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- EMDB-22279: CryoEM structure of human presequence protease in partial open state 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-22279
TitleCryoEM structure of human presequence protease in partial open state 2
Map data
Sample
  • Complex: CryoEM map of Human Presequence Protease in partial open state 1
    • Protein or peptide: Presequence protease, mitochondrial
KeywordsPartial open state / HYDROLASE
Function / homology
Function and homology information


Mitochondrial protein import / protein targeting to mitochondrion / Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases / enzyme activator activity / protein processing / metalloendopeptidase activity / metallopeptidase activity / mitochondrial matrix / mitochondrion / proteolysis / zinc ion binding
Similarity search - Function
Peptidase M16C associated / Peptidase M16C associated / Peptidase M16C associated / Peptidase M16, C-terminal / Peptidase M16 inactive domain / Peptidase M16, N-terminal / Insulinase (Peptidase family M16) / Metalloenzyme, LuxS/M16 peptidase-like
Similarity search - Domain/homology
Presequence protease, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsLiang WG / Zhao M
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Aging (NIH/NIA)R01 GM121964 United States
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis for the mechanisms of human presequence protease conformational switch and substrate recognition.
Authors: Wenguang G Liang / Juwina Wijaya / Hui Wei / Alex J Noble / Jordan M Mancl / Swansea Mo / David Lee / John V Lin King / Man Pan / Chang Liu / Carla M Koehler / Minglei Zhao / Clinton S ...Authors: Wenguang G Liang / Juwina Wijaya / Hui Wei / Alex J Noble / Jordan M Mancl / Swansea Mo / David Lee / John V Lin King / Man Pan / Chang Liu / Carla M Koehler / Minglei Zhao / Clinton S Potter / Bridget Carragher / Sheng Li / Wei-Jen Tang /
Abstract: Presequence protease (PreP), a 117 kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other ...Presequence protease (PreP), a 117 kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3-4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, these structures reveal the key stages of the PreP catalytic cycle and how the binding of substrates or PreP inhibitor drives a rigid body motion of the protein for substrate binding and catalysis. Together, our studies provide key mechanistic insights into M16C metalloproteases for future therapeutic innovations.
History
DepositionJul 7, 2020-
Header (metadata) releaseJul 7, 2021-
Map releaseJul 7, 2021-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xot
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22279.png

Downloads & links

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Map

FileDownload / File: emd_22279.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.856 Å
Density
Contour LevelBy AUTHOR: 0.005 / Movie #1: 0.008
Minimum - Maximum-0.015979938 - 0.033516403
Average (Standard dev.)-0.000000850273 (±0.0012903472)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 219.136 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8560.8560.856
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z219.136219.136219.136
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0160.034-0.000

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Supplemental data

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Mask #1

Fileemd_22279_msk_1.map
Projections & Slices
AxesZYX

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Additional map: #1

Fileemd_22279_additional_1.map
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Half map: #1

Fileemd_22279_half_map_1.map
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Half map: #2

Fileemd_22279_half_map_2.map
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Sample components

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Entire : CryoEM map of Human Presequence Protease in partial open state 1

EntireName: CryoEM map of Human Presequence Protease in partial open state 1
Components
  • Complex: CryoEM map of Human Presequence Protease in partial open state 1
    • Protein or peptide: Presequence protease, mitochondrial

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Supramolecule #1: CryoEM map of Human Presequence Protease in partial open state 1

SupramoleculeName: CryoEM map of Human Presequence Protease in partial open state 1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Presequence protease, mitochondrial

MacromoleculeName: Presequence protease, mitochondrial / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 114.901461 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MHHHHHHAAA CERALQYKLG DKIHGFTVNQ VTSVPELFLT AVKLTHDDTG ARYLHLARED TNNLFSVQFR TTPMDSTGVP HILEHTVLC GSQKYPCRDP FFKMLNRSLS TFMNAFTASD YTLYPFSTQN PKDFQNLLSV YLDATFFPCL RELDFWQEGW R LEHENPSD ...String:
MHHHHHHAAA CERALQYKLG DKIHGFTVNQ VTSVPELFLT AVKLTHDDTG ARYLHLARED TNNLFSVQFR TTPMDSTGVP HILEHTVLC GSQKYPCRDP FFKMLNRSLS TFMNAFTASD YTLYPFSTQN PKDFQNLLSV YLDATFFPCL RELDFWQEGW R LEHENPSD PQTPLVFKGV VFNEMKGAFT DNERIFSQHL QNRLLPDHTY SVVSGGDPLC IPELTWEQLK QFHATHYHPS NA RFFTYGN FPLEQHLKQI HEEALSKFQK IEPSTVVPAQ TPWDKPREFQ ITCGPDSFAT DPSKQTTVSV SFLLPDITDT FEA FTLSLL SSLLTSGPNS PFYKALIESG LGTDFSPDVG YNGYTREAYF SVGLQGIVEK DIETVRSLID RTIDEVVEKG FEDD RIEAL LHKIEIQMKH QSTSFGLMLT SYIASCWNHD GDPVELLKLG NQLAKFRQCL QENPKFLQEK VKQYFKNNQH KLTLS MRPD DKYHEKQAQV EATKLKQKVE ALSPGDRQQI YEKGLELRSQ QSKPQDASCL PALKVSDIEP TIPVTELDVV LTAGDI PVQ YCAQPTNGMV YFRAFSSLNT LPEELRPYVP LFCSVLTKLG CGLLDYREQA QQIELKTGGM SASPHVLPDD SHMDTYE QG VLFSSLCLDR NLPDMMQLWS EIFNNPCFEE EEHFKVLVKM TAQELANGIP DSGHLYASIR AGRTLTPAGD LQETFSGM D QVRLMKRIAE MTDIKPILRK LPRIKKHLLN GDNMRCSVNA TPQQMPQTEK AVEDFLRSIG RSKKERRPVR PHTVEKPVP SSSGGDAHVP HGSQVIRKLV MEPTFKPWQM KTHFLMPFPV NYVGECIRTV PYTDPDHASL KILARLMTAK FLHTEIREKG GAYGGGAKL SHNGIFTLYS YRDPNTIETL QSFGKAVDWA KSGKFTQQDI DEAKLSVFST VDAPVAPSDK GMDHFLYGLS D EMKQAHRE QLFAVSHDKL LAVSDRYLGT GKSTHGLAIL GPENPKIAKD PSWIIR

UniProtKB: Presequence protease, mitochondrial

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.7
Details: 20 mM Tris, pH 7.7, 150 mM NaCl, 10mM KCl, 20 mM EDTA and 1 mM 2-mercaptoethanol
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 308 K / Instrument: SPOTITON

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average exposure time: 6.0 sec. / Average electron dose: 62.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 139127
FSC plot (resolution estimation)

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