National Institute of General Medical Sciences
National Institutes of Health
December 10-11, 2008
Report of the PSI Advisory Committee
Introduction
This report summarizes the progress that has been made by the large-scale centers and other components of PSI-2. Recommendations of the PSI Advisory Committee regarding the future of the PSI are presented separately.
Large-Scale Centers
Progress of the four large-scale centers was reviewed by the respective Directors, Ian Wilson, Andrzej Joachimiak, Stephen Burley and Guy Montelione.
Dr. Wilson noted that the pipelines for rapid structure determination have now been in place for some time and are working extremely well. The use of “salvage pathways” to permit structure determination of proteins that are initially recalcitrant continue to be developed. These include the use of multiple orthologs, sequence-based truncations, proteolytically-defined truncations, surface mutations, reductive methylation and the use of crystallization additives. Through the efforts of the PSI it is now possible to estimate the likelihood that such approaches will be successful. This information will be useful to the structural biology community in general. It is now generally agreed that high-pressure refolding of proteins is not useful in facilitating structure determination.
An encouraging finding is that the effectiveness of X-ray crystallography and NMR spectroscopy in structure determination tends to be complementary, i.e. where one may fail the other may be successful.
Each of the large-scale centers is now contributing around 200 structures to the PDB each year. Dr. Joachimiak’s group, for example, has contributed 607 structures in PSI-2. The group of Dr. Burley has contributed 440. (Dr. Montelione noted that the PSI is depositing two structures per day in the PDB, 10% of these coming from NMR.) It is anticipated that the overall goal of having the PSI-2 centers contribute 3000 novel structures to the PDB will be comfortably met.
The PSIAC continues to be very impressed with the ability of the large-scale centers to maintain efficient structure determination pipelines and to contribute a sizable fraction of all of the “novel” structures being deposited in the PDB. The large-scale centers have also continued to contribute to technology development, although “the slope is lower” than it was in the early stages of the PSI.
Specialized Centers
(i) Accelerated Technologies Center for Gene to 3D Structure (Lance Stewart)
Dr. Stewart reviewed progress with the microfluidics system for nanovolume crystallization. In this system very small volumes or “plugs” of protein solution are introduced sequentially into a single microcapillary tube arranged on a “crystal card” (not unlike a conventional credit card). The system is being commercialized by Emerald Biosystems. The use of small amounts of protein makes this system potentially attractive, especially if it proves possible to collect X-ray data from crystals “in situ” (as is currently being tested).
In another part of his presentation, Dr. Stewart noted that there has been an approximately two-year delay in the development of the Tuneable X-ray Source due to defective components and challenges in technology development. It is no longer anticipated that an X-ray source will be installed at Scripps Research Institute prior to the end of PSI-2. These delays were not anticipated during the May 2007 site visit by members of the PSIAC and are reason for concern.
(ii) Center for Eukaryotic Structural Genomics (John Markley)
Dr. Markley reviewed ongoing development with the LIMS information management system which was developed in Madison and is being used by a number of other groups. He also noted that in PSI-2 the Madison group has received 359 requests from the community for structural studies. Of these, 290 were approved, 55 are still in progress and 8 structures have been determined.
Experience with this center suggests that the cost of supplies for protein production by cell-free protein synthesis for NMR structure determination of a eukaryotic protein is about $5000 and requires about five person-weeks. Using conventional cell-based protein production, the cost for supplies is about $15,000 and requires about seven person-weeks per structure.
(iii) Center for High-Throughput Structural Biology (George DeTitta/Michael Malkowski)
It has been noted previously that the development of a system to permit the purification of selenomethionine-containing proteins from yeast has been one of the major accomplishments of this group.
A more recent contribution has been the identification of “silver bullets,” i.e. additives to help protein crystallization. These should be of benefit to the structural biology community at large.
Another very interesting new development has been the development of 1500 or so cocktails based on the principle that membrane proteins tend to crystallize at phase boundaries. A preliminary test with a limited number of membrane proteins appears to be extremely promising. Developments with this approach will be followed with great interest.
(iv) Center for Structures of Membrane Proteins (Robert Stroud)
Perhaps the most encouraging comment from Dr. Stroud was that his group has to date determined 13 membrane protein structures. His experience is that the main problem in membrane proteins is expression in a stable active form. He noted that it is necessary to prove that the constructs being used for structural studies are well-behaved in solution and retain activity. He also noted that it is often necessary to “tune” crystallization conditions to obtain crystals suitable for structural analysis. Because of the need to know the function of the protein, and also because of the need for individual treatment for each protein, it raises the question as to whether membrane proteins can be solved in a high-throughput manner analogous to the pipelines currently used for soluble proteins.
(v) Integrated Center for Structure and Function Innovation (Tom Terwilliger)
Dr. Terwilliger provided a brief update on the work of Tony Kossiakoff’s group in developing antibody fragment crystallization chaperones. It is a highly innovative and potentially very effective approach, although Dr. Terwilliger noted that the technology is not to the point that it can be routinely used by the community at large.
