EMD-5723 Experiments and Validation

Negative stain Electron Microscopy of Bg505 SOSIP.664 in complex with sCD4 and 17b

Single particle reconstruction
Overview of EMD-5723
Sample name: HIV spike protein 664G construct in complex with sCD4 and Fab fragment of 17b monoclonal antibody
Organisms: Human immunodeficiency virus, Escherichia coli, Cricetulus griseus
Related EM entry by publication: EMD-2427

Map parameters

Recommended contour level: 4.00 (author)
Number of grid points: 192 × 192 × 192
Voxel size: 2.05 × 2.05 × 2.05 Å
Minimum density: -5.033
Maximum density: 16.133
Average density: 0.00
Standard deviation: 0.919

Sample information

Sample name: HIV spike protein 664G construct in complex with sCD4 and Fab fragment of 17b monoclonal antibody
Proteins: HIV spike protein BG505 SOSIP.664, sCD4, 17b Fab

Validation information

Experimental information

 

Image processing

Applied symmetry: C3
Software: EMAN2, EMAN1
Number of particles: 22145
Number of class averages: 128
Reconstruction protocol: common lines
Details: The particles were selected using a DoG Picker, and cleaned using reference-free class averaging. The final map was calculated from a single dataset. Particles were picked automatically using DoG Picker and put into a particle stack using the Appion software package. Initial, reference-free, two-dimensional (2D) class averages were calculated using particles binned by five via the Xmipp Clustering 2D Alignment and sorted into classes. EMAN was used for the 3D reconstruction.

Fitting

Initial atomic model: 1RZK
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: Correlation, Fit in Map, Chimera
Refinement space: REAL

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: LAB6
Electron dose (e/A**2): 30.0
Nominal CS (mm): 2.00
Nominal magnification: 52000.0
Defocus max (nm): 1,300.00
Defocus min (nm): 900.00
Tilt max (degrees): 50.00
Tilt min (degrees): 0.00
Holder model: SIDE ENTRY, EUCENTRIC
Acquisition
Number of images: 176

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.03
Buffer: 50 mM TRIS-HCl, 150 mM NaCl
Staining procedure: 2% w/v Uranyl Formate for 25 seconds
Specimen support: 400 mesh copper grid
Vitrification
Apparatus: NONE
Cryogen: NONE