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- PDB-5fn5: Cryo-EM structure of gamma secretase in class 3 of the apo- state... -

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Basic information

Entry
Database: PDB / ID: 5fn5
TitleCryo-EM structure of gamma secretase in class 3 of the apo- state ensemble
Components
  • Gamma-secretase subunit APH-1A
  • Gamma-secretase subunit PEN-2
  • Nicastrin
  • Presenilin-1
KeywordsHYDROLASE
Function / homology
Function and homology information


positive regulation of endopeptidase activity / gamma-secretase complex / aspartic endopeptidase activity, intramembrane cleaving / Noncanonical activation of NOTCH3 / protein catabolic process at postsynapse / TGFBR3 PTM regulation / Notch receptor processing / skin morphogenesis / membrane protein intracellular domain proteolysis / NOTCH4 Activation and Transmission of Signal to the Nucleus ...positive regulation of endopeptidase activity / gamma-secretase complex / aspartic endopeptidase activity, intramembrane cleaving / Noncanonical activation of NOTCH3 / protein catabolic process at postsynapse / TGFBR3 PTM regulation / Notch receptor processing / skin morphogenesis / membrane protein intracellular domain proteolysis / NOTCH4 Activation and Transmission of Signal to the Nucleus / ciliary rootlet / neural retina development / Regulated proteolysis of p75NTR / mitochondria-associated endoplasmic reticulum membrane contact site / aggresome / amyloid precursor protein metabolic process / endoplasmic reticulum calcium ion homeostasis / regulation of synaptic vesicle cycle / regulation of postsynapse organization / astrocyte activation involved in immune response / regulation of neuron projection development / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / regulation of canonical Wnt signaling pathway / Golgi cisterna membrane / growth factor receptor binding / azurophil granule membrane / positive regulation of amyloid fibril formation / positive regulation of dendritic spine development / amyloid-beta formation / amyloid precursor protein catabolic process / membrane protein ectodomain proteolysis / smooth endoplasmic reticulum / positive regulation of receptor recycling / nuclear outer membrane / EPH-ephrin mediated repulsion of cells / cerebellum development / Notch signaling pathway / negative regulation of ubiquitin-dependent protein catabolic process / Nuclear signaling by ERBB4 / endopeptidase activator activity / calcium ion homeostasis / Degradation of the extracellular matrix / rough endoplasmic reticulum / neuron projection maintenance / astrocyte activation / positive regulation of glycolytic process / NOTCH2 Activation and Transmission of Signal to the Nucleus / NRIF signals cell death from the nucleus / Activated NOTCH1 Transmits Signal to the Nucleus / dendritic shaft / protein processing / PDZ domain binding / neuromuscular junction / NOTCH3 Activation and Transmission of Signal to the Nucleus / cell-cell adhesion / memory / synapse organization / sarcolemma / beta-catenin binding / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / cellular response to amyloid-beta / calcium channel activity / kinetochore / positive regulation of tumor necrosis factor production / melanosome / regulation of gene expression / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / negative regulation of neuron apoptotic process / synaptic vesicle / nuclear membrane / ATPase binding / growth cone / presynaptic membrane / early endosome membrane / endopeptidase activity / cell cortex / aspartic-type endopeptidase activity / molecular adaptor activity / early endosome / learning or memory / protein-macromolecule adaptor activity / postsynapse / endosome membrane / neuron projection / mitochondrial inner membrane / intracellular signal transduction / cadherin binding / membrane raft / Amyloid fiber formation / negative regulation of gene expression / Golgi membrane / lysosomal membrane / focal adhesion / apoptotic process / neuronal cell body / centrosome / positive regulation of gene expression / negative regulation of apoptotic process / Neutrophil degranulation
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 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 11, 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 1.3Oct 23, 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)172,2534
Polymers172,2534
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: 52713.535 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: Oct 25, 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: 3200 nm / Nominal defocus min: 700 nm / Cs: 2.7 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: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 66720 / Refinement type: HALF-MAPS REFINED INDEPENDENTLY / Symmetry type: POINT
RefinementHighest resolution: 4.3 Å
Refinement stepCycle: LAST / Highest resolution: 4.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9645 0 0 0 9645

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