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- PDB-5fn2: Cryo-EM structure of gamma secretase in complex with a drug DAPT -

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Basic information

Entry
Database: PDB / ID: 5fn2
TitleCryo-EM structure of gamma secretase in complex with a drug DAPT
Components
  • Gamma-secretase subunit APH-1A
  • Gamma-secretase subunit PEN-2
  • Nicastrin
  • Presenilin-1
KeywordsHYDROLASE
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 coagulation / gamma-secretase complex / aspartic endopeptidase activity, intramembrane cleaving / short-term synaptic potentiation / positive regulation of endopeptidase activity / positive regulation of amyloid precursor protein biosynthetic process ...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 coagulation / gamma-secretase complex / aspartic endopeptidase activity, intramembrane cleaving / short-term synaptic potentiation / positive regulation of endopeptidase activity / positive regulation of amyloid precursor protein biosynthetic process / protein catabolic process at postsynapse / Noncanonical activation of NOTCH3 / TGFBR3 PTM regulation / sequestering of calcium ion / Notch receptor processing / central nervous system myelination / synaptic vesicle targeting / negative regulation of axonogenesis / membrane protein intracellular domain proteolysis / regulation of resting membrane potential / choline transport / T cell activation involved in immune response / skin morphogenesis / NOTCH4 Activation and Transmission of Signal to the Nucleus / 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 / metanephros development / regulation of phosphorylation / locomotion / brain morphogenesis / endoplasmic reticulum calcium ion homeostasis / nuclear outer membrane / amyloid precursor protein metabolic process / smooth endoplasmic reticulum calcium ion homeostasis / regulation of long-term synaptic potentiation / astrocyte activation involved in immune response / regulation of canonical Wnt signaling pathway / embryonic limb morphogenesis / aggresome / cell fate specification / skeletal system morphogenesis / glutamate receptor signaling pathway / myeloid cell homeostasis / azurophil granule membrane / regulation of postsynapse organization / ciliary rootlet / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / G protein-coupled dopamine receptor signaling pathway / Golgi cisterna membrane / positive regulation of amyloid fibril formation / mitochondrial transport / positive regulation of dendritic spine development / positive regulation of receptor recycling / adult behavior / blood vessel development / regulation of neuron projection development / heart looping / amyloid precursor protein catabolic process / cerebral cortex cell migration / protein glycosylation / amyloid-beta formation / negative regulation of apoptotic signaling pathway / membrane protein ectodomain proteolysis / endopeptidase activator activity / autophagosome assembly / EPH-ephrin mediated repulsion of cells / smooth endoplasmic reticulum / hematopoietic progenitor cell differentiation / neuron development / somitogenesis / negative regulation of ubiquitin-dependent protein catabolic process / calcium ion homeostasis / Nuclear signaling by ERBB4 / T cell proliferation / rough endoplasmic reticulum / Notch signaling pathway / regulation of synaptic transmission, glutamatergic / neuron projection maintenance / Degradation of the extracellular matrix / NOTCH2 Activation and Transmission of Signal to the Nucleus / positive regulation of glycolytic process / cellular response to calcium ion / NRIF signals cell death from the nucleus / Activated NOTCH1 Transmits Signal to the Nucleus / cerebellum development / post-embryonic development / thymus development / epithelial cell proliferation / negative regulation of protein phosphorylation / dendritic shaft / apoptotic signaling pathway / astrocyte activation / NOTCH3 Activation and Transmission of Signal to the Nucleus / PDZ domain binding / neuron migration
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsBai, X.C. / Rajendra, E. / Yang, G.H. / Shi, Y.G. / Scheres, S.H.W.
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, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 16, 2015Provider: repository / Type: Initial release
Revision 1.1Dec 21, 2016Group: Database references
Revision 1.2Sep 4, 2019Group: Data collection / Database references ...Data collection / Database references / Source and taxonomy / Structure summary
Category: entity / entity_name_com ...entity / entity_name_com / entity_src_gen / struct_ref
Item: _entity.pdbx_description / _entity_src_gen.gene_src_common_name ..._entity.pdbx_description / _entity_src_gen.gene_src_common_name / _entity_src_gen.pdbx_beg_seq_num / _entity_src_gen.pdbx_end_seq_num / _entity_src_gen.pdbx_gene_src_gene / _entity_src_gen.pdbx_gene_src_scientific_name / _entity_src_gen.pdbx_seq_type / _struct_ref.pdbx_align_begin / _struct_ref.pdbx_seq_one_letter_code
Revision 1.3Nov 6, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Nicastrin
B: Presenilin-1
C: Gamma-secretase subunit APH-1A
D: Gamma-secretase subunit PEN-2


Theoretical massNumber of molelcules
Total (without water)172,1914
Polymers172,1914
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

#1: Protein Nicastrin


Mass: 78483.570 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: A DRUG DAPT WAS BOUND TO GAMMA SECRETASE COMPLEX / Source: (gene. exp.) Homo sapiens (human) / Cell line: HEK293F / Gene: NCSTN, KIAA0253, UNQ1874/PRO4317 / Plasmid: PMLINK / Production host: HOMO SAPIENS (human) / References: UniProt: Q92542
#2: Protein Presenilin-1 / PS-1 / Protein S182


Mass: 52651.465 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: A DRUG DAPT WAS BOUND TO GAMMA SECRETASE COMPLEX / Source: (gene. exp.) Homo sapiens (human) / Cell line: HEK293F / Gene: PSEN1, AD3, PS1, PSNL1 / Plasmid: PMLINK / Production host: HOMO SAPIENS (human)
References: UniProt: P49768, Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases
#3: Protein Gamma-secretase subunit APH-1A / APH-1a / Aph-1alpha / Presenilin-stabilization factor


Mass: 29017.943 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: A DRUG DAPT WAS BOUND TO GAMMA SECRETASE COMPLEX / Source: (gene. exp.) Homo sapiens (human) / Cell line: HEK293F / Gene: APH1A, PSF, CGI-78, UNQ579/PRO1141 / Plasmid: PMLINK / Production host: HOMO SAPIENS (human) / References: UniProt: Q96BI3
#4: Protein Gamma-secretase subunit PEN-2 / Presenilin enhancer protein 2


Mass: 12038.029 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: A DRUG DAPT WAS BOUND TO GAMMA SECRETASE COMPLEX / Source: (gene. exp.) Homo sapiens (human) / Cell line: HEK293F / Gene: PSENEN, PEN2, MDS033 / Plasmid: PMLINK / Production host: HOMO SAPIENS (human) / References: UniProt: Q9NZ42
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: GAMMA SECRETASE / Type: COMPLEX
Buffer solutionName: 25 MM HEPES, PH 7.4, 150 MM NACL AND AMPHIPOL A8-35 / pH: 7.4
Details: 25 MM HEPES, PH 7.4, 150 MM NACL AND AMPHIPOL A8-35
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Details: LIQUID ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Dec 31, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 35714 X / Nominal defocus max: 2700 nm / Nominal defocus min: 800 nm / Cs: 2 mm
Specimen holderTemperature: 85 K
Image recordingElectron dose: 38 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)
Image scansNum. digital images: 2000

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Processing

SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51366 / Refinement type: HALF-MAPS REFINED INDEPENDENTLY / Symmetry type: POINT
RefinementHighest resolution: 4.2 Å
Refinement stepCycle: LAST / Highest resolution: 4.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10276 0 0 0 10276

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