Engineering Crystal Packing in RNA-Protein Complexes II: A Historical Perspective from the Structural Studies of the Spliceosome
, Yasushi Kondo 2,3, Daniel A. Pomeranz Krummel 4, Jade Li 5, Stephen R. Price 6 and Anne-Marie M. van Roon 5Round 1
Reviewer 1 Report
This paper gives an overview on the engineering of crystal packing that were used in the Nagai group to solve crystal structures of various spliceosomal RNA-protein complexes. I recommend this paper for publication. I only have small revisions:
l42: "these structures" is underlined
l86: Figure 2E is cited before Figure 2B
l102: basepairing
l255: Muto/Pomeranz/Krummel it lacks a reference or is it a personal communication?
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript by Leung et al describes the history of engineering crystal packing contacts in spliceosomal RNA-protein complexes whose structures were solved over the years in the late Kiyoshi Nagai's lab.
This historical overview is very specific and detailed, starting with the U1A protein/RNA and U2A'B"/RNA complexes, which are fragments of the U1 and U2 SnRNPs, respectively. Both required rather insightful and unique approaches that were a testament to the creativity of the late Chris Oubridge (for whom the manuscript is also dedicated, in addition to Kiyoshi Nagai) and Stephen Price. It is very easy to lose sight of the fact that these were two of original protein-RNA complex crystal structures that involved complexes other than those formed by tRNA (an RNA whose tertiary structural attributes differ considerably from many other RNAs). The exposition is very detailed and informative. One thing I do think should be mentioned is that the idea for scanning a large variety of nucleic acid potential crystal contact sequences originated with Schultz, Shields and Steitz (in the context of protein/DNA interactions). This should be included for completeness.
The manuscript then describes how these principles were applied for obtaining crystals of the U4 core domain, and the U1 snRNP. The latter initially had a rather modest diffraction limit, and it would have been helpful to learn more about the potential pitfalls as well in terms of engineering. Chris Oubridge once remarked that he found it a bit ironic (his words) that the U1A portion had to be deleted in order to get crystals, and although the manuscript mentions that indeed the U1 snRNP is functional with this deleted, it would be helpful to understand what it might be contributing in terms of structural flexibility and whether this provides some sort of biologically relevant functionality (presumably it has not been retained purely as an evolutionary accident).
Overall, this exposition is a fitting tribute to Kiyoshi and Chris, and I believe it will be a valued reference when published. Having said this, the section describing its relevance in the age of cryo-EM seems a bit weak and apologetic. It should either be strengthened, or deleted.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
