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- EMDB-8595: CryoEM Structure Refinement by Integrating NMR Chemical Shifts wi... -

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Entry
Database: EMDB / ID: 8595
TitleCryoEM Structure Refinement by Integrating NMR Chemical Shifts with Molecular Dynamics Simulations
Map dataHIV-1 CA hexamer
SampleHIV-1 Capsid Protein Assembly
  • Gag polyproteinGroup-specific antigen
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
SourceHuman immunodeficiency virus type 1 (NEW YORK-5 ISOLATE) / virus
Methodhelical reconstruction / cryo EM / 5 Å resolution
AuthorsZhao G / Zhang P
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 ReportPDB-ID: 5upw

SummaryFull reportAbout validation report
DateDeposition: Feb 4, 2017 / Header (metadata) release: Mar 1, 2017 / Map release: Mar 1, 2017 / Last update: Dec 6, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5upw
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_8595.map.gz (map file in CCP4 format, 379276 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
456 pix
1.22 Å/pix.
= 556.32 Å
456 pix
1.22 Å/pix.
= 556.32 Å
456 pix
1.22 Å/pix.
= 556.32 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.22 Å
Density
Contour Level:0.035 (by author), 0.035 (movie #1):
Minimum - Maximum-0.025930129 - 0.092899546
Average (Standard dev.)3.2771706E-5 (0.0012119777)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions456456456
Origin000
Limit455455455
Spacing456456456
CellA=B=C: 556.32 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.221.221.22
M x/y/z456456456
origin x/y/z0.0000.0000.000
length x/y/z556.320556.320556.320
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS456456456
D min/max/mean-0.0260.0930.000

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Supplemental data

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Sample components

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Entire HIV-1 Capsid Protein Assembly

EntireName: HIV-1 Capsid Protein Assembly / Number of components: 2

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Component #1: protein, HIV-1 Capsid Protein Assembly

ProteinName: HIV-1 Capsid Protein Assembly / Recombinant expression: No
SourceSpecies: Human immunodeficiency virus type 1 (NEW YORK-5 ISOLATE) / virus
Source (engineered)Expression System: Escherichia coli / / bacteria /

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Component #2: protein, Gag polyprotein

ProteinName: Gag polyproteinGroup-specific antigen / Recombinant expression: No
MassTheoretical: 24.654268 kDa
Source (engineered)Expression System: Human immunodeficiency virus type 1 (NEW YORK-5 ISOLATE) / virus

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Experimental details

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Sample preparation

SpecimenSpecimen state: helical array / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 6.94 Å / Delta phi: -31.13 deg.
Sample solutionSpecimen conc.: 2 mg/ml / pH: 8
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 295 K / Humidity: 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
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 41 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 31000 X (nominal) / Cs: 2.2 mm / Imaging mode: BRIGHT FIELD / Defocus: 700 - 2200 nm
Specimen HolderModel: SIDE ENTRY, EUCENTRIC
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 523

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution assessment)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Input PDB model: 4XFX
Output model

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