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Title:Negative stain reconstruction of PG9 Fab in complex with HIV-1 SOSIP gp140 trimer
Authors:Lee JH, Julien JP, Cupo A, Moore JP, Wilson IA, Ward AB
Sample:Fab fragment of broadly neutralizing antibody PG9 bound to BG505 SOSIP gp140 HIV-1 trimer
Method:Single particle reconstruction (18.1 angstroms resolution)
Red flagLatest update:2013-03-20
Sample
Sample name: Fab fragment of broadly neutralizing antibody PG9 bound to BG505 SOSIP gp140 HIV-1 trimer
Oligomeric state: One Fab binds one SOSIP trimer
Experimental molecular weight of the sample: 0.387
Components:
ID Type Name Exp. MW (MDa) Theo. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinBroadly Neutralizing Antibody PG90.050.05monomertrueHomo sapiensRecombinantly expressed antibody Fab fragment. Co-transfected with gene encoding tyroslprotein sulfotransferase 1 (TPST1)
2proteinHIV-1 SOSIP.6640.120.14trimertrueHuman immunodeficiency virus 1Cotransfected with pcDNA3.1 Furin.
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.40.03 mg/mL50 mM Tris-HCl, 150 mM NaCl30 sec Nano-W400 Cu mesh grid with carbon support, glow discharged at 20 mA for 30 seconds
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
NONE% KNONE ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI F20120 kVFLOOD BEAMBRIGHT FIELD mm500 nm900 nm100000FIELD EMISSION GUNGENERIC GATAN (4k x 4k) mmObjective astigmatism corrected at 100,000x

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
SIDE ENTRY, EUCENTRIC55° eV K293 K293 K mrad30 e/Å2Images taken from 0 to -55 degrees in 5 degree tilt increments.31-JUL-2012
Processing
Software:Xmipp, EMAN
CTF correction:Not corrected
Number of particles:33431
Imposed symmetry:C1
Resolution by author:18.1 Å
Resolution method:FSC 0.5
Processing details:Particles were picked using DogPicker then processed with Xmipp and EMAN.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
12830.109 μm/pixel16Images recorded on CCD
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails