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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 / Peptidase M42, domain 2 / M42 glutamyl aminopeptidase / Aminopeptidases / aminopeptidase activity / metallopeptidase activity / metal ion binding / Tetrahedral aminopeptidase
Function and homology information
Specimen sourcePyrococcus horikoshii (archaea)
MethodELECTRON MICROSCOPY / SOLUTION NMR / SOLID-STATE NMR / single particle reconstruction / cryo EM / 4.1 Å resolution
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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Structure viewerMolecule:
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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
  • defined by software
  • 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)
Strain: ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3
Gene: frvX, PH1527 / Production host: Escherichia coli (E. coli) / References: UniProt: O59196, 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 reconstruction
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 MDa / Experimental value: NO
Source (natural)Organism: Pyrococcus horikoshii (archaea)
Source (recombinant)Organism: Escherichia coli (E. 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.
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
Sample
Conc.ComponentIsotopic labelingSolution ID
20 mMTRISnone1
50 mMsodium chloridenone1
20 mMTRISnone5
50 mMsodium chloridenone5
20 mMTrisnone3
50 mMNaClnone3
20 mMTRISnone4
50 mMsodium chloridenone4
50 mMTrisnone2
50 mMNaClnone2
sample conditions

Ionic strength: 50 mM / Pressure: 1 atm

Conditions IDLabelpHTemperatureTemperature err
1MAS_50 kHz7.5 300 K2
315kHz MAS7.5 300 K
2solution8 328 K

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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 FIELDBright-field microscopy / 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
TypeManufacturerModelField strengthSpectrometer IDDetails
Bruker AvanceIIBrukerAvanceII6001solid-state NMR
Varian INOVAVarianINOVA8002solution-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
12RELION1.43D reconstruction
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
NameDeveloperClassification
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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