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

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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 / M42 glutamyl aminopeptidase / Peptidase M42 / Hydrolases; Acting on peptide bonds (peptidases); Aminopeptidases / aminopeptidase activity / metallopeptidase activity / proteolysis / metal ion binding / Tetrahedral aminopeptidase
Function and homology information
Biological speciesPyrococcus horikoshii (archaea)
MethodELECTRON MICROSCOPY / SOLUTION NMR / SOLID-STATE NMR / single particle reconstruction / cryo EM / Resolution: 4.1 Å
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. ...Gauto, 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.
Funding support France, 3items
OrganizationGrant numberCountry
European Research Council311318
French National Research AgencyANR-10-INSB-05-02 France
French National Research AgencyANR-10-LABX-49-01 France
CitationJournal: Nat Commun / Year: 2019
Title: Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex.
Authors: Diego F Gauto / Leandro F Estrozi / Charles D Schwieters / Gregory Effantin / Pavel Macek / Remy Sounier / Astrid C Sivertsen / Elena Schmidt / Rime Kerfah / Guillaume Mas / Jacques-Philippe ...Authors: Diego F Gauto / Leandro F Estrozi / Charles D Schwieters / Gregory Effantin / Pavel Macek / Remy Sounier / Astrid C Sivertsen / Elena Schmidt / Rime Kerfah / Guillaume Mas / Jacques-Philippe Colletier / Peter Güntert / Adrien Favier / Guy Schoehn / Paul Schanda / Jerome Boisbouvier /
Abstract: Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely ...Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enables structure determination of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and ILV methyl groups, and a 4.1 Å resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution cryo-EM data are available.
History
DepositionNov 28, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 14, 2018Provider: repository / Type: Initial release
Revision 1.1May 8, 2019Group: Data collection / Derived calculations
Category: em_admin / pdbx_database_proc ...em_admin / pdbx_database_proc / pdbx_struct_assembly_gen / pdbx_struct_oper_list
Item: _em_admin.last_update / _pdbx_struct_assembly_gen.oper_expression
Revision 1.2Aug 21, 2019Group: Data collection / Category: pdbx_nmr_software / pdbx_nmr_spectrometer
Item: _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model
Revision 1.3Aug 28, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.4Sep 13, 2023Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation

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Assembly

Deposited unit
A: Tetrahedral aminopeptidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,2023
Polymers39,0711
Non-polymers1312
Water0
1
A: Tetrahedral aminopeptidase
hetero molecules
x 12


Theoretical massNumber of molelcules
Total (without water)470,42236
Polymers468,85212
Non-polymers1,57024
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation11
Buried area0 Å2
ΔGint0 kcal/mol
Surface area16540 Å2
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / -
Representative

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Components

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


Mass: 39071.027 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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, Hydrolases; Acting on peptide bonds (peptidases); Aminopeptidases
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Experimental details

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Experiment

Experiment
Method
ELECTRON MICROSCOPY
SOLUTION NMR
SOLID-STATE NMR
EM experimentAggregation state: PARTICLE / 3D 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 / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / 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. (mg/ml)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
Conditions-IDIonic strengthLabelpHPressure (kPa)Temperature (K)Temperature err
150 mMMAS_50 kHz7.5 1 atm300 K2
350 mM15kHz MAS7.5 1 atm300 K
250 mMsolution8 1 atm328 K

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Data collection

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: 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 X / Calibrated magnification: 25773 X / 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) / Num. of real images: 90
Image scansMovie frames/image: 40
NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Bruker AVANCE IIBrukerAVANCE II6001solid-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 selectionNum. of particles selected: 30407
SymmetryPoint symmetry: T (tetrahedral)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27130 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
NMR software
NameDeveloperClassification
CcpNmr AnalysisCCPNrefinement
CYANAGuntert, Mumenthaler and Wuthrichstructure calculation
CcpNmr AnalysisCCPNchemical shift assignment
CcpNmr AnalysisCCPNpeak picking
Xplor-NIHSchwieters, Kuszewski, Tjandra and Clorestructure calculation
NMR ensembleConformers submitted total number: 10

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