{"title":"Low-cooling-rate freezing in biomolecular cryo-electron microscopy for recovery of initial frames.","authors":"Chunling Wu, Huigang Shi, Dongjie Zhu, Kelong Fan, Xinzheng Zhang","doi":"10.1017/qrd.2021.8","DOIUrl":"https://doi.org/10.1017/qrd.2021.8","url":null,"abstract":"<p><p>When biological samples are first exposed to electrons in cryo-electron microcopy (cryo-EM), proteins exhibit a rapid 'burst' phase of beam-induced motion that cannot be corrected with software. This lowers the quality of the initial frames, which are the least damaged by the electrons. Hence, they are commonly excluded or down-weighted during data processing, reducing the undamaged signal and the resolution in the reconstruction. By decreasing the cooling rate during sample preparation, either with a cooling-rate gradient or by increasing the freezing temperature, we show that the quality of the initial frames for various protein and virus samples can be recovered. Incorporation of the initial frames in the reconstruction increases the resolution by an amount equivalent to using ~60% more data. Moreover, these frames preserve the high-quality cryo-EM densities of radiation-sensitive residues, which is often damaged or very weak in canonical three-dimensional reconstruction. The improved freezing conditions can be easily achieved using existing devices and enhance the overall quality of cryo-EM structures.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"2 ","pages":"e11"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/df/9c/S2633289221000089a.PMC10392635.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9917603","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
QRB DiscoveryPub Date : 2021-01-01DOI: 10.1017/qrd.2021.5
Sara Linse, Kyrre Thalberg, Tuomas P J Knowles
{"title":"The unhappy chaperone.","authors":"Sara Linse, Kyrre Thalberg, Tuomas P J Knowles","doi":"10.1017/qrd.2021.5","DOIUrl":"https://doi.org/10.1017/qrd.2021.5","url":null,"abstract":"<p><p>Chaperones protect other proteins against misfolding and aggregation, a key requirement for maintaining biological function. Experimental observations of changes in solubility of amyloid proteins in the presence of certain chaperones are discussed here in terms of thermodynamic driving forces. We outline how chaperones can enhance amyloid solubility through the formation of heteromolecular aggregates (co-aggregates) based on the second law of thermodynamics and the flux towards equal chemical potential of each compound in all phases of the system. Higher effective solubility of an amyloid peptide in the presence of chaperone implies that the chemical potential of the peptide is higher in the aggregates formed under these conditions compared to peptide-only aggregates. This must be compensated by a larger reduction in chemical potential of the chaperone in the presence of peptide compared to chaperone alone. The driving force thus relies on the chaperone being very unhappy on its own (high chemical potential), thus gaining more free energy than the amyloid peptide loses upon forming the co-aggregate. The formation of heteromolecular aggregates also involves the kinetic suppression of the formation of homomolecular aggregates. The unhappiness of the chaperone can explain the ability of chaperones to favour an increased population of monomeric client protein even in the absence of external energy input, and with broad client specificity. This perspective opens for a new direction of chaperone research and outlines a set of outstanding questions that aim to provide additional cues for therapeutic development in this area.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"2 ","pages":"e7"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/qrd.2021.5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10301862","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
QRB DiscoveryPub Date : 2021-01-01DOI: 10.1017/qrd.2021.7
Amirhossein Mafi, Soo-Kyung Kim, William A Goddard
{"title":"The G protein-first activation mechanism of opioid receptors by Gi protein and agonists.","authors":"Amirhossein Mafi, Soo-Kyung Kim, William A Goddard","doi":"10.1017/qrd.2021.7","DOIUrl":"https://doi.org/10.1017/qrd.2021.7","url":null,"abstract":"<p><p>We report the G protein-first mechanism for activation of G protein-coupled receptors (GPCR) for the three closest subtypes of the opioid receptors (OR), μOR, κOR and δOR. We find that they couple to the inactive Gi protein-bound guanosine diphosphate (GDP) <i>prior</i> to agonist binding. The inactive Gi protein forms anchors to the intracellular loops of the <i>inactive</i> apo-μOR, apo-κOR and apo-δOR, inducing opening of the cytoplasmic region to form a pre-activated state that holds Gi protein in place until agonist binds. Then, agonist binds to μOR, κOR and δOR already complexed with Gi protein, to trigger the Gαi to open up the tightly coupled GDP binding site, making GDP accessible for GTP exchange, an essential step for Gi signalling. We show that the agonist alone <i>cannot</i> open the intracellular region of μOR and κOR, requiring Gi protein to open the cytoplasmic region by itself. We consider that this G protein-first mechanism may apply to activation of other Class A GPCRs. However, for δOR, agonist binding can open up the intracellular region to encourage Gi protein recruitment. Thus, activation of Gi protein mediated by δOR favourably may proceed with either ligand-first or G protein-first activation mechanisms.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"2 ","pages":"e9"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10392629/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9917607","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
