Showing papers by "Michael Levitt published in 2010"

Super-resolution biomolecular crystallography with low-resolution data

Abstract: X-ray crystallography has become the most common method used by structural biologists to obtain three-dimensional structures of proteins and protein–protein complexes. However, crystals of large macromolecular complexes often diffract only weakly — yielding a resolution of less than about 4 A — so it is important to develop new methods that work at such low resolution. Here, the authors show that information from comparative modelling can be combined in a statistically controlled fashion with the observed diffraction data in order to achieve a structure from low-resolution diffraction data that has a similar quality as a high-resolution structure. X-ray crystallography has become the most common way for structural biologists to obtain the three-dimensional structures of proteins and protein complexes. However, crystals of large macromolecular complexes often diffract only weakly (yielding a resolution worse than 4 A), so new methods that work at such low resolution are needed. Here a new method is described by which to obtain higher-quality electron density maps and more accurate molecular models of weakly diffracting crystals. X-ray diffraction plays a pivotal role in the understanding of biological systems by revealing atomic structures of proteins, nucleic acids and their complexes, with much recent interest in very large assemblies like the ribosome. As crystals of such large assemblies often diffract weakly (resolution worse than 4 A), we need methods that work at such low resolution. In macromolecular assemblies, some of the components may be known at high resolution, whereas others are unknown: current refinement methods fail as they require a high-resolution starting structure for the entire complex1. Determining the structure of such complexes, which are often of key biological importance, should be possible in principle as the number of independent diffraction intensities at a resolution better than 5 A generally exceeds the number of degrees of freedom. Here we introduce a method that adds specific information from known homologous structures but allows global and local deformations of these homology models. Our approach uses the observation that local protein structure tends to be conserved as sequence and function evolve. Cross-validation with Rfree (the free R-factor) determines the optimum deformation and influence of the homology model. For test cases at 3.5–5 A resolution with known structures at high resolution, our method gives significant improvements over conventional refinement in the model as monitored by coordinate accuracy, the definition of secondary structure and the quality of electron density maps. For re-refinements of a representative set of 19 low-resolution crystal structures from the Protein Data Bank, we find similar improvements. Thus, a structure derived from low-resolution diffraction data can have quality similar to a high-resolution structure. Our method is applicable to the study of weakly diffracting crystals using X-ray micro-diffraction2 as well as data from new X-ray light sources3. Use of homology information is not restricted to X-ray crystallography and cryo-electron microscopy: as optical imaging advances to subnanometre resolution4,5, it can use similar tools.

Mechanism of folding chamber closure in a group II chaperonin

Abstract: Chaperonins are large, cylindrical complexes that assist in the folding of cellular proteins in an ATP-dependent manner Group II chaperonins are present in eukaryotes and archaea and consist of two back-to-back rings and a lid segment extending from an apical domain In this study, Wah Chiu and colleagues determine the cryo-electron microscopy structure of an archael chaperonin called Mm-cpn in the nucleotide-free (open) and nucleotide-induced (closed) states The structure provides details on conformational changes triggered by ATP hydrolysis leading to rearrangements in inter-ring subunits that differ from other classes of chaperonins Group II chaperonins are present in eukaryotes and archaea and are essential mediators of cellular protein folding This process is critically dependent on the closure of a built-in lid, which is triggered by ATP hydrolysis, but the structural rearrangements and molecular events leading to lid closure are unknown Using cryo-electron microscopy, the structures of an archaeal group II chaperonin in the open and closed states are now reported, providing details of this mechanism Group II chaperonins are essential mediators of cellular protein folding in eukaryotes and archaea These oligomeric protein machines, ∼1 megadalton, consist of two back-to-back rings encompassing a central cavity that accommodates polypeptide substrates1,2,3 Chaperonin-mediated protein folding is critically dependent on the closure of a built-in lid4,5, which is triggered by ATP hydrolysis6 The structural rearrangements and molecular events leading to lid closure are still unknown Here we report four single particle cryo-electron microscopy (cryo-EM) structures of Mm-cpn, an archaeal group II chaperonin5,7, in the nucleotide-free (open) and nucleotide-induced (closed) states The 43 A resolution of the closed conformation allowed building of the first ever atomic model directly from the single particle cryo-EM density map, in which we were able to visualize the nucleotide and more than 70% of the side chains The model of the open conformation was obtained by using the deformable elastic network modelling with the 8 A resolution open-state cryo-EM density restraints Together, the open and closed structures show how local conformational changes triggered by ATP hydrolysis lead to an alteration of intersubunit contacts within and across the rings, ultimately causing a rocking motion that closes the ring Our analyses show that there is an intricate and unforeseen set of interactions controlling allosteric communication and inter-ring signalling, driving the conformational cycle of group II chaperonins Beyond this, we anticipate that our methodology of combining single particle cryo-EM and computational modelling will become a powerful tool in the determination of atomic details involved in the dynamic processes of macromolecular machines in solution

Systematic Review: Effective Management Strategies for Lactose Intolerance

Abstract: This systematic review provides background for the NIH consensus statement on lactose intolerance. The review includes evidence from 36 trials that evaluated a variety of dietary, behavioral, and p...

