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Title:Cryo-electron tomography reconstruction of native HIV-1 core
Authors:Zhao G, Perilla JR, Yufenyuy E, Meng X, Chen B, Ning J, Ahn J, Gronenborn AM, Schulten K, Aiken C, Zhang P
Sample:HIV-1 core with A14C/E45C cross-linked capsid protein
Method:Tomography
Red flagLatest update:2013-06-12
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Sample
Sample name: HIV-1 core with A14C/E45C cross-linked capsid protein
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1virusHuman immunodeficiency virus 1Human immunodeficiency virus 1HIV-1 core was isolated from virions carrying A14C/E45C mutation in capsid protein.
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
8.00.011 mg/mL10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTAQuantifoil R2/2 200 mesh holey carbon copper grids
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE70%90 KHOMEMADE PLUNGERPurified HIV-1 A14C/E45C cores (3 uL) were applied to the carbon side of glow-discharged, perforated R2/2 Quantifoil grids and quickly mixed with 3 uL of a 15 nM fiducial gold bead solution before plunge-freezing. ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI POLARA 300300 kVFLOOD BEAMBRIGHT FIELD2 mm8000 nm8000 nm39000FIELD EMISSION GUNGATAN ULTRASCAN 4000 (4k x 4k) mmObjective lens astigmatism was corrected at 115,000 times magnification

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
SIDE ENTRY, EUCENTRIC-70°66° eV82 K80 K85 K mrad120 e/Å211-SEP-2010
Processing
Protocol:SIRT
Software:tomo3d
Number of tilts:53
Tilt angle increment:
Imposed symmetry:C1
Processing details:The raw tomogram was denoised using the nonlinear anisotropic diffusion edge-enhancing algorithm available in IMOD. The core density was segmented out using Volume Tracer in UCSF Chimera.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
53 μm/pixel16
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
3J34 flexibleMDREALThe model was built using hexamer of hexamers (PDB entry 3J34) and pentamer of hexamers (computer-based MD model available upon request).