QRB DiscoveryPub Date : 2021-01-01DOI: 10.1017/qrd.2021.10
Ying Wang, Chaoyi Gu, Andrew G Ewing
{"title":"A multimodal electrochemical approach to measure the effect of zinc on vesicular content and exocytosis in a single cell model of ischemia.","authors":"Ying Wang, Chaoyi Gu, Andrew G Ewing","doi":"10.1017/qrd.2021.10","DOIUrl":"https://doi.org/10.1017/qrd.2021.10","url":null,"abstract":"<p><p>Zinc ion is essential for normal brain function that modulates synaptic activity and neuronal plasticity and it is associated with memory formation. Zinc is considered to be a contributing factor to the pathogenesis of ischemia, but the association between zinc and ischemia on vesicular exocytosis is unclear. In this study, we used a combination of chemical analysis methods and a cell model of ischemia/reperfusion to investigate exocytotic release and vesicular content, as well as the effect of zinc alteration on vesicular exocytosis. Oxygen-glucose deprivation and reperfusion (OGDR) was used as an <i>in vitro</i> model of ischemia in a model cell line. Exocytotic release and vesicular storage of catecholamine content were increased following OGDR, resulting in a higher fraction of release during exocytosis. However, zinc eliminated these increases following OGDR and the fraction of release remained unchanged. Understanding the consequences of zinc accumulation on vesicular exocytosis at the early stage of OGDR should aid in the development of therapeutic strategies to reduce ischemic brain injury. As the fraction released has been suggested to be related to presynaptic plasticity, insights are gained towards deciphering ischemia related memory impairment.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"2 ","pages":"e12"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10392633/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9923419","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
QRB DiscoveryPub Date : 2021-01-01DOI: 10.1017/qrd.2021.1
Moon Young Yang, Amirhossein Mafi, Soo-Kyung Kim, William A Goddard, Brian Guthrie
{"title":"Predicted structure of fully activated human bitter taste receptor TAS2R4 complexed with G protein and agonists.","authors":"Moon Young Yang, Amirhossein Mafi, Soo-Kyung Kim, William A Goddard, Brian Guthrie","doi":"10.1017/qrd.2021.1","DOIUrl":"https://doi.org/10.1017/qrd.2021.1","url":null,"abstract":"<p><p>Bitter taste is sensed by bitter taste receptors (TAS2Rs) that belong to the G protein-coupled receptor (GPCR) superfamily. In addition to bitter taste perception, TAS2Rs have been reported recently to be expressed in many extraoral tissues and are now known to be involved in health and disease. Despite important roles of TAS2Rs in biological functions and diseases, no crystal structure is available to help understand the signal transduction mechanism or to help develop selective ligands as new therapeutic targets. We report here the three-dimensional structure of the fully activated TAS2R4 human bitter taste receptor predicted using the GEnSeMBLE complete sampling method. This TAS2R4 structure is coupled to the gustducin G protein and to each of several agonists. We find that the G protein couples to TAS2R4 by forming strong salt bridges to each of the three intracellular loops, orienting the activated Gα5 helix of the Gα subunit to interact extensively with the cytoplasmic region of the activated receptor. We find that the TAS2Rs exhibit unique motifs distinct from typical Class A GPCRs, leading to a distinct activation mechanism and a less stable inactive state. This fully activated bitter taste receptor complex structure provides insight into the signal transduction mechanism and into ligand binding to TAS2Rs.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"2 ","pages":"e3"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/qrd.2021.1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9926488","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
QRB DiscoveryPub Date : 2020-11-17DOI: 10.1017/qrd.2020.16
Kaiming Zhang, Shanshan Li, Grigore Pintilie, David Chmielewski, Michael F Schmid, Graham Simmons, Jing Jin, Wah Chiu
