Tag Archives: proteins

A celebration of cryo-EM

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Here at Nature Methods, we were quite excited yesterday to wake up to the news that the Nobel Prize in Chemistry had been awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson for their seminal developments in cryo-electron microscopy (better known as cryo-EM) which now enable high-resolution biomolecule structure determination. This is a technique we have been watching closely since 2013, when the first papers (including one of our own) realizing the capability of near-atomic-resolution structure determination with cryo-EM were published.

Though much of the excitement about cryo-EM is quite recent, the Nobel Prize is a good reminder to us all that the essential foundations of this technology were laid decades ago. We celebrated such developments, both old and new, in our 2015 Method of the Year issue featuring cryo-EM.

To commemorate this well-deserved Nobel Prize, Nature Research presents an editorially curated collection of papers published in our pages – including methods and protocols, biological results generated using cryo-EM technology, and reviews, news and comment. Check it out!

Sunset on the PSI

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As discussed in this month’s Editorial, the Protein Structure Initiative (PSI), a 15-year, nearly $1 billion structural genomics project funded by the National Institute of General Medical Sciences (NIGMS), will be coming to an end in 2015. The impact of ending this project should be minimized to avoid the loss of valuable resources and expertise.

The PSI was begun in 2000 when the US NIH budget was in the midst of substantial growth, spurring the creation of many new “big science” projects. At that time, protein structure determination was painfully slow. The PSI’s initial goals were to develop tools and methods to improve the speed and ability to solve protein structures, as well as to generate a large resource of novel and unique protein structures to facilitate homology modeling and to promote follow-up functional studies.

Protein Structures

{credit}Image Credit: Erin Boyle{/credit}

The PSI drew criticisms from many in the structural biology community from its inception, however. Critics of the first two phases of the PSI pointed out that it focused mainly on solving small bacterial protein structures that were not biologically very interesting, simply because they were relatively easy to express, purify and crystallize. This led the PSI to substantially change course in the third and current phase, PSI-Biology, which began in 2010. The emphasis on throughput was diminished and shifted to solving important, difficult structures like human membrane proteins and drug targets. This change of course has led to several successful structures for highly interesting yet difficult proteins such as GPCRs.

In 2013, a scientific advisory panel produced a mid-point evaluation report of PSI-Biology for NIGMS (PDF), assessing its strengths and weaknesses. The panel commended the impressive number of high-quality structures and methodological advances of the PSI centers, but noted that outreach to the broader biological community was inadequate. The panel found that the PSI’s community resources – the Structural Biology Knowledgebase, a portal for research, news and resources produced by the PSI (in collaboration with Nature Publishing Group), and the Materials Repository, which provides over 80,000 plasmids and 106 empty vectors to the community – were not widely taken advantage of by researchers outside the PSI. The panel also noted that NIGMS should start planning to transition the PSI from its current set-aside funding structure to a different funding model that maintains its unique resources and capabilities – but that it should also extend PSI-Biology for another 3 to 5 years past 2015 to allow it to reach its full potential.

However, budget cuts and a reassessment of NIGMS’s large-scale research initiatives by its new director, Jon Lorsch, has led the institute to prematurely cut the program after the current phase, PSI-Biology, ends in 2015.

The loss of this large-scale program will certainly shake up structural biology in the US, particularly for all those involved in the PSI but also for the many researchers – even outside of traditional structural biology – who benefit from PSI resources. As we discuss in this month’s Editorial, NIGMS now has the opportunity to set an example for other funding agencies in how to wind down a big science project with minimal negative impact. Internal and external transition planning committees have been created by NIGMS to determine which resources and capabilities developed by the PSI should be preserved and how this can be done. NIGMS has also put out a Request for Information seeking community input on the utility of resources developed by the PSI; the response date (May 23, 2014) has only just passed.

NIGMS is expected to make a decision before the end of 2014 as to what will be done with the substantial high-throughput expression, purification and crystallization facilities developed during the PSI’s tenure. In the Editorial we argue that this infrastructure – and the large-scale raw data and metadata generated that is not in any database, but is valuable for mining and algorithm development – should be preserved as much as possible. We argue that a project to systematically sample protein folds should be continued on a smaller scale. We also argue that NIGMS should continue to facilitate team research – as the internal transition planning committee chair Douglas Sheeley has said it will – to tackle particularly challenging structural biology research problems that require hybrid methods to solve.

