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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 polyprotein
KeywordsVIRAL PROTEIN / Cryo-EM / HIV capsid / Chemical shift / Molecular Dynamics / hydrolase
Function / homology
Function and homology information


viral budding via host ESCRT complex / host multivesicular body / ISG15 antiviral mechanism / viral nucleocapsid / viral translational frameshifting / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / RNA binding ...viral budding via host ESCRT complex / host multivesicular body / ISG15 antiviral mechanism / viral nucleocapsid / viral translational frameshifting / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / RNA binding / zinc ion binding / membrane
Similarity search - Function
Gag protein p6 / Gag protein p6 / : / gag protein p24 N-terminal domain / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retrovirus capsid, C-terminal ...Gag protein p6 / Gag protein p6 / : / gag protein p24 N-terminal domain / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retrovirus capsid, C-terminal / Retroviral matrix protein / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile.
Similarity search - Domain/homology
Biological speciesHuman immunodeficiency virus type 1
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 5 Å
AuthorsPerilla, J.R.
Funding support United States, 6items
OrganizationGrant numberCountry
National Institutes of HealthP50 GM082251 United States
National Institutes of HealthP41 GM104601 United States
National Institutes of HealthR01 GM067887 United States
National Science Foundation (NSF, United States)OCI-0725070 United States
National Science Foundation (NSF, United States)ACI-1238993 United States
National Science Foundation (NSF, United States)ACI-1440026 United States
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.
History
DepositionFeb 4, 2017Deposition site: RCSB / Processing site: PDBE
Revision 1.0Mar 1, 2017Provider: repository / Type: Initial release
Revision 1.1May 3, 2017Group: Database references
Revision 1.2Aug 2, 2017Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.3Dec 6, 2017Group: Structure summary / Category: struct_keywords
Item: _struct_keywords.pdbx_keywords / _struct_keywords.text
Revision 1.4Mar 30, 2022Group: Author supporting evidence / Database references / Category: database_2 / pdbx_audit_support
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization
Revision 1.5Nov 13, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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Structure viewerMolecule:
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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
Polymers147,9266
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14440 Å2
ΔGint-77 kcal/mol
Surface area66770 Å2
MethodPISA

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Components

#1: Protein
Gag polyprotein / Pr55Gag


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

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: HIV-1 Capsid Protein Assembly / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 24 kDa/nm / 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.NameFormulaBuffer-ID
11 Msodium chlorideNaCl1
250 mMTrisTris1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K
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 ...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
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 31000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm / Cs: 2.2 mm / C2 aperture diameter: 100 µm
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) / Num. of real images: 523
Image scansMovie frames/image: 30

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Processing

EM software
IDNameVersionCategory
1EMAN2particle selection
4CTFFIND3CTF correction
7MDFF2.1model fitting
9Rosetta3model refinement
10IHRSRinitial Euler assignment
11RELION1.4final Euler assignment
13RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -31.13 ° / Axial rise/subunit: 6.94 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 39712
3D reconstructionResolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 38452 / Algorithm: FOURIER SPACE / Num. of class averages: 3 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 4XFX

4xfx


Accession code: 4XFX / Pdb chain residue range: 1-220 / Source name: PDB / Type: experimental model

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