Cryo EM structures of maturation-intermediate Prohead I of bacteriophage HK97 with (PhIPro+) and without (PhIPro-) the viral protease packaged have been reported (Veesler et al., 2014). In spite of PhIPro+ containing an additional similar to 100 x 24 kD of protein, the two structures appeared identical although the two particles have substantially different biochemical properties, e.g., PhIPro- is less stable to disassembly conditions such as urea. Here the same cryo EM images are used to characterize the spatial heterogeneity of the particles at 17 A resolution by variance analysis and show that PhIPro- has roughly twice the standard deviation of PhIPro+. Furthermore, the greatest differences in standard deviation are present in the region where the delta-domain, not seen in X-ray crystallographic structures or fully seen in cryo EM, is expected to be located. Thus presence of the protease appears to stabilize the delta-domain which the protease will eventually digest. (C) 2016 Elsevier Inc. All rights reserved.