To further quantify assembly formation, the inter-QR distances were analyzed by measuring the perimeterperimeter nearest neighbor distances (ppNNDs) between each QR (i.e. the shortest distance between the perimeter of one QR with that of its nearest neighboring QR). The ppNNDs distribution histograms were fitted with Gaussian distribution curves. This analysis revealed three welldefined species for QR-DiMan•DC-SIGNR assemblies with ppNNDs of 1.9 ± 0.8, 4.0 ± 1.1 and 6.7 ± 1.0 nm (mean ± ½FWHM; Figure ; Table ), respectively. DC-SIGN, on the other hand, displayed only one well-defined ppNND distribution at 2.3 ± 1.1 nm (Figure ; Table ). These results allowed us to draw three conclusions. (1) The smallest ppNND species (~2 nm) were observed in similar amounts for both DC-SIGN and DC-SIGNR (20-27%). Such distances are comparable to the thickness of the glycan ligand coating, thus these QR assemblies are deemed to result from non-specific interactions. (2) The species with ppNNDs ≳ 7.7 nm were found in 71% of QR-DiMan•DC-SIGN and 31% of QR-DiMan•DC-SIGNR complexes and were randomly distributed (Figure ). These corresponded nicely to single QR-DiMan particles bound with a layer of proteins as observed by DLS, thus confirming that both DC-SIGN and DC-SIGNR were able to bind tetravalently with all CRDs with one QR-DiMan. (3) The well-defined species for DC-SIGNR binding with ppNNDs of 4.0 ± 1.1 and 6.7 ± 1.0 nm were not observed in significant amount with DC-SIGN (Figure ), such distances were consistent with the discrete distances expected for DC-SIGNR-bridged QRs. In addition, 49% of QRs with ppNNDs below ~7 nm contained Histograms of the center-center nearest neighbor distances (ccNNDs, for 2.9 < ppNND < 7.7 nm), fitted with trimodal Gaussian fits (where applicable), for QR-DiMan incubated with (G) DC-SIGN, (H) DC-SIGNR or (I) preincubated QR-DiMan•DC-SIGNR with 2 mM Man. Statistical analysis of the (J) numbers of QRs per cluster (i.e. the number of interconnected QRs with a ppNND < 7.7 nm) and (K) QR nearest neighbor orientations (individual: ppNND > 7.7 nm; parallel: the area of the first ccNND peak (or ccNND <10 nm for DC-SIGN); non-parallel: the sum of the second and third ccNND peak areas (or ccNND >10 nm for DC-SIGN); non-specific: ppNND < 2.9 nm). Schematics of (L) simultaneous binding of DC-SIGN (blue) to all sections of QR or DC-SIGNR (red) to the cylindrical section only, and (M) DC-SIGNR crosslinking QR-DiMan into parallel stacks.