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- PDB-5upw: CryoEM Structure Refinement by Integrating NMR Chemical Shifts wi... -

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Basic information

Entry
Database: PDB / ID: 5upw
TitleCryoEM Structure Refinement by Integrating NMR Chemical Shifts with Molecular Dynamics Simulations
ComponentsGag polyproteinGroup-specific antigen
KeywordsVIRAL PROTEIN / Cryo-EM / HIV capsid / Chemical shift / Molecular Dynamics / hydrolase / viral protein
Function / homologyRetrovirus capsid, C-terminal / Zinc finger CCHC-type profile. / gag gene protein p24 (core nucleocapsid protein) / gag gene protein p17 (matrix protein) / Zinc knuckle / Zinc finger, CCHC-type superfamily / Gag protein p6 / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral matrix protein / Retrovirus capsid, N-terminal ...Retrovirus capsid, C-terminal / Zinc finger CCHC-type profile. / gag gene protein p24 (core nucleocapsid protein) / gag gene protein p17 (matrix protein) / Zinc knuckle / Zinc finger, CCHC-type superfamily / Gag protein p6 / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral matrix protein / Retrovirus capsid, N-terminal / Zinc finger, CCHC-type / Retroviral nucleocapsid protein Gag / Immunodeficiency lentiviral matrix, N-terminal / ISG15 antiviral mechanism / Gag protein p6 / viral budding via host ESCRT complex / host multivesicular body / viral nucleocapsid / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / RNA binding / zinc ion binding / Gag polyprotein
Function and homology information
Specimen sourceHuman immunodeficiency virus type 1 (HIV)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / 5 Å resolution
AuthorsPerilla, J.R.
CitationJournal: J Phys Chem B / Year: 2017
Title: CryoEM Structure Refinement by Integrating NMR Chemical Shifts with Molecular Dynamics Simulations.
Authors: Juan R Perilla / Gongpu Zhao / Manman Lu / Jiying Ning / Guangjin Hou / In-Ja L Byeon / Angela M Gronenborn / Tatyana Polenova / Peijun Zhang
Abstract: Single particle cryoEM has emerged as a powerful method for structure determination of proteins and complexes, complementing X-ray crystallography and NMR spectroscopy. Yet, for many systems, the ...Single particle cryoEM has emerged as a powerful method for structure determination of proteins and complexes, complementing X-ray crystallography and NMR spectroscopy. Yet, for many systems, the resolution of cryoEM density map has been limited to 4-6 Å, which only allows for resolving bulky amino acids side chains, thus hindering accurate model building from the density map. On the other hand, experimental chemical shifts (CS) from solution and solid state MAS NMR spectra provide atomic level data for each amino acid within a molecule or a complex; however, structure determination of large complexes and assemblies based on NMR data alone remains challenging. Here, we present a novel integrated strategy to combine the highly complementary experimental data from cryoEM and NMR computationally by molecular dynamics simulations to derive an atomistic model, which is not attainable by either approach alone. We use the HIV-1 capsid protein (CA) C-terminal domain as well as the large capsid assembly to demonstrate the feasibility of this approach, termed NMR CS-biased cryoEM structure refinement.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 4, 2017 / Release: Mar 1, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Mar 1, 2017Structure modelrepositoryInitial release
1.1May 3, 2017Structure modelDatabase references
1.2Aug 2, 2017Structure modelData collectionem_software_em_software.name
1.3Dec 6, 2017Structure modelStructure summarystruct_keywords_struct_keywords.pdbx_keywords / _struct_keywords.text

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Structure visualization

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Assembly

Deposited unit
A: Gag polyprotein
B: Gag polyprotein
C: Gag polyprotein
D: Gag polyprotein
E: Gag polyprotein
F: Gag polyprotein


Theoretical massNumber of molelcules
Total (without water)147,9266
Polyers147,9266
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)14440
ΔGint (kcal/M)-77
Surface area (Å2)66770
MethodPISA

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Components

#1: Protein/peptide
Gag polyprotein / Group-specific antigen / Pr55Gag


Mass: 24654.268 Da / Num. of mol.: 6 / Fragment: UNP residues 133-353 / Mutation: A92E
Source: (gene. exp.) Human immunodeficiency virus type 1 (NEW YORK-5 ISOLATE)
Gene: gag / Production host: Escherichia coli (E. coli) / References: UniProt:P12493

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / Reconstruction method: helical reconstruction

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Sample preparation

ComponentName: HIV-1 Capsid Protein Assembly / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 24 deg. / Units: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Human immunodeficiency virus type 1 (NEW YORK-5 ISOLATE)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.UnitsNameFormulaBuffer ID
11Msodium chlorideNaCl1
250mMTrisTris1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 / Grid type: Quantifoil R2/1
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 kelvins
Details: The assembled sample (1.5 microliter) was applied to the carbon side of a glow discharged perforated Quantifoil grid, followed by application of 3 microliter of low salt buffer (100 milimolar NaCl, 50 milimolar Tris pH 8.0) on the back side of the grid, and blotting, from the back side, with a filter paper, before plunge-freezing in liquid ethane

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Electron microscopy imaging

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: 31000 / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm / Cs: 2.2 mm / C2 aperture diameter: 100 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: SIDE ENTRY, EUCENTRIC
Image recordingAverage exposure time: 6 sec. / Electron dose: 41 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of real images: 523
Image scansMovie frames/image: 30

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Processing

EM software
IDNameVersionCategory
1EMAN2particle selection
4CTFFIND3CTF correction
7MDFF2.10model fitting
9Rosetta3.0model refinement
10IHRSRinitial Euler assignment
11RELION1.4final Euler assignment
13RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -31.13 deg. / Axial rise/subunit: 6.94 Å / Axial symmetry: C1
Particle selectionNumber of particles selected: 39712
3D reconstructionResolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 38452 / Algorithm: FOURIER SPACE / Number of class averages: 3 / Symmetry type: HELICAL
Atomic model buildingRef protocol: FLEXIBLE FIT / Ref space: REAL
Atomic model buildingPDB-ID: 4XFX
Pdb chain residue range: 1-220

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