EMDB-2478: Negative stain electron microscopy structure of compound E-bound ...

Basic information

• Entry: Database: EMDB / ID: EMD-2478
• Title: Negative stain electron microscopy structure of compound E-bound human Presenilin 1 (PS1) complex
• Map data: Native human PS1 complex

Sample

• Sample: Compound E-bound human Presenilin 1 (PS1) complex
• Protein or peptide: Presenilin-1
• Protein or peptide: Nicastrin
• Protein or peptide: Gamma-secretase subunit APH-1A
• Protein or peptide: Gamma-secretase subunit PEN-2
• Ligand: E ((S,S)- 2-[2-(3,5-Difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide)

Function / homology

Function:

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

Domain/homology:

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

Component:

Presenilin-1 / Nicastrin / Gamma-secretase subunit APH-1A / Gamma-secretase subunit PEN-2

• Biological species: Homo sapiens (human) / synthetic construct (others)
• Method: single particle reconstruction / negative staining / Resolution: 17.4 Å
• Authors: Li Y / Lu S / Tsai CJ / Bohm C / Qamar S / Dodd RB / Meadows W / Jeon A / McLeod A / Chen F / Arimon M / Berezovska O / Hyman BT / Tomita T / Iwatsubod T / Johnsof CM / Farrer L / Schmitt-Ulms G / Fraser P / St George-Hyslop P
• Citation: Journal: Structure / Year: 2014; Title: Structural interactions between inhibitor and substrate docking sites give insight into mechanisms of human PS1 complexes.; Authors: Yi Li / Stephen Hsueh-Jeng Lu / Ching-Ju Tsai / Christopher Bohm / Seema Qamar / Roger B Dodd / William Meadows / Amy Jeon / Adam McLeod / Fusheng Chen / Muriel Arimon / Oksana Berezovska / Bradley T Hyman / Taisuke Tomita / Takeshi Iwatsubo / Christopher M Johnson / Lindsay A Farrer / Gerold Schmitt-Ulms / Paul E Fraser / Peter H St George-Hyslop / ; Abstract: Presenilin-mediated endoproteolysis of transmembrane proteins plays a key role in physiological signaling and in the pathogenesis of Alzheimer disease and some cancers. Numerous inhibitors have been found via library screens, but their structural mechanisms remain unknown. We used several biophysical techniques to investigate the structure of human presenilin complexes and the effects of peptidomimetic γ-secretase inhibitors. The complexes are bilobed. The head contains nicastrin ectodomain. The membrane-embedded base has a central channel and a lateral cleft, which may represent the initial substrate docking site. Inhibitor binding induces widespread structural changes, including rotation of the head and closure of the lateral cleft. These changes block substrate access to the catalytic pocket and inhibit the enzyme. Intriguingly, peptide substrate docking has reciprocal effects on the inhibitor binding site. Similar reciprocal shifts may underlie the mechanisms of other inhibitors and of the "lateral gate" through which substrates access to the catalytic site.

History

Deposition	Sep 23, 2013	-
Header (metadata) release	Nov 13, 2013	-
Map release	Nov 20, 2013	-
Update	Mar 2, 2016	-
Current status	Mar 2, 2016	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_2478.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Native human PS1 complex
• Voxel size: X=Y=Z: 2.04 Å

Density

Contour Level	By AUTHOR: 0.0155 / Movie #1: 0.0155
Minimum - Maximum	-0.0298597 - 0.05378654
Average (Standard dev.)	-0.00013971 (±0.00514203)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 128 128 128 Spacing 128 128 128
Cell	A=B=C: 261.12 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	2.04	2.04	2.04
M x/y/z	128	128	128
origin x/y/z	0.000	0.000	0.000
length x/y/z	261.120	261.120	261.120
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	0	0	-40
NX/NY/NZ	55	55	81
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	128	128	128
D min/max/mean	-0.030	0.054	-0.000

