Computational protocol: The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein

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Protocol publication

[…] Crystals of HAP2e were obtained using in situ proteolysis as described before (). Briefly, subtilisin dissolved in 10mM Tris pH8, 30mM NaCl at 10mg/mL was added to protein solution (12-14 mg/ml in 10 mM bicine, pH 9.3) on ice immediately prior to crystallization trials in a 1/100w:w ratio. Crystals of HAP2e were grown at 293K using the hanging-drop vapor-diffusion method in drops containing 1 μL protein/protease solution mixed with 1 μL reservoir solution containing 100 mM HEPES pH7.5, 2% 2-Propanol, 100 mM sodium acetate, and 12%–14%w/v PEG 8000. Diffraction quality rod-like crystals appeared after 1 week and were flash-frozen in mother liquor containing 30% (v/v) MPD.Data collection was carried out at the Swiss Light Source (PX I), the ESRF (ID30A-3), and the Synchrotron Soleil (Proxima1). Data were processed, scaled and reduced with XDS (), Pointless () and programs from the CCP4 suite (). A single-wavelength anomalous dispersion (SAD) dataset was collected from a single crystal of HAP2e from C. reinhardtii soaked for 6 hr in 2 mM K2PtCl4 solution in cryo buffer. Data were collected at the LIII edge of Platinum (1.072 Å) on a single crystal using low-dose (0.5 MGy per 360°), high-redundancy (5 × 360°) fine φ-sliced collection strategy using five crystal orientations by means of a high-precision multi-axis PRIGo goniometer (). An initial set of experimental phases was obtained by the Single Isomorphous Replacement method using autoSHARP () with the Platinum derivative and a highly isomorphous native dataset. Starting phases were improved by consecutive cycles of manual building and combination with phases derived from molecular replacement using the partial model as search model in Phaser (MR-SAD) (). After building an initial poly-alanine model accounting for ∼50% of the Cα atoms these phases were further refined using the anomalous signal of a highly redundant Sulfur-SAD dataset collected at a wavelength of 2.06641 Å on crystals of the native protein following a similar collection strategy as mentioned above (). Model building was performed using Coot (), and refinement was done using AutoBuster () with repeated validation using MolProbity (). The final model includes amino acids 24 to 581 (see linear diagram in B), with internal breaks at loops 69-97, 152-156, 167-182, 194-205, 238-283 and 330-345, corresponding to disordered loops that are marked with a gray background in the C. rheinhardtii sequence in D (top sequence) and as dashed tubes in the ribbon diagrams ( and A–5C). Clear electron density was observed for one N-linked and one O-linked glycan chain (attached to N497 and T577 in domain III). […]

Pipeline specifications

Software tools XDS, CCP4, PHENIX, Coot, MolProbity
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Homo sapiens, Chlamydomonas reinhardtii, Human poliovirus 1 Mahoney