

confidence: 99%

Structural basis of MsbA-mediated lipopolysaccharide transport

Yoon

et al. 2017

Nature

230

294

View full text Add to dashboard Cite

Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for their cell envelope assembly. LPS synthesized in the cytoplasmic leaflet of the inner membrane is flipped to the periplasmic leaflet by MsbA, an ATP-binding cassette transporter. Despite substantial efforts, the structural mechanisms underlying MsbA-driven LPS flipping remain elusive. Here, we use single-particle cryo-EM to elucidate the structures of lipid nanodisc-embedded MsbA in three functional states. The 4.2 Å-resolution structure of the transmembrane domains of nucleotide-free MsbA reveals that LPS binds deeply inside MsbA at the height of the periplasmic leaflet, establishing extensive hydrophilic and hydrophobic interactions with MsbA. Two subnanometer-resolution structures of MsbA with ADP-vanadate and ADP reveal an unprecedented closed and an inward-facing conformation, respectively. Our study uncovers the long-sought-after structural basis for LPS recognition, delineates the conformational transitions of MsbA to flip LPS, and paves the way for structural characterization of other lipid flippases.

show abstract

Section: Methodsmentioning

confidence: 99%

Structural basis of MsbA-mediated lipopolysaccharide transport

Yoon

et al. 2017

Nature

230

294

View full text Add to dashboard Cite

show abstract

“…However, part of the transmembrane domain showed poor density, indicative of local structural heterogeneity. Therefore, we performed a focused 3D classification with density subtraction 35 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.…”

Section: Methodsmentioning

confidence: 99%

Structure of mammalian endolysosomal TRPML1 channel in nanodiscs

Chen

She

Zeng

et al. 2017

Nature

Self Cite

256

170

View full text Add to dashboard Cite

Transient receptor potential mucolipin 1 (TRPML1) is an endo/lysosomal cation channel ubiquitously expressed in mammalian cells1,2 and its loss-of-function mutations are the direct cause of Type IV mucolipidosis (MLIV), an autosomal recessive lysosomal storage disease3-6. Here we present the single particle cryo-electron microscopy (cryo-EM) structure of the mouse TRPML1 channel embedded in nanodiscs. Combined with mutagenesis, the TRPML1 structure reveals that phosphatidylinositol bisphosphate (PIP2) binds to the N-terminus of the channel – distal from the pore – and the helix-turn-helix extension between S2 and S3 likely couples ligand binding to pore opening. The tightly packed selectivity filter contains multiple ion binding sites and the conserved acidic residues form the luminal Ca2+ blocking site that confers luminal pH and Ca2+ modulation on channel conductance. A luminal linker domain forms a fenestrated canopy atop the channel, providing multiple luminal ion passages to the pore and also creating a negative electrostatic trap – preferably for divalent cations at the luminal entrance. The structure also reveals two equally distributed S4-S5 linker conformations in the closed channel, providing structural implication for the S4-S5 linker-mediated PIP2 gating mechanism among TRPML channels7,8.

show abstract

“…7d,e). Therefore, masked 3D classification and refinement after signal subtraction 56 was carried out, resulting in slightly improved resolution but no substantial differences in the electron density map. Map generation from coordinates as well as filtering and file type conversion was done using EMAN2 tools 49 .…”

Section: Methodsmentioning

confidence: 99%

The structural organization of substrate loading in iterative polyketide synthases

et al. 2018

View full text Add to dashboard Cite

Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.

show abstract

Sampling the conformational space of the catalytic subunit of human γ-secretase

Cited by 572 publications

References 54 publications

Structural basis of MsbA-mediated lipopolysaccharide transport

Structural basis of MsbA-mediated lipopolysaccharide transport

Structure of mammalian endolysosomal TRPML1 channel in nanodiscs

The structural organization of substrate loading in iterative polyketide synthases

Contact Info

Product

Resources

About