Computational protocol: A mechanism of viral immune evasion revealed by cryo-EM analysis of the TAP transporter

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Protocol publication

[…] Vitrified specimens of TAP/ICP47 complex were prepared on glow-discharged Quantifoil holey carbon grids by plunge-freezing into liquid ethane using a Vitrobot (FEI). Cryo-EM data were collected at liquid-nitrogen temperature using a K2 Summit direct electron detector camera (Gatan Inc.) on a Tecnai F20 electron microscope (FEI) operating at 200 keV. Dose-fractionated image stacks were recorded with UCSF Image 4 in super-resolution counting mode at a calibrated magnification of 40410x (nominal magnification of 29,000x) with a dose rate of 8 electrons/pixel/s (5.2 electrons/Å2/s). Frames were read out every 200 ms and 30 frames were collected, resulting in an exposure time of 6 s and a total dose of 31.2 electrons/Å2. Dose-fractionated image stacks were 2x binned and motion-corrected, as described. The defocus was determined with CTER. BOXER was used to interactively pick 28,813 particles from ~750 images. The particle images were subjected to the iterative stable alignment and clustering (ISAC) procedure implemented in SPARX. Four ISAC generations specifying 100 particles-per-group and a pixel error threshold of 0.7 yielded 324 averages. Of these, 270 averages were used to calculate an initial 3D density map with the validation of individual parameter reproducibility (VIPER) procedure in SPARX. [...] Cryo-EM grids were prepared by pipetting 3 μl freshly purified TAP/ICP47 (2 mg/ml) onto glow-discharged C-flat holey carbon CF-1.2/1.3-4C grids (Protochips) and letting the sample adsorb for 20 s. The grids were blotted for 4 s at 90% humidity using a Vitrobot Mark IV (FEI) and immediately plunge-frozen in liquid nitrogen-cooled liquid ethane. The grids were imaged using a FEI Titan Krios electron microscope operating at an acceleration voltage of 300 keV. Images were recorded using a K2 Summit direct electron detector (Gatan Inc.) set to super-resolution counting mode with a super-resolution pixel size of 0.675 Å using the program SerialEM. In addition, a Gatan Imaging filter with a slit width of 20 eV was used to remove inelastically scattered electrons. Movie frames were recorded with an exposure time of 200 ms using a dose rate of 10 electrons/pixel/s or 5.5 electrons/Å2/s (1.35 Å at the image plane). Three datasets were recorded using different total doses and defocus ranges ( and ). [...] Movie frames were corrected for gain reference and binned by a factor of 2, giving a pixel size of 1.35 Å. Drift correction was performed using the program Unblur,. Next, the drift-corrected frames were summed into single micrographs, which were used to estimate the Contrast Transfer Function (CTF) using CTFFIND4. The program Summovie was used to recalculate the summed images, first with a low-pass filter for autopicking and then with the noise power restored after filtering for particle extraction. Autopicking, particle extraction, 2D classification, 3D classification, and initial 3D refining were all performed in Relion. In order to achieve a more robust classification of the extracted particles, 2D classification was performed with a particle mask diameter of 145 Å while ignoring the effects of the CTF until the first zero transition. 3D classification was performed on particles from selected 2D classes using the initial model calculated in SPARX as a reference map. Particles from selected 3D classes from both datasets were combined for 3D refinement. Using the orientation parameters determined by Relion, 3D refinement in FREALIGN was also performed. The final map, reconstructed from 139,293 particles, had a resolution of 6.5 Å as determined by Fourier shell correlation (FSC) of independently refined half-datasets using the 0.143 cut-off criterion (). [...] We used a model of the TAP1 nucleotide-binding domain (NBD) from a previously reported structure of the isolated domain (PDBcode 1JJ7) to generate a homology model for the TAP2 NBD using the program Modeller. We also generated a homology model of the TAP1 and TAP2 transmembrane domains (TMDs) using the half-transporter subunit of human ABCB10 (PDBcode 4AYT) as the source structure . We manually docked these poly-alanine models into our final cryo-EM map and rebuilt each model in Coot. […]

Pipeline specifications

Software tools SPARX, SerialEM, Unblur, CTFFIND, Summovie, RELION, Frealign, MODELLER, Coot
Applications cryo-EM, Protein structure analysis
Organisms Homo sapiens
Diseases Immunologic Deficiency Syndromes, Neoplasms, Virus Diseases
Chemicals Adenosine Triphosphate, Nucleotides