Computational protocol: Observation of gold sub-nanocluster nucleation within a crystalline protein cage

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Protocol publication

[…] The crystal structures of the Au-ferritin composites were determined by molecular replacement method (MOLREP) using the apo-rHLFr structure (pdbID: 1DAT) as initial model. The structures were refined by using REFMAC5 in CCP4 suit and re-built in COOT using sigma-weighted (2Fo-Fc) and (Fo-Fc) electron-density maps. Water molecules were positioned to fit residual (Fo-Fc) density peaks with a lower cutoff of 3σ. The positions of the Au atoms were determined from the observed anomalous difference Fourier maps with a lower cutoff of 4σ. The Cd binding sites were assigned by comparing previous reported structures. The Au atoms were distinguished from Cd atoms by comparison with the control ferritin structure as well as the anomalous density at Au peak (1.03 Å) and remote (1.05 Å) wavelengths. The occupancy values of Au and Cd atoms were adjusted manually by considering the surrounding negative density and refined with fractional occupancy (). The B-factors of the Au atoms are in the range of 26–56 Å2, except that the Au atoms at the three-fold channels of the structures Au0(M)·CL-apo-E45C/R52C-rHLFr and Au0(E)·CL-apo-E45C/R52C-rHLFr, which are in the range of 77–96 Å2. Ser1, Ser2, His173 and Asp174 of all the structures and Lys172 for the structures Au0(M)·CL-apo-E45C/R52C-rHLFr and Au0(E)·CL-apo-E45C/R52C-rHLFr were not decided due to disordered electron density. Arg18, Asp135, Ser157 and Lys172 of Au·CL-apo-E45C/R52C-rHLFr; Lys97 and Lys172 of Au0(L)·CL-apo-E45C/R52C-rHLFr; Arg18, Glu53, Glu56, Ser131, Asp135, Ser157, Gln158 of Au0(M)·CL-apo-E45C/R52C-rHLFr; and His49 and Ser157 of Au0(E)·CL-apo-E45C/R52C-rHLFr were replaced by Ala due to low electron density of the side chain. The models were subjected to quality analysis during the various refinement stages with omit maps and RAMPAGE. Two experiments were performed separately to test the reproducibility. Atomic coordinates of the crystal structures of Au·CL-apo-E45C/R52C-rHLFr, Au0(L)·CL-apo-E45C/R52C-rHLFr, Au0(M)·CL-apo-E45C/R52C-rHLFr and Au0(E)·CL-apo-E45C/R52C-rHLFr were deposited in the Protein Data Bank under accession codes of 5GU0, 5GU1, 5GU2 and 5GU3, respectively. […]

Pipeline specifications

Software tools Molrep, REFMAC5
Application Protein structure analysis