|Entry||Database: PDB / ID: 5upw|
|Title||CryoEM Structure Refinement by Integrating NMR Chemical Shifts with Molecular Dynamics Simulations|
|Components||Gag polyproteinGroup-specific antigen|
|Keywords||VIRAL PROTEIN / Cryo-EM / HIV capsid / Chemical shift / Molecular Dynamics / hydrolase / viral protein|
|Function / homology||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 ...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 source||Human immunodeficiency virus type 1 (HIV)|
|Method||ELECTRON MICROSCOPY / helical reconstruction / cryo EM / 5 Å resolution|
|Citation||Journal: 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.
SummaryFull reportAbout validation report
|Date||Deposition: Feb 4, 2017 / Release: Mar 1, 2017|
|Structure viewer||Molecule: |
Downloads & links
A: Gag polyprotein
B: Gag polyprotein
C: Gag polyprotein
D: Gag polyprotein
E: Gag polyprotein
F: Gag polyprotein
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
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: HELICAL ARRAY / Reconstruction method: helical reconstruction|
|Component||Name: HIV-1 Capsid Protein Assembly / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT|
|Molecular weight||Value: 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 solution||pH: 8|
|Specimen||Conc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid material: COPPER / Grid mesh size: 300 / Grid type: Quantifoil R2/1|
|Vitrification||Instrument: 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
-Electron microscopy imaging
Model: Tecnai Polara / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI POLARA 300|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: 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 holder||Cryogen: NITROGEN / Specimen holder model: SIDE ENTRY, EUCENTRIC|
|Image recording||Average 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 scans||Movie frames/image: 30|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Helical symmerty||Angular rotation/subunit: -31.13 deg. / Axial rise/subunit: 6.94 Å / Axial symmetry: C1|
|Particle selection||Number of particles selected: 39712|
|3D reconstruction||Resolution: 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 building||Ref protocol: FLEXIBLE FIT / Ref space: REAL|
|Atomic model building||PDB-ID: 4XFX|
Pdb chain residue range: 1-220
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