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- PDB-6f3k: Combined solid-state NMR, solution-state NMR and EM data for stru... -

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Basic information

Entry
Database: PDB / ID: 6f3k
TitleCombined solid-state NMR, solution-state NMR and EM data for structure determination of the tetrahedral aminopeptidase TET2 from P. horikoshii
ComponentsTetrahedral aminopeptidase
KeywordsPEPTIDE BINDING PROTEIN / peptidase / protein quality control / oligomer / aminopeptidase
Function/homologyPeptidase M42, domain 2 / Peptidase M42 / M42 glutamyl aminopeptidase / Aminopeptidases / aminopeptidase activity / metallopeptidase activity / metal ion binding / Tetrahedral aminopeptidase
Function and homology information
Specimen sourcePyrococcus horikoshii / archaea / thermophilic /
MethodElectron microscopy + Solution NMR + Solid-state NMR (4.1 Å resolution / Particle / Single particle) / Transmission electron microscopy / Nuclear magnetic resonance / Solid-state nuclear magnetic resonance
AuthorsGauto, D.F. / Estrozi, L.F. / Schwieters, C.D. / Effantin, G. / Macek, P. / Sounier, R. / Kerfah, R. / Sivertsen, A.C. / Colletier, J.P. / Boisbouvier, J. / Schoehn, G. / Favier, A. / Schanda, P.
CitationJournal: To Be Published
Title: Atomic-resolution structure of the 468 kDa TET2 protease machinery from an integrated NMR and cryo-EM approach
Authors: Gauto, D.F. / Estrozi, L. / Macek, P. / Effantin, G. / Sounier, R. / Kerfah, R. / Colletier, J.P. / Schwieters, C.D. / Boisbouvier, J. / Schoehn, G. / Favier, A. / Schanda, P.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 28, 2017 / Release: Mar 14, 2018

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Assembly

Deposited unit
A: Tetrahedral aminopeptidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,2023
Polyers39,0711
Non-polymers1312
Water0
1


  • idetical with deposited unit
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  • Evidence: gel filtration, microscopy, CryoEM, negative staining EM
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)0
ΔGint (kcal/M)0
Surface area (Å2)16540
MethodPISA
NMR ensembles
Datacriteria
Number of conformers (submitted / calculated)10 / -
Representative

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Components

#1: Protein/peptide Tetrahedral aminopeptidase / TET aminopeptidase / Leucyl aminopeptidase / PhTET2


Mass: 39071.027 Da / Num. of mol.: 1
Source: (gene. exp.) Pyrococcus horikoshii (strain atcc 700860 / dsm 12428 / jcm 9974 / nbrc 100139 / ot-3) / archaea / thermophilic /
Strain: ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3
Gene: frvX, PH1527 / Production host: Escherichia coli / References: UniProt:O59196, EC:3.4.11.- (Aminopeptidases)
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Formula: Zn / : Zinc

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Experimental details

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Experiment

Experiment
Method
ELECTRON MICROSCOPY
SOLUTION NMR
SOLID-STATE NMR
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE
NMR experiment
Conditions IDExperiment IDSolution IDSample stateSpectrometer IDType
111isotropic1RFDR 3D
323isotropic1DARR
232isotropic23D 1H-13C NOESY aliphatic
344isotropic2CCdream (CC)
354isotropic2NCO
364isotropic2NCOCX
374isotropic2NCACX
384isotropic2CONCACB
394isotropic2CANCOCX
1105isotropic1hCANH
1115isotropic1hCONH
1125isotropic1hcoCAcoNH

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Sample preparation

ComponentName: tetrahedral amino-peptidase from P. horikoshii / Type: COMPLEX / Details: H78C/H248C mutant / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 0.49 deg. / Units: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Pyrococcus horikoshii
Source (recombinant)Organism: Escherichia coli
Buffer solutionpH: 7.4
SpecimenConc.: 1 mg/ml / Details: This sample was monodisperse. / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 kelvins / Details: Blotting time 2s, force 1, drain time 0.
NMR sample details
TypeSolution IDContentsDetailsLabelSolvent system
solid120 mM TRIS, 50 mM sodium chloride, 100% H2Oprotein solution at 10 mg/ml was supplemented by methyl-pentanediol, resulting in microcrystalline precipitate. Labeling 2H15N and 13C at methyls of ILV.2H15N_ILVCHD2100% H2O
solid520 mM TRIS, 50 mM sodium chloride, 100% H2O2H13C15N100% H2O
solid320 mM Tris, 50 mM NaCl, 100% H2Oprotein solution at 10 mg/ml was supplemented by methyl-pentanediol, resulting in microcrystalline precipitate. Labeling 13C at all carbons of LKP residuesLKP-labeled100% H2O
solid420 mM TRIS, 50 mM sodium chloride, 100% H2OH2O13C15N TET2100% H2O
solution250 mM Tris, 50 mM NaCl, 100% D2O2H15N_ILV-CH3100% D2O
NMR experiment sample
Conc.UnitsComponentIsotopic labelingSolution ID
20mMTRISnone1
50mMsodium chloridenone1
20mMTRISnone5
50mMsodium chloridenone5
20mMTrisnone3
50mMNaClnone3
20mMTRISnone4
50mMsodium chloridenone4
50mMTrisnone2
50mMNaClnone2
NMR experiment sample conditions

Ionic strength: 50 / Ionic strength units: mM / PH units: pH / Pressure: 1 Pa / Pressure units: atm / Temperature units: K

Conditions IDLabelpHTemperatureTemperature err
1MAS_50 kHz7.53002
315kHz MAS7.5300
2solution8328

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Data collection

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 20000 / Calibrated magnification: 25773 / Calibrated defocus min: 800 nm / Calibrated defocus max: 3500 nm / Cs: 2 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of real images: 90
Image scansMovie frames/image: 40
NMR spectrometer
ManufacturerModelField strengthSpectrometer IDDetails
BrukerAvanceII6001solid-state NMR
VarianINOVA8002solution-NMR NOEs

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Processing

EM software
IDNameVersionCategory
1RELION1.4PARTICLE SELECTION
2Latitude3IMAGE ACQUISITION
4CTFFIND3CTF CORRECTION
7iMODFIT1.44MODEL FITTING
9RELION1.4INITIAL EULER ASSIGNMENT
10RELION1.4FINAL EULER ASSIGNMENT
11RELION1.4CLASSIFICATION
12RELION1.4RECONSTRUCTION
13X-PLOR4.39MODEL REFINEMENT
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 30407
SymmetryPoint symmetry: T
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 27130 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingRef protocol: OTHER
NMR software
NameAuthorsClassification
CcpNMRCCPNrefinement
CYANAGuntert, Mumenthaler and Wuthrichstructure calculation
AnalysisCCPNchemical shift assignment
AnalysisCCPNpeak picking
X-PLOR NIHSchwieters, Kuszewski, Tjandra and Clorestructure calculation
NMR ensembleConformers submitted total number: 10

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