26 December 1988 Dear Joe, This past weekend in Nice was really quite nice and I took advantage of the relaxing environment to read through the paper in detail. while I have written most of my comments on the paper itself, I'11 summarize some of the more important ones below. First of all, I think that the paper is very good (my bias notwithstanding). 1 certainly agree with Jean-Luc and you that the statistics (and their extensive description) is a necessary part of a work which stands on so few reflections as well as on such small intensity differences among these reflections. Apart from a concern that the difference fourier method may not have been emphasized enough, I agree with the general approach of the paper. As such, most of my comments are oriented towards either points of grammer or style. TITLE PAGE (page 1) Since Columbia’s medical school is entirely separate from the main campus, it would be more accurate to describe my present address as: Columbia University, College of Physicians & Surgeons, New York, New York ABSTRACT (page 2) only grammatical/stylistic comments here. INTRODUCTION (pages 3-4) The last sentence of the first paragraph is tricky. I’ve included my version (which I am not entirely satisfied with) at the top of the page. Otherwise, there are only grammatical/stylistic comments. MATERIALS & METHODS (pages 5-11) With respect to the error analysis, are the errors in the model attributable to the arbitrary side chain conformations and the kinking significant enough to be discussed? If so, we should include them here (page 9 end of paragraph 3 as indicated). Otherwise, only grammatical/stylistic comments.RESULTS (pages 12-15) Since we include the difference fourier results in the DISCUSSION as well as refer to the difference fourier work of the EMBO '86 paper, it might be useful to describe the present difference fourier results in more detail under section 3.2.1. Perhaps under this section we might include a third paragraph reading as follows: The present results allow the A and B helices to be distinguished from each other and consequently assigned to corresponding peaks in the difference fourier map (fig 2). This is most easily done for helix A which has a considerably greater level of deuteration that helix B. Initially, then, one might assign helix A to the strong peak located near map position 1. This result is further substantiated upon considering the fact that the tilt of helix A with respect to the membrane normal allows the majority of label to fall towards the perimeter of map position 1 close to the strongest peak in the difference fourier map. Helix B, then, would appear to fall in one of the secondary peaks near map position 7 but on the basis of this qualitative analysis such a result must be regarded as highly preliminary. More systematic and quantifiable results were obtained with the model building approach. Other than this there are only grammatical/stylistic comments. DISCUSSION (pages 16-20) This section was well done and very complete. While this may be contrary to JMB protocol, I have suggested subtitling the DISCUSSION section as indicated in the paper. Such an addition would enhance what is already a well-organized presentation. As we discussed, we may have to completely rewrite the section on page 17 concerning the rotational orientation of helix B. I have put in a number of modifications concerning our response to the Lys-41-PITC data. These modifications are based on Jean-Luc’s most recent rotational assignment for helix B. I quote from the paper with my changes incorporated (page 18): Even assuming Lys-41 to be part of helix B, we note that the tentative assignment we find for the rotational orientation of this helix would place Lys-41 turned approximately towards the center of the molecular projection. with an appropriately extended lysyl chain, the center of gravity of the almost equally long PITC label would lie 5-6 A away from the peak located by Seiff et al. Let's look at the final rotation again to see if this needs to be modified more. Otherwise, only grammatical/stylistic comments.LEGENDS (pages 21-22) only grammatical and stylistic comments. ACKNOWLEDGEMENTS: (page 26) Would it be possible to include my summer ILL grant in the acknowledgements? In addition, is there some way I could acknowledge Jeremy Smith and his "introducing" me to the ILL as well as Dave Wild and his help with the MRC fast fourier programs? FIGURES (pages 27-33) My suggestions are: Figure 2 (654 diff + native) in black and white Figure 4 (3 or 4 contours) in color; otherwise much of the points of this figure may be lost. Figure 5 (655/660 diff + native) in black and white Figure 6 in black and white Overall, the paper was excellent and I look forward to discussing these comments with you soon. Based on your conclusions, we can then proceed with preparing the figures in a reasonably final manner. Yours very truly,