Climate Change and the Integrity of Science

Abstract: We are deeply disturbed by the recent escalation of political assaults on scientists in general and on climate scientists in particular. All citizens should understand some basic scientific facts. There is always some uncertainty associated with scientific conclusions; science never absolutely proves anything. When someone says that society should wait until scientists are absolutely certain before taking any action, it is the same as saying society should never take action. For a problem as potentially catastrophic as climate change, taking no action poses a dangerous risk for our planet.

Lactose Intolerance and Health

Abstract: Objectives We systematically reviewed evidence to determine lactose intolerance (LI) prevalence, bone health after dairy-exclusion diets, tolerable dose of lactose in subjects with diagnosed LI, and management. Data sources We searched multiple electronic databases for original studies published in English from 1967-November 2009. Review methods We extracted patient and study characteristics using author's definitions of LI and lactose malabsorption. We compared outcomes in relation to diagnostic tests, including lactose challenge, intestinal biopsies of lactase enzyme levels, genetic tests, and symptoms. Fractures, bone mineral content (BMC) and bone mineral density (BMD) were compared in categories of lactose intake. Reported symptoms, lactose dose and formulation, timing of lactose ingestion, and co-ingested food were analyzed in association with tolerability of lactose. Symptoms were compared after administration of probiotics, enzyme replacements, lactose-reduced milk and increasing lactose load. Results Prevalence was reported in 54 primarily nonpopulation based studies (15 from the United States). Studies did not directly assess LI and subjects were highly selected. LI magnitude was very low in children and remained low into adulthood among individuals of Northern European descent. For African American, Hispanic, Asian, and American Indian populations LI rates may be 50 percent higher in late childhood and adulthood. Small doses of lactose were well tolerated in most populations. Low level evidence from 55 observational studies of 223,336 subjects indicated that low milk consumers may have increased fracture risk. Strength and significance varied depended on exposure definitions. Low level evidence from randomized controlled trials (RCTs) of children (seven RCTs) and adult women (two RCTs) with low lactose intake indicated that dairy interventions may improve BMC in select populations. Most individuals with LI can tolerate up to 12 grams of lactose, though symptoms became more prominent at doses above 12 grams and appreciable after 24 grams of lactose; 50 grams induced symptoms in the vast majority. A daily divided dose of 24 grams was generally tolerated. We found insufficient evidence that use of lactose reduced solution/milk, with lactose content of 0-2 grams, compared to a lactose dose of greater than 12 grams, reduced symptoms of lactose intolerance. Evidence was insufficient for probiotics (eight RCTs), colonic adaptation (two RCTs) or varying lactose doses (three RCTs) or other agents (one RCT). Inclusion criteria, interventions, and outcomes were variable. Yogurt and probiotic types studied were variable and results either showed no difference in symptom scores or small differences in symptoms that may be of low clinical relevance. Conclusions There are race and age differences in LI prevalence. Evidence is insufficient to accurately assess U.S. population prevalence of LI. Children with low lactose intake may have beneficial bone outcomes from dairy interventions. There was evidence that most individuals with presumed LI or LM can tolerate 12-15 grams of lactose (approximately 1 cup of milk). There was insufficient evidence regarding effectiveness for all evaluated agents. Additional research is needed to determine LI treatment effectiveness.

Endogenous production of H2S in the gastrointestinal tract: still in search of a physiologic function.

Abstract: Hydrogen sulfide (H2S) has long been associated with the gastrointestinal tract, especially the bacteria-derived H2S present in flatus. Along with evidence from other organ systems, the finding that gastrointestinal tissues are capable of endogenous production of H2S has led to the hypothesis that H2S is an endogenous gaseous signaling molecule. In this review, the criteria of gasotransmitters are reexamined, and evidence from the literature regarding H2S as a gaseous signaling molecule is discussed. H2S is produced enzymatically by gastrointestinal tissues, but evidence is lacking on whether H2S production is regulated. H2S causes well-defined physiologic effects in gastrointestinal tissues, but evidence for a receptor for H2S is lacking. H2S is inactivated through enzymatic oxidation, but evidence is lacking on whether manipulating H2S oxidation alters endogenous cell signaling. Remaining questions regarding the role of H2S as a gaseous signaling molecule in the gastrointestinal tract suggest t...

