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- EMDB-3237: Cryo-EM structure of gamma secretase in complex with a drug DAPT -

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Basic information

Entry
Database: EMDB / ID: EMD-3237
TitleCryo-EM structure of gamma secretase in complex with a drug DAPT
Map dataReconstruction of gamma secretase in complex with a drug DAPT
Sample
  • Sample: human gamma secretase in complex with a drug DAPT
  • Protein or peptide: gamma-secretaseGamma secretase
Function / homology
Function and homology information


positive regulation of L-glutamate import across plasma membrane / Cajal-Retzius cell differentiation / amyloid precursor protein biosynthetic process / positive regulation of coagulation / protein catabolic process at postsynapse / negative regulation of core promoter binding / gamma-secretase complex / aspartic endopeptidase activity, intramembrane cleaving / short-term synaptic potentiation / positive regulation of amyloid precursor protein biosynthetic process ...positive regulation of L-glutamate import across plasma membrane / Cajal-Retzius cell differentiation / amyloid precursor protein biosynthetic process / positive regulation of coagulation / protein catabolic process at postsynapse / negative regulation of core promoter binding / gamma-secretase complex / aspartic endopeptidase activity, intramembrane cleaving / short-term synaptic potentiation / positive regulation of amyloid precursor protein biosynthetic process / choline transport / Noncanonical activation of NOTCH3 / positive regulation of endopeptidase activity / Notch receptor processing / central nervous system myelination / sequestering of calcium ion / membrane protein intracellular domain proteolysis / synaptic vesicle targeting / negative regulation of axonogenesis / regulation of resting membrane potential / T cell activation involved in immune response / skin morphogenesis / NOTCH4 Activation and Transmission of Signal to the Nucleus / growth factor receptor binding / neural retina development / regulation of synaptic vesicle cycle / dorsal/ventral neural tube patterning / L-glutamate import across plasma membrane / myeloid dendritic cell differentiation / Regulated proteolysis of p75NTR / amyloid precursor protein metabolic process / regulation of phosphorylation / metanephros development / locomotion / brain morphogenesis / endoplasmic reticulum calcium ion homeostasis / glutamate receptor signaling pathway / nuclear outer membrane / smooth endoplasmic reticulum calcium ion homeostasis / astrocyte activation involved in immune response / regulation of canonical Wnt signaling pathway / regulation of long-term synaptic potentiation / aggresome / embryonic limb morphogenesis / skeletal system morphogenesis / positive regulation of amyloid fibril formation / cell fate specification / regulation of postsynapse organization / positive regulation of dendritic spine development / ciliary rootlet / myeloid cell homeostasis / azurophil granule membrane / dopamine receptor signaling pathway / adult behavior / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / Golgi cisterna membrane / positive regulation of receptor recycling / mitochondrial transport / positive regulation of catalytic activity / heart looping / regulation of neuron projection development / blood vessel development / neuron development / smooth endoplasmic reticulum / amyloid precursor protein catabolic process / cerebral cortex cell migration / protein glycosylation / amyloid-beta formation / negative regulation of apoptotic signaling pathway / autophagosome assembly / membrane protein ectodomain proteolysis / endopeptidase activator activity / EPH-ephrin mediated repulsion of cells / Nuclear signaling by ERBB4 / hematopoietic progenitor cell differentiation / somitogenesis / amyloid-beta metabolic process / T cell proliferation / rough endoplasmic reticulum / Notch signaling pathway / regulation of synaptic transmission, glutamatergic / NOTCH2 Activation and Transmission of Signal to the Nucleus / negative regulation of ubiquitin-dependent protein catabolic process / cellular response to calcium ion / neuron projection maintenance / NRIF signals cell death from the nucleus / Activated NOTCH1 Transmits Signal to the Nucleus / cerebellum development / Degradation of the extracellular matrix / positive regulation of glycolytic process / post-embryonic development / dendritic shaft / thymus development / negative regulation of protein phosphorylation / epithelial cell proliferation / PDZ domain binding / NOTCH3 Activation and Transmission of Signal to the Nucleus / astrocyte activation / apoptotic signaling pathway / synapse organization
Similarity search - Function
Peptidase A22A, presenilin 1 / Gamma-secretase subunit Aph-1 / Gamma-secretase aspartyl protease complex, presenilin enhancer-2 subunit / Aph-1 protein / Presenilin enhancer-2 subunit of gamma secretase / Peptidase A22A, presenilin / Presenilin, C-terminal / Presenilin / Nicastrin / Nicastrin, small lobe ...Peptidase A22A, presenilin 1 / Gamma-secretase subunit Aph-1 / Gamma-secretase aspartyl protease complex, presenilin enhancer-2 subunit / Aph-1 protein / Presenilin enhancer-2 subunit of gamma secretase / Peptidase A22A, presenilin / Presenilin, C-terminal / Presenilin / Nicastrin / Nicastrin, small lobe / Nicastrin / Nicastrin small lobe / Presenilin/signal peptide peptidase / Presenilin, signal peptide peptidase, family
Similarity search - Domain/homology
Presenilin-1 / Nicastrin / Gamma-secretase subunit APH-1A / Gamma-secretase subunit PEN-2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 4.2 Å
AuthorsBai XC / Rajendra E / Yang GH / Shi YG / Scheres SHW
CitationJournal: Elife / Year: 2015
Title: Sampling the conformational space of the catalytic subunit of human γ-secretase.
Authors: Xiao-chen Bai / Eeson Rajendra / Guanghui Yang / Yigong Shi / Sjors H W Scheres /
Abstract: Human γ-secretase is an intra-membrane protease that cleaves many different substrates. Aberrant cleavage of Notch is implicated in cancer, while abnormalities in cutting amyloid precursor protein ...Human γ-secretase is an intra-membrane protease that cleaves many different substrates. Aberrant cleavage of Notch is implicated in cancer, while abnormalities in cutting amyloid precursor protein lead to Alzheimer's disease. Our previous cryo-EM structure of γ-secretase revealed considerable disorder in its catalytic subunit presenilin. Here, we describe an image classification procedure that characterizes molecular plasticity at the secondary structure level, and apply this method to identify three distinct conformations in our previous sample. In one of these conformations, an additional transmembrane helix is visible that cannot be attributed to the known components of γ-secretase. In addition, we present a γ-secretase structure in complex with the dipeptidic inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Our results reveal how conformational mobility in the second and sixth transmembrane helices of presenilin is greatly reduced upon binding of DAPT or the additional helix, and form the basis for a new model of how substrate enters the transmembrane domain.
History
DepositionNov 10, 2015-
Header (metadata) releaseNov 18, 2015-
Map releaseDec 9, 2015-
UpdateDec 16, 2015-
Current statusDec 16, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5fn2
  • Surface level: 0.05
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3237.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of gamma secretase in complex with a drug DAPT
Voxel sizeX=Y=Z: 1.4 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.05
Minimum - Maximum-0.12731156 - 0.21583737
Average (Standard dev.)0.00013833 (±0.01161833)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 252.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z252.000252.000252.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-190-190-189
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.1270.2160.000