We will be watching closely to see whether the negative fallout from the end of the PSI can indeed be minimized.

An all-encompassing term to describe protein complexity

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Neil Kelleher and Lloyd Smith propose that the scientific community adopt the term ‘proteoform’ to refer to all the different forms that a protein can take. Will the community adopt it?

The field of top-down proteomics, in which intact proteins are analyzed by a mass spectrometer, provides rich information about the genetic variations, alternative splicing and post-translational modifications that can be lost in a bottom-up proteomics approach (where proteins are digested into peptides prior to analysis). An unsolved problem in the top-down field, however, has been what exactly to call these various protein forms. Besides ‘protein forms’, a handful of other terms have been batted around in the literature, including ‘protein variants’, ‘protein isoforms’ and ‘protein species.’

In a Correspondence in the March issue of Nature Methods, Neil Kelleher and Lloyd Smith lay out the reasons why none of these terms are satisfactory. What is needed, they argue, is a novel, unique, intuitive, single-word term with a precise definition that is all-encompassing in describing protein complexity, and is also compatible with a gene-centric approach to protein naming. They believe that they have the perfect term: proteoform.

“It’s not just a term, it’s a movement,” says Kelleher. Kelleher has been one of the key drivers of top down methodology development, and argues that using a controlled vocabulary to describe proteins will serve a catalytic role in moving the field forward. “The implicit thing about this term is that it puts a focal point on the fact that [the proteoforms] are the functional players, insofar as protein primary structure is concerned,” he says. Especially in clinical research, he notes, different proteoforms are tied strongly to function and phenotype.

Kelleher and Smith have been gathering support for their term over the last several months by introducing it at conferences and inviting researchers to comment on a LinkedIn forum. The term also has the full support of the Consortium for Top Down Proteomics. At their latest conference in Florida, about a month ago, Kelleher says that “everyone” was using “proteoform” in their talks. “It just catches on…it fills a void the rolls right off the tongue at conferences and sits well in the gut while digesting text,” he says. The consortium website maintains a repository of proteoforms, which they hope will grow. Kelleher also notes that the term is being embraced by key protein informatics players at UniProt and the Protein Information Resource, both of which have adopted a gene-centric approach to protein naming.

What do you think about the term “proteoform”? Will you adopt it? We’d love to hear from you!

A different kind of Method of the Year for 2012

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Our choice of Method of the Year in prior years has tended to be methods that generally didn’t even exist only a few years earlier but which had quickly bounded onto the scientific stage and attracted the attention of a large portion of the scientific community. Targeted proteomics, our choice for 2012, on the other hand has existed for years in scaled-down forms using methods based on antibodies. Western blotting, immunofluorescence, antibody arrays, etc. can all be used to detect and measure targeted subsets the proteins expressed in cells and tissues.

During this time the workhorse of proteomics, the mass spectrometer, has been used mostly for shotgun proteomics experiments in which the goal was to analyze all the proteins in a sample. But the means to use these machines for targeted detection of defined subsets of proteins and obtain more reproducible measurements than shotgun experiments can typically provide have been around for decades.

Shotgun methods have been mostly confined to specialist laboratories as many biologists have been intimidated by the complexity of implementing and analyzing these experiments properly. Targeted proteomics on the other hand offers a tantalizing opportunity to bring a sampling of the power of mass spectrometry to the wider community of biologists. The assays are simpler, easier to run and well suited to the hypothesis-driven experiments that are the mainstay of biological research.

The ubiquitous Western blot has long filled a central role or functioned as a crucial control in many research studies. Unfortunately performing a high-quality Western blot can feel a bit like roulette. Sometimes you get a fantastic looking blot with an accurate antibody but other times either the blot is blank, the bands may look like they ran through some carnival ride or it might suffer from any number of other problems. This might prompt people to either look for a goat to appease the Western blot gods or take unscientific liberties with the presentation of the data in order to make it look like they are believe it should. It also lessens the likelihood that important replicates are performed or reported.

Targeted mass spectrometry offers the possibility for thousands of labs to move away from, or supplement, Western blots; and improve the quality and quantity of their protein measurements. This is not as sexy as next-generation sequencing, super-resolution imaging or optogenetics, some of our prior choices of Method of the Year, but the potential for revolutionizing an arguably mundane but indispensable technique was compelling enough that it played no small role in our decision. Only time will tell what impact the method has and we eagerly look forward to the answer.