RNA polymerase II trigger loop residues stabilize and position the incoming nucleotide triphosphate in transcription

Abstract: A structurally conserved element, the trigger loop, has been suggested to play a key role in substrate selection and catalysis of RNA polymerase II (pol II) transcription elongation. Recently resolved X-ray structures showed that the trigger loop forms direct interactions with the β-phosphate and base of the matched nucleotide triphosphate (NTP) through residues His1085 and Leu1081, respectively. In order to understand the role of these two critical residues in stabilizing active site conformation in the dynamic complex, we performed all-atom molecular dynamics simulations of the wild-type pol II elongation complex and its mutants in explicit solvent. In the wild-type complex, we found that the trigger loop is stabilized in the “closed” conformation, and His1085 forms a stable interaction with the NTP. Simulations of point mutations of His1085 are shown to affect this interaction; simulations of alternative protonation states, which are inaccessible through experiment, indicate that only the protonated form is able to stabilize the His1085-NTP interaction. Another trigger loop residue, Leu1081, stabilizes the incoming nucleotide position through interaction with the nucleotide base. Our simulations of this Leu mutant suggest a three-component mechanism for correctly positioning the incoming NTP in which (i) hydrophobic contact through Leu1081, (ii) base stacking, and (iii) base pairing work together to minimize the motion of the incoming NTP base. These results complement experimental observations and provide insight into the role of the trigger loop on transcription fidelity.

Consistent refinement of submitted models at CASP using a knowledge-based potential.

Abstract: Protein structure refinement is an important but unsolved problem; it must be solved if we are to predict biological function that is very sensitive to structural details. Specifically, Critical Assessment of Techniques for Protein Structure Prediction (CASP) shows that the accuracy of predictions in the comparative modeling category is often worse than that of the template on which the homology model is based. Here we describe a refinement protocol that is able to consistently refine submitted predictions for all categories at CASP7. The protocol uses direct energy minimization of the knowledge-based potential of mean force that is based on the interaction statistics of 167 atom types (Summa and Levitt, Proc Natl Acad Sci USA 2007; 104:3177–3182). Our protocol is thus computationally very efficient; it only takes a few minutes of CPU time to run typical protein models (300 residues). We observe an average structural improvement of 1% in GDT_TS, for predictions that have low and medium homology to known PDB structures (Global Distance Test score or GDT_TS between 50 and 80%). We also observe a marked improvement in the stereochemistry of the models. The level of improvement varies amongst the various participants at CASP, but we see large improvements (>10% increase in GDT_TS) even for models predicted by the best performing groups at CASP7. In addition, our protocol consistently improved the best predicted models in the refinement category at CASP7 and CASP8. These improvements in structure and stereochemistry prove the usefulness of our computationally inexpensive, powerful and automatic refinement protocol.

Conformational Optimization with Natural Degrees of Freedom: A Novel Stochastic Chain Closure Algorithm

Abstract: The present article introduces a set of novel methods that facilitate the use of “natural moves” or arbitrary degrees of freedom that can give rise to collective rearrangements in the structure of biological macromolecules. While such “natural moves” may spoil the stereochemistry and even break the bonded chain at multiple locations, our new method restores the correct chain geometry by adjusting bond and torsion angles in an arbitrary defined molten zone. This is done by successive stages of partial closure that propagate the location of the chain break backwards along the chain. At the end of these stages, the size of the chain break is generally reduced so much that it can be repaired by adjusting the position of a single atom. Our chain closure method is efficient with a computational complexity of O(Nd), where Nd is the number of degrees of freedom used to repair the chain break. The new method facilitates the use of arbitrary degrees of freedom including the “natural” degrees of freedom inf...

Insights into the intra-ring subunit order of tric/cct: a structural and evolutionary analysis

Abstract: TRiC is an important group II chaperonin that facilitates the folding of many eukaryotic proteins. The TRiC complex consists of two stacked rings, each comprised of eight paralogous subunits with a mutual sequence identity of 30-35%. Each subunit has unique functional roles that are manifested by corresponding sequence conservation. It is generally assumed that the subunit order within each ring is fixed, but this order is still uncertain. Here we address the problem of the intra-ring subunit order by combining two sources of information: evolutionary conservation and a structural hypothesis. Specifically, we identify residues in the TRiC subunits that are likely to be part of the intra-unit interface, based on homology modeling to the solved thermosome structure. Within this set of residues, we search for a subset that shows an evolutionary conservation pattern that is indicative of the subunit order key. This pattern shows considerable conservation across species, but large variation across the eight subunits. By this approach we were able to locate two parts of the interface where complementary interactions seem to favor certain pairing of subunits. This knowledge leads to restrictions on the 5,040 (=7!) possible subunits arrangements in the ring, and limits them to just 72. Although our findings give only partial understanding of the inter-subunit interactions that determine their order, we conclude that they are comprised of complementary charged, polar and hydrophobic interactions that occur in both the equatorial and middle domains of each subunit.