Computational protocol: Structural and immunologic correlates of chemically stabilized HIV-1 envelope glycoproteins

Similar protocols

Protocol publication

[…] Micrographs were collected on an FEI Titan Krios operating at 300 KeV coupled with Gatan K2 direct electron detector via the Leginon interface []. Each exposure image was collected in counting mode at 29000 x nominal magnification resulting in a pixel size of 1.02 Å/pixel, using a dose rate of ~10 e-/pix/sec, and 200 ms exposure per frame. A total of 1329 micrographs were collected in ~72 h. The total dose received for each movie micrograph was 67 e-/Å2. The nominal defocus range used was -1.5 to -4.0 μm. [...] Movie micrograph frames were aligned using MotionCorr [] and CTF models were calculated using CTFFIND3 []. The resulting motion-corrected and signal-integrated micrographs were subjected to automated difference-of-Gaussian particle selection []. The resulting set of molecular projection-image candidates were binned by a factor of 4 and subjected to reference-free class averaging in Relion 1.4b1 []. Projection images belonging to structural classes were selected for angular refinement and reconstruction using a low-pass filtered unliganded HIV-1 Env trimer density map as a reference []. The projection images were then subjected to 3D classification and split by K-means clustering into six classes. Two classes resulted in stoichiometrically identical maps (Env trimer with three PGV04 Fabs bound) and projection images sorted into these two classes were combined followed by refinement and reconstruction. This data pool was then subjected to particle polishing and the resulting aligned, B-factor-corrected and signal-integrated projection images were refined and reconstructed to a final average resolution of ~4.2 Å (0.143 FSC cut-off). [...] An initial model was generated based on PDB ID 5CEZ [] and PDB ID 3SE9 [] using UCSF Chimera [], Modeler [] and Coot []. A library of 200 homologous 7-mer peptide fragment coordinates per relevant residue were compiled and used in iterative centroid-representation density-guided rebuild-and-refinement in the Rosetta software suite []. Rosetta models were manually adjusted in Coot followed by evaluation based on geometry (MolProbity []) and cryo-EM density fit (EMRinger []). Upon convergence, the best model underwent iterative all-atom Rosetta refinement and manual rebuilding constrained by the cryo-EM density map and with distance constraints introduced for observed cross-links. The final model was selected based on MolProbity, EMRinger, Privateer [] and CARP [] validation (). […]

Pipeline specifications

Software tools Leginon, MotionCorr, CTFFIND, RELION, UCSF Chimera, Coot, Rosetta software suite, MolProbity, EMRinger
Applications cryo-EM, Protein structure analysis
Organisms Viruses
Diseases HIV Infections
Chemicals Glutaral