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Supplemental data

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Sample components

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Entire : human gamma secretase in complex with a drug DAPT

EntireName: human gamma secretase in complex with a drug DAPT
Components
  • Sample: human gamma secretase in complex with a drug DAPT
  • Protein or peptide: gamma-secretaseGamma secretase

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Supramolecule #1000: human gamma secretase in complex with a drug DAPT

SupramoleculeName: human gamma secretase in complex with a drug DAPT / type: sample / ID: 1000 / Oligomeric state: Heterotetramer / Number unique components: 1
Molecular weightExperimental: 170 KDa / Theoretical: 170 KDa

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Macromolecule #1: gamma-secretase

MacromoleculeName: gamma-secretase / type: protein_or_peptide / ID: 1 / Details: a drug DAPT was bound to gamma scretase complex / Number of copies: 1 / Oligomeric state: Heterotetramer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: membrane
Molecular weightExperimental: 170 KDa / Theoretical: 170 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293F / Recombinant plasmid: pMLink

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration6 mg/mL
BufferpH: 7.4
Details: 25 mM HEPES, pH 7.4, 150 mM NaCl and amphipol A8-35
StainingType: NEGATIVE / Details: cryo-EM
GridDetails: 300 mesh Au 1.2/1.3 Quantifoil grid, glow discharged for 1 minute
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 85 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 4 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 35714 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: Gatan Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 80 K / Max: 90 K / Average: 85 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 81000 magnification
DateDec 31, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 2206 / Average electron dose: 38 e/Å2
Details: Use a newly developed statistical movie processing and particle polishing approach to compensate for beam-induced movement and reduce the effect of radiation-damage
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: OTHER / Software - Name: CTFFIND4, RELION
Details: Use a newly developed statistical movie processing and particle polishing approach to compensate for beam-induced movement and reduce the effect of radiation-damage
Number images used: 51366

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