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

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Basic information

Entry
Database: EMDB / ID: EMD-8595
TitleCryoEM Structure Refinement by Integrating NMR Chemical Shifts with Molecular Dynamics Simulations
Map dataHIV-1 CA hexamer
Sample
  • Complex: HIV-1 Capsid Protein Assembly
    • Protein or peptide: Gag polyproteinGroup-specific antigen
Function / homology
Function and homology information


viral budding via host ESCRT complex / ISG15 antiviral mechanism / host multivesicular body / viral nucleocapsid / host cell nucleus / structural molecule activity / host cell plasma membrane / virion membrane / 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) / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retrovirus capsid, C-terminal / Retroviral matrix protein ...Gag protein p6 / Gag protein p6 / gag protein p24 N-terminal domain / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / 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 (NEW YORK-5 ISOLATE)
Methodhelical reconstruction / cryo EM / Resolution: 5.0 Å
AuthorsZhao G / Zhang P
Funding support United States, 6 items
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, 2017-
Header (metadata) releaseMar 1, 2017-
Map releaseMar 1, 2017-
UpdateMar 30, 2022-
Current statusMar 30, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • 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
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8595.png

Downloads & links

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Map

FileDownload / File: emd_8595.map.gz / Format: CCP4 / Size: 361.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHIV-1 CA hexamer
Voxel sizeX=Y=Z: 1.22 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.025930129 - 0.092899546
Average (Standard dev.)3.2771706e-05 (±0.0012119777)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions456456456
Spacing456456456
CellA=B=C: 556.32 Å
α=β=γ: 90.0 °

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
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
Components
  • Complex: HIV-1 Capsid Protein Assembly
    • Protein or peptide: Gag polyproteinGroup-specific antigen

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Supramolecule #1: HIV-1 Capsid Protein Assembly

SupramoleculeName: HIV-1 Capsid Protein Assembly / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Human immunodeficiency virus type 1 (NEW YORK-5 ISOLATE)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 24 kDa/nm

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Macromolecule #1: Gag polyprotein

MacromoleculeName: Gag polyprotein / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus type 1 (NEW YORK-5 ISOLATE)
Molecular weightTheoretical: 24.654268 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: PIVQNLQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG GHQAAMQMLK ETINEEAAEW DRLHPVHAG PIAPGQMREP RGSDIAGTTS TLQEQIGWMT HNPPIPVGEI YKRWIILGLN KIVRMYSPTS ILDIRQGPKE P FRDYVDRF ...String:
PIVQNLQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG GHQAAMQMLK ETINEEAAEW DRLHPVHAG PIAPGQMREP RGSDIAGTTS TLQEQIGWMT HNPPIPVGEI YKRWIILGLN KIVRMYSPTS ILDIRQGPKE P FRDYVDRF YKTLRAEQAS QEVKNWMTET LLVQNANPDC KTILKALGPG ATLEEMMTAC QGV

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
1.0 MNaClSodium chloridesodium chloride
50.0 mMTrisTris
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 10.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: HOMEMADE PLUNGER
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

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 31000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 523 / Average exposure time: 6.0 sec. / Average electron dose: 41.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 39712 / Software - Name: EMAN (ver. 2)
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: OTHER / Details: IHRSR
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 1.4)
Final reconstructionNumber classes used: 3
Applied symmetry - Helical parameters - Δz: 6.94 Å
Applied symmetry - Helical parameters - Δ&Phi: -31.13 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 38452
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4xfx


Chain - Residue range: 1-220
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5upw:
CryoEM Structure Refinement by Integrating NMR Chemical Shifts with Molecular Dynamics Simulations

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