Supplemental data

Sample components

Entire : Compound E-bound human Presenilin 1 (PS1) complex

• Entire: Name: Compound E-bound human Presenilin 1 (PS1) complex

Components

• Sample: Compound E-bound human Presenilin 1 (PS1) complex
• Protein or peptide: Presenilin-1
• Protein or peptide: Nicastrin
• Protein or peptide: Gamma-secretase subunit APH-1A
• Protein or peptide: Gamma-secretase subunit PEN-2
• Ligand: E ((S,S)- 2-[2-(3,5-Difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide)

Supramolecule #1000: Compound E-bound human Presenilin 1 (PS1) complex

• Supramolecule: Name: Compound E-bound human Presenilin 1 (PS1) complex / type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: 1:1:1:1 / Number unique components: 5
• Molecular weight: Theoretical: 200 KDa

Macromolecule #1: Presenilin-1

• Macromolecule: Name: Presenilin-1 / type: protein_or_peptide / ID: 1 / Name.synonym: Protein S182; Details: N-terminus as tagged with TAP tag composed of Protein G and Streptavidin binding peptide tags separated by tobacco etch virus protease cleavage site; Number of copies: 1 / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Recombinant expression: Organism: Homo sapiens (human) / Recombinant cell: HEK293
• Sequence: UniProtKB: Presenilin-1

Macromolecule #2: Nicastrin

• Macromolecule: Name: Nicastrin / type: protein_or_peptide / ID: 2 / Name.synonym: KIAA0253 / Number of copies: 1 / Recombinant expression: No
• Source (natural): Organism: Homo sapiens (human) / synonym: Human / Cell: HEK293
• Sequence: UniProtKB: Nicastrin

Macromolecule #3: Gamma-secretase subunit APH-1A

• Macromolecule: Name: Gamma-secretase subunit APH-1A / type: protein_or_peptide / ID: 3 / Name.synonym: Aph-1alpha / Number of copies: 1 / Recombinant expression: No
• Source (natural): Organism: Homo sapiens (human) / synonym: Human / Cell: HEK293
• Sequence: UniProtKB: Gamma-secretase subunit APH-1A

Macromolecule #4: Gamma-secretase subunit PEN-2

• Macromolecule: Name: Gamma-secretase subunit PEN-2 / type: protein_or_peptide / ID: 4 / Name.synonym: Presenilin enhancer protein 2 / Number of copies: 1 / Recombinant expression: No
• Source (natural): Organism: Homo sapiens (human) / synonym: Human / Cell: HEK293
• Sequence: UniProtKB: Gamma-secretase subunit PEN-2

Macromolecule #5: E ((S,S)- 2-[2-(3,5-Difluorophenyl)-acetylamino]-N-(1-methyl-2-ox...

• Macromolecule: Name: E ((S,S)- 2-[2-(3,5-Difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide); type: ligand / ID: 5 / Name.synonym: gamma-Secretase Inhibitor XXI / Number of copies: 1 / Recombinant expression: No
• Source (natural): Organism: synthetic construct (others)

Experimental details

Structure determination

• Method: negative staining
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.02 mg/mL
• Buffer: pH: 7.4 ; Details: 50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 5 mM MgCl2, 5 mM CaCl2
• Staining: Type: NEGATIVE; Details: Grids with adsorbed protein floated on 1% w/v uranyl acetate for 2-10 seconds
• Grid: Details: Carbon-coated 400-mesh copper grids were glow discharged in air at 600-700 V for 30-60 seconds on an Edward S150B sputter coater.
• Vitrification: Cryogen name: NONE / Instrument: OTHER

Electron microscopy

• Microscope: FEI TECNAI 12
• Date: Sep 19, 2009
• Image recording: Category: CCD / Film or detector model: TVIPS TEMCAM-F224 (2k x 2k) / Number real images: 300
• Electron beam: Acceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 1.0 µm
• Sample stage: Specimen holder model: OTHER

Image processing

• Details: The particles were selected using EMAN2
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.4 Å / Resolution method: OTHER / Software - Name: EMAN2, RELION / Number images used: 10651