Building on the idea that many structures determined by PSI labs have been observed to have ligands bound, Dr. Terwilliger described an approach in which test ligands are used to displace individual proteins from a mixture bound to a suitable resin. If a ligand displaces a given protein the ligand can be used as a crystallization additive. Likewise, it may provide a lead in identifying the function of the protein.
(vi) New York Consortium on Membrane Protein Structure (Wayne Hendrickson)
Dr. Hendrickson re-emphasized not only the difficulty of crystallizing membrane proteins (1.3 crystallization hits per 100 processed targets), but especially in obtaining crystals that diffract to reasonably high resolution. As noted by Dr. Hendrickson, “there are a lot of complications still”. During the past 12 months (12/2007-12/2008) the NYCOMPS pipeline has yielded 18 membrane proteins that give crystals, of which Dr. Hendrickson characterized nine as being “promising” for X-ray diffraction analysis. This result tends to support the conclusion of Dr. Stroud (see above) that it is often necessary to “tune” the crystallization conditions, taking into account the functional characteristics of each protein.
Crystals of the TrkG bacterial K+ uptake protein, obtained previously via the NYCOMPS pipeline, have given a 3.4 Å resolution electron density map that can be partially interpreted. Dr. Hendrickson showed structures of the M. Jannaschi APC transporter alone and in complex with an Fab. These were determined by Drs. Shafer and Gouaux (Vollum Institute) as one of the “Biological Theme” projects.
As part of NYCOMPS technology development, Drs. Inouye and Montelione are developing methods to greatly simplify the production of isotopically-enriched proteins for NMR.
Molecular Modeling
(i) Joint Center for Molecular Modeling (Adam Godzik)
Dr. Godzik primarily discussed comparison of protein structures in different crystal forms and/or different functional states. The relevance of such comparisons to the PSI seemed questionable.
(ii) New Methods for High-Resolution Comparative Modeling (Roland Dunbrack/David Baker)
Dr. Dunbrack focused particularly on the development of better potential functions for inclusion in the Rosetta program developed by Dr. Baker’s group. Dr. Baker (by video conference) gave selected examples showing how the accuracy of modeling had been improved, although the results need to be evaluated in a more controlled manner to assess the overall benefits.
PSI Materials Repository (Joshua Labaer)
The materials transfer agreements with most of the PSI centers have now been established, and the remainder should be executed shortly.
Dr. Labaer noted that approximately 11,000 plasmids from the PSI are now available through the Materials Repository. This is expected to expand to 100,000 or so.
PSI Knowledgebase (Helen Berman)
Dr. Berman described ongoing rapid progress in developing the PSI Knowledgebase. This is a key component of the PSI and Dr. Berman has clearly demonstrated both her enthusiasm for this initiative as well as her willingness to modify and improve the knowledgebase in response to suggestions and comments from inside and outside the PSI community.
PSI-2 Network Steering Committee Meeting (Wayne Hendrickson et al.)
A number of brief reports were presented from the different subcommittees of the Network Steering Committee. One of the most encouraging aspects of the PSI has been the willingness of the large-scale centers to exchange information and to coordinate target selection.
Summary
The large-scale centers have been extremely effective in introducing new technologies and developing pipelines to permit highly efficient structure determination for soluble proteins. The overall objective of determining 3000 novel structures in PSI-2 is well within reach. At the same time, and as emphasized in the recent Future Structural Genomics Initiatives Meeting Report, these structures are not providing information that is seen to have broad biological and/or medical relevance. A way needs to be found to harness the power of the large-scale centers to determine structures of more immediate biomedical impact.
The success of the specialized centers of PSI-2 has been mixed.
The knowledgebase and the materials repository are clearly essential and need to be maintained. Both are central to the mission of the PSI.
The modeling centers are, at best, making incremental contributions to the success of the PSI. Some of the work in progress may be of interest in its own right, but it does not clearly add value to the PSI initiative as a whole.
With regard to the structural biology of membrane proteins, there is no question that this is a critical area that needs ongoing support. One of the main conclusions of the Center for Structures of Membrane Proteins at UCSF is that the main problem in membrane structural biology is to obtain expressions that are stable and retain activity. To do so it is necessary to know the activity of the protein before protein purification is attempted. Also, in order to obtain crystals that diffract to sufficiently high resolution for structure determination, it is necessary to treat each protein on a case-by-case basis (e.g. finding conditions to trap the protein in one of several functional states). These conclusions tend to be supported by the experience of the New York Consortium on Membrane Protein Structure, where the development of a more “generic” pipeline for membrane proteins has not (yet) led to a significant number of structure determinations. Rather than having large-scale “pipelines” for membrane protein structure determination (similar to the PSI-2 large-scale centers for soluble proteins), it may be more productive to have smaller groups working on subsets of related membrane proteins. There could still be “specialized centers” for membrane proteins, but experience to date inside and outside the PSI in solving membrane protein structures suggests that it may be more productive to have a larger number of smaller centers, each focused on a biological theme, rather than on “pipeline development.”
Report submitted by Protein Structure Initiative Advisory Committee Chair Brian Matthews
