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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-secretase
Function / homology
Function and homology information


Cajal-Retzius cell differentiation / positive regulation of L-glutamate import across plasma membrane / amyloid precursor protein biosynthetic process / negative regulation of core promoter binding / positive regulation of endopeptidase activity / gamma-secretase complex / short-term synaptic potentiation / aspartic endopeptidase activity, intramembrane cleaving / positive regulation of amyloid precursor protein biosynthetic process / smooth endoplasmic reticulum calcium ion homeostasis ...Cajal-Retzius cell differentiation / positive regulation of L-glutamate import across plasma membrane / amyloid precursor protein biosynthetic process / negative regulation of core promoter binding / positive regulation of endopeptidase activity / gamma-secretase complex / short-term synaptic potentiation / aspartic endopeptidase activity, intramembrane cleaving / positive regulation of amyloid precursor protein biosynthetic process / smooth endoplasmic reticulum calcium ion homeostasis / Noncanonical activation of NOTCH3 / protein catabolic process at postsynapse / TGFBR3 PTM regulation / sequestering of calcium ion / Notch receptor processing / synaptic vesicle targeting / positive regulation of coagulation / central nervous system myelination / negative regulation of axonogenesis / membrane protein intracellular domain proteolysis / skin morphogenesis / choline transport / T cell activation involved in immune response / NOTCH4 Activation and Transmission of Signal to the Nucleus / dorsal/ventral neural tube patterning / ciliary rootlet / neural retina development / regulation of resting membrane potential / L-glutamate import across plasma membrane / Regulated proteolysis of p75NTR / regulation of phosphorylation / myeloid dendritic cell differentiation / metanephros development / endoplasmic reticulum calcium ion homeostasis / brain morphogenesis / locomotion / amyloid precursor protein metabolic process / regulation of synaptic vesicle cycle / regulation of long-term synaptic potentiation / regulation of postsynapse organization / astrocyte activation involved in immune response / embryonic limb morphogenesis / cell fate specification / regulation of canonical Wnt signaling pathway / aggresome / myeloid cell homeostasis / growth factor receptor binding / skeletal system morphogenesis / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / azurophil granule membrane / G protein-coupled dopamine receptor signaling pathway / Golgi cisterna membrane / glutamate receptor signaling pathway / positive regulation of amyloid fibril formation / : / blood vessel development / amyloid-beta formation / mitochondrial transport / amyloid precursor protein catabolic process / heart looping / regulation of neuron projection development / positive regulation of dendritic spine development / adult behavior / positive regulation of receptor recycling / cerebral cortex cell migration / smooth endoplasmic reticulum / nuclear outer membrane / membrane protein ectodomain proteolysis / negative regulation of apoptotic signaling pathway / negative regulation of ubiquitin-dependent protein catabolic process / EPH-ephrin mediated repulsion of cells / autophagosome assembly / endopeptidase activator activity / neuron development / somitogenesis / hematopoietic progenitor cell differentiation / T cell proliferation / regulation of synaptic transmission, glutamatergic / Nuclear signaling by ERBB4 / calcium ion homeostasis / rough endoplasmic reticulum / Degradation of the extracellular matrix / Notch signaling pathway / neuron projection maintenance / astrocyte activation / NOTCH2 Activation and Transmission of Signal to the Nucleus / NRIF signals cell death from the nucleus / cellular response to calcium ion / Activated NOTCH1 Transmits Signal to the Nucleus / cerebellum development / thymus development / positive regulation of glycolytic process / dendritic shaft / epithelial cell proliferation / post-embryonic development / PDZ domain binding / NOTCH3 Activation and Transmission of Signal to the Nucleus / neuromuscular junction / apoptotic signaling pathway / cell-cell adhesion
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 large lobe / 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

EMD-3237.jpg

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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.4 Å/pix.
x 180 pix.
= 252. Å		1.4 Å/pix.
x 180 pix.
= 252. Å		1.4 Å/pix.
x 180 pix.
= 252. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.4 Å
Density

Histogram

Histogram (log scale)
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-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
TemperatureMin: 80 K / Max: 90 K / Average: 85 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 81000 magnification
Specialist opticsEnergy filter - Name: Gatan Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 35714 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
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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