{"title":"A 3.4-Å cryo-electron microscopy structure of the human coronavirus spike trimer computationally derived from vitrified NL63 virus particles.","authors":"Kaiming Zhang, Shanshan Li, Grigore Pintilie, David Chmielewski, Michael F Schmid, Graham Simmons, Jing Jin, Wah Chiu","doi":"10.1017/qrd.2020.16","DOIUrl":"10.1017/qrd.2020.16","url":null,"abstract":"<p><p>Human coronavirus NL63 (HCoV-NL63) is an enveloped pathogen of the family <i>Coronaviridae</i> that spreads worldwide and causes up to 10% of all annual respiratory diseases. HCoV-NL63 is typically associated with mild upper respiratory symptoms in children, elderly and immunocompromised individuals. It has also been shown to cause severe lower respiratory illness. NL63 shares ACE2 as a receptor for viral entry with SARS-CoV-1 and SARS-CoV-2. Here, we present the <i>in situ</i> structure of HCoV-NL63 spike (S) trimer at 3.4-Å resolution by single-particle cryo-EM imaging of vitrified virions without chemical fixative. It is structurally homologous to that obtained previously from the biochemically purified ectodomain of HCoV-NL63 S trimer, which displays a three-fold symmetric trimer in a single conformation. In addition to previously proposed and observed glycosylation sites, our map shows density at other sites, as well as different glycan structures. The domain arrangement within a protomer is strikingly different from that of the SARS-CoV-2 S and may explain their different requirements for activating binding to the receptor. This structure provides the basis for future studies of spike proteins with receptors, antibodies or drugs, in the native state of the coronavirus particles.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"1 ","pages":"e11"},"PeriodicalIF":0.0,"publicationDate":"2020-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737156/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10138338","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
QRB DiscoveryPub Date : 2020-06-02eCollection Date: 2020-01-01DOI: 10.1017/qrd.2020.8
Birger Sørensen, Andres Susrud, Angus George Dalgleish
{"title":"Biovacc-19: A Candidate Vaccine for Covid-19 (SARS-CoV-2) Developed from Analysis of its General Method of Action for Infectivity.","authors":"Birger Sørensen, Andres Susrud, Angus George Dalgleish","doi":"10.1017/qrd.2020.8","DOIUrl":"https://doi.org/10.1017/qrd.2020.8","url":null,"abstract":"<p><p>This study presents the background, rationale and method of action of Biovacc-19, a candidate vaccine for corona virus disease 2019 (Covid-19), now in advanced preclinical development, which has already passed the first acute toxicity testing. Unlike conventionally developed vaccines, Biovacc-19's method of operation is upon nonhuman-like (NHL) epitopes in 21.6% of the composition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)'s spike protein, which displays distinct distributed charge including the presence of a charged furin-like cleavage site. The logic of the design of the vaccine is explained, which starts with empirical analysis of the aetiology of SARS-CoV-2. Mistaken assumptions about SARS-CoV-2's aetiology risk creating ineffective or actively harmful vaccines, including the risk of antibody-dependent enhancement. Such problems in vaccine design are illustrated from past experience in the human immunodeficiency viruses domain. We propose that the dual effect general method of action of this chimeric virus's spike, including receptor binding domain, includes membrane components other than the angiotensin-converting enzyme 2 receptor, which explains clinical evidence of its infectivity and pathogenicity. We show the nonreceptor dependent phagocytic general method of action to be specifically related to cumulative charge from insertions placed on the SARS-CoV-2 spike surface in positions to bind efficiently by salt bridge formations; and from blasting the spike we display the NHL epitopes from which Biovacc-19 has been down-selected.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":" ","pages":"e6"},"PeriodicalIF":0.0,"publicationDate":"2020-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/qrd.2020.8","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39125272","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
QRB DiscoveryPub Date : 2020-04-09DOI: 10.1017/qrd.2020.4
Shilei Hao, David Jin, Shuguang Zhang, Rui Qing
{"title":"QTY Code-designed Water-soluble Fc-fusion Cytokine Receptors Bind to their Respective Ligands.","authors":"Shilei Hao, David Jin, Shuguang Zhang, Rui Qing","doi":"10.1017/qrd.2020.4","DOIUrl":"https://doi.org/10.1017/qrd.2020.4","url":null,"abstract":"<p><p>Cytokine release syndrome (CRS), or 'cytokine storm', is the leading side effect during chimeric antigen receptor (CAR)-T therapy that is potentially life-threatening. It also plays a critical role in viral infections such as Coronavirus Disease 2019 (COVID-19). Therefore, efficient removal of excessive cytokines is essential for treatment. We previously reported a novel protein modification tool called the QTY code, through which hydrophobic amino acids Leu, Ile, Val and Phe are replaced by Gln (Q), Thr (T) and Tyr (Y). Thus, the functional detergent-free equivalents of membrane proteins can be designed. Here, we report the application of the QTY code on six variants of cytokine receptors, including interleukin receptors IL4Rα and IL10Rα, chemokine receptors CCR9 and CXCR2, as well as interferon receptors IFNγR1 and IFNλR1. QTY-variant cytokine receptors exhibit physiological properties similar to those of native receptors without the presence of hydrophobic segments. The receptors were fused to the Fc region of immunoglobulin G (IgG) protein to form an antibody-like structure. These QTY code-designed Fc-fusion receptors were expressed in <i>Escherichia coli</i> and purified. The resulting water-soluble fusion receptors bind to their respective ligands with <i>K</i> <sub>d</sub> values affinity similar to isolated native receptors. Our cytokine receptor-Fc-fusion proteins potentially serve as an antibody-like decoy to dampen the excessive cytokine levels associated with CRS and COVID-19 infection.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"1 ","pages":"e4"},"PeriodicalIF":0.0,"publicationDate":"2020-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/qrd.2020.4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9909376","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
