Use of computerized multidimensional scaling to compare immunoelectron microscopy data with protein near-neighbor information: application to the 30S ribosome from Escherichia coli.

Abstract

A three-dimensional model of the protein arrangement in the Escherichia coli 30S ribosome was constructed by using computerized multidimensional scaling of immunoelectron microscope data. This enabled data comparison between the new electron microscope technique and other methods such as crosslinking, chemical protection, affinity labeling, energy transfer, and assembly interactions. The immunoelectron microscopy data are reasonably consistent with those from other sources. Reasons for some inconsistent data are discussed and our calculation of the dimensions of the proteins, both globular and elongated, are summarized. One of the most interesting recent developments in the analysis of ribosome structure is the use of immunoelectron microscopy (immuno-EM) to determine the relative positions of the individual proteins on the surface of the subunit particle (1)(2)(3)(4)(5) We have built such a model by using the following two-step procedure. First, a three-dimensional plaster model was made based on the immuno-EM diagrams of Tischendorf et al. (1, 2) and the antigenic sites were marked on its surface. Second, the shortest distances between all pairs of these antigenic sites were measured and used as input for a multidimensional scaling program. Its output gives the three-dimensional coordinates for a "naked" protein model-one without the outline of the ribosome. The model provides a unique and insightful view of the 30S ribosome. The uneven distribution of proteins within the subunit and the elongation of 12 of them are strikingly apparent. Questions on the relationship of structure to function take on fresh meaning but, more importantly, the model allows us to make a systematic comparison of all the relevant published data regarding protein-protein relationships.Computer program Multidimensional scaling is a statistical technique that has been used for approximately 15 years in psychology and is now being used extensively in other fields, including an early attempt to generate a three-dimensional model of the 30S ribosome (19). It constructs a configuration of points in space from information about the distances between points. For example, if a matrix were made of the 703 distances in miles between any 38 cities in the United States, and used as input in a multidimensional scaling program, the output would be 38 points representing the cities in the correct two-dimensional configuration. The orientation (north, south, east, west) would be left for the subjective decision of the user. Missing data and tied data can be accommodated but present some problems.The program places N points in a space of a given dimension so as to minimize STRESS, which measures the badness-of-fit between the configuration of points and the data. It represents the extent to which the data deviate in the least squares sense from the monotonic curve (see Fig. 3 A and B)

Analysis of protein-protein relationships in 30S ribosome assembly intermediates using protection from proteolytic digestion

“…to be very close by fluorescence transfer measurements (Huang et al, 1975), have centers of mass approximately 34 A apart (Langer et al, 1978), reside together on a S'-proximal fragment of 16S RNA (Morgan & Brimacombe, 1973), are cross-linked by the bifunctional reagent bis(dimethyl suberimidate) (Lutter et al, 1972), and are involved in chemical protection effects (Changchien & Craven, 1977). Furthermore, in both immunoelectron microscopic models of the 30S particle, sections of S7 and S9 have been found to have antigenic sites very near one another (Lake, 1977;Tischendorf et al, 1975;Gaffney & Craven, 1978). In this case the protection of S7 from trypsin digestion by the presence of S9 most likely is due to the close physical proximity of the two proteins.…”

“…Of the other proteins capable of individually binding 16S RNA, proteins S7, S13, S20, and Sll were observed to be completely exposed to the action of trypsin or chymotrypsin despite being tightly associated with the RNA (Changchien & Craven, 1976). However, we then discovered that three of these proteins, S7, Sll, and S20, become pro- Gaffney & Craven (1978). teolysis resistant at early phases in ribosome assembly.…”

A computer model for the 30S ribosome subunit

“…In the past few years several numerical procedures have been developed with specific application to the geometry of the 30S subunit. Among these are the triangulation and second moment methods used by Moore and Engelman (2, footnote 1) and the multidimensional scaling approach employed by Bolin (3) and most recently by Craven (4).…”