QRB DiscoveryPub Date : 2020-01-01DOI: 10.1017/qrd.2020.13
Bengt Nordén, Masayuki Takahashi
{"title":"Understanding Rad51 function is a prerequisite for progress in cancer research.","authors":"Bengt Nordén, Masayuki Takahashi","doi":"10.1017/qrd.2020.13","DOIUrl":"https://doi.org/10.1017/qrd.2020.13","url":null,"abstract":"<p><p>The human protein Rad51 is double-edged in cancer contexts: on one hand, preventing tumourigenesis by eliminating potentially carcinogenic DNA damage and, on the other, promoting tumours by introducing new mutations. Understanding mechanistic details of Rad51 in homologous recombination (HR) and repair could facilitate design of novel methods, including CRISPR, for Rad51-targeted cancer treatment. Despite extensive research, however, we do not yet understand the mechanism of HR in sufficient detail, partly due to complexity, a large number of Rad51 protein units being involved in the exchange of long DNA segments. Another reason for lack of understanding could be that current recognition models of DNA interactions focus only on hydrogen bond-directed base pair formation. A more complete model may need to include, for example, the kinetic effects of DNA base stacking and unstacking ('longitudinal breathing'). These might explain how Rad51 can recognize sequence identity of DNA over several bases long stretches with high accuracy, despite the fact that a single base mismatch could be tolerated if we consider only the hydrogen bond energy. We here propose that certain specific hydrophobic effects, recently discovered destabilizing stacking of nucleobases, may play a central role in this context for the function of Rad51.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"1 ","pages":"e9"},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/qrd.2020.13","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10300873","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
QRB DiscoveryPub Date : 2020-01-01DOI: 10.1017/qrd.2020.3
Bengt Norden
{"title":"Editorial.","authors":"Bengt Norden","doi":"10.1017/qrd.2020.3","DOIUrl":"https://doi.org/10.1017/qrd.2020.3","url":null,"abstract":"On the 51st anniversary of Quarterly Reviews of Biophysics (QRB) and on my 4th year as Editorin-Chief, it is with pleasure that I announce the new open access journal from Cambridge University Press will provide an outlet for exciting new discoveries in the burgeoning field of biophysics. The section called Discovery, which was tested in previous years as part of Quarterly Reviews of Biophysics (QRB), is now upgraded and relaunched as a journal on its own right. The launch of QRBDiscovery, which coincided with the annual conference of the Biophysical Society, promises those working in the field a fast, transparent way to publish cutting edge results. The focus for QRB Discovery will be on biological phenomena that can be described and analysed from a molecular angle. Biophysics applies approaches and methods traditionally used in physics and maths to study the living world, from molecules and cells right up to populations of animals and plants. This interdisciplinary approach has a huge number of applications and has the potential to address some of the biggest challenges facing our species and our planet. It is vital that discoveries with the potential to benefit society are published quickly and transparently. The field has beenmissing a dedicated place to publish ground-breaking results – ‘discoveries’ – that point towards an exciting direction, rather than presenting of a traditional comprehensive study. This is the gap QRB Discovery will seek to fill. Authors are encouraged to elaborate on the potential consequences and wider impact of their discoveries. If the research is of high quality and it is a sound result that points in an exciting direction – even if it is speculative – we will publish. This transparency is further extended by publishing open peer review reports. This will, expectantly, promote a more constructive type of review for authors but it will also contribute to the recognition of reviewers. I look forward to see what exciting research will be submitted next! QRB Discovery","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"1 ","pages":"e1"},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/qrd.2020.3","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9921033","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}