Basic Life Science Research Associate, Bioengineering
Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR-Cas complex revealed by cryo-EM.
Proceedings of the National Academy of Sciences of the United States of America
Prokaryotes and viruses have fought a long battle against each other. Prokaryotes use CRISPR-Cas-mediated adaptive immunity, while conversely, viruses evolve multiple anti-CRISPR (Acr) proteins to defeat these CRISPR-Cas systems. The type I-F CRISPR-Cas system in Pseudomonas aeruginosa requires the crRNA-guided surveillance complex (Csy complex) to recognize the invading DNA. Although some Acr proteins against the Csy complex have been reported, other relevant Acr proteins still need studies to understand their mechanisms. Here, we obtain three structures of previously unresolved Acr proteins (AcrF9, AcrF8, and AcrF6) bound to the Csy complex using electron cryo-microscopy (cryo-EM), with resolution at 2.57 A, 3.42 A, and 3.15 A, respectively. The 2.57-A structure reveals fine details for each molecular component within the Csy complex as well as the direct and water-mediated interactions between proteins and CRISPR RNA (crRNA). Our structures also show unambiguously how these Acr proteins bind differently to the Csy complex. AcrF9 binds to key DNA-binding sites on the Csy spiral backbone. AcrF6 binds at the junction between Cas7.6f and Cas8f, which is critical for DNA duplex splitting. AcrF8 binds to a distinct position on the Csy spiral backbone and forms interactions with crRNA, which has not been seen in other Acr proteins against the Csy complex. Our structure-guided mutagenesis and biochemistry experiments further support the anti-CRISPR mechanisms of these Acr proteins. Our findings support the convergent consequence of inhibiting degradation of invading DNA by these Acr proteins, albeit with different modes of interactions with the type I-F CRISPR-Cas system.
View details for DOI 10.1073/pnas.1922638117
View details for PubMedID 32170016
Measurement of atom resolvability in cryo-EM maps with Q-scores.
Cryogenic electron microscopy (cryo-EM) maps are now at the point where resolvability of individual atoms can be achieved. However, resolvability is not necessarily uniform throughout the map. We introduce a quantitative parameter to characterize the resolvability of individual atoms in cryo-EM maps, the map Q-score. Q-scores can be calculated for atoms in proteins, nucleic acids, water, ligands and other solvent atoms, using models fitted to or derived from cryo-EM maps. Q-scores can also be averaged to represent larger features such as entire residues and nucleotides. Averaged over entire models, Q-scores correlate very well with the estimated resolution of cryo-EM maps for both protein and RNA. Assuming the models they are calculated from are well fitted to the map, Q-scores can be used as a measure of resolvability in cryo-EM maps at various scales, from entire macromolecules down to individual atoms. Q-score analysis of multiple cryo-EM maps of the same proteins derived from different laboratories confirms the reproducibility of structural features from side chains down to water and ion atoms.
View details for DOI 10.1038/s41592-020-0731-1
View details for PubMedID 32042190
Accelerated cryo-EM-guided determination of three-dimensional RNA-only structures.
2020; 17 (7): 699–707
The discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. Here, we demonstrate that cryo-electron microscopy can routinely resolve maps of RNA-only systems and that these maps enable subnanometer-resolution coordinate estimation when complemented with multidimensional chemical mapping and Rosetta DRRAFTER computational modeling. This hybrid 'Ribosolve' pipeline detects and falsifies homologies and conformational rearrangements in 11 previously unknown 119- to 338-nucleotide protein-free RNA structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length Vibrio cholerae and Fusobacterium nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and the computer-designed ATP-TTR-3 aptamer with and without AMP. Simulation benchmarks, blind challenges, compensatory mutagenesis, cross-RNA homologies and internal controls demonstrate that Ribosolve can accurately resolve the global architectures of RNA molecules but does not resolve atomic details. These tests offer guidelines for making inferences in future RNA structural studies with similarly accelerated throughput.
View details for DOI 10.1038/s41592-020-0878-9
View details for PubMedID 32616928
Structural basis of amino acid surveillance by higher-order tRNA-mRNA interactions.
Nature structural & molecular biology
Amino acid availability in Gram-positive bacteria is monitored by T-box riboswitches. T-boxes directly bind tRNAs, assess their aminoacylation state, and regulate the transcription or translation of downstream genes to maintain nutritional homeostasis. Here, we report cocrystal and cryo-EM structures of Geobacillus kaustophilus and Bacillus subtilis T-box-tRNA complexes, detailing their multivalent, exquisitely selective interactions. The T-box forms a U-shaped molecular vise that clamps the tRNA, captures its 3' end using an elaborate 'discriminator' structure, and interrogates its aminoacylation state using a steric filter fashioned from a wobble base pair. In the absence of aminoacylation, T-boxes clutch tRNAs and form a continuously stacked central spine, permitting transcriptional readthrough or translation initiation. A modeled aminoacyl disrupts tRNA-T-box stacking, severing the central spine and blocking gene expression. Our data establish a universal mechanism of amino acid sensing on tRNAs and gene regulation by T-box riboswitches and exemplify how higher-order RNA-RNA interactions achieve multivalency and specificity.
View details for DOI 10.1038/s41594-019-0326-7
View details for PubMedID 31740854
Segmentation and Comparative Modeling in an 8.6-angstrom Cryo-EM Map of the Singapore Grouper Iridovirus
2019; 27 (10): 1561-+
SGIV, or Singapore grouper iridovirus, is a large double-stranded DNA virus, reaching a diameter of 220 nm and packaging a genome of 140 kb. We present a 3D cryoelectron microscopy (cryo-EM) icosahedral reconstruction of SGIV determined at 8.6-Å resolution. It reveals several layers including a T = 247 icosahedral outer coat, anchor proteins, a lipid bilayer, and the encapsidated DNA. A new segmentation tool, iSeg, was applied to extract these layers from the reconstructed map. The outer coat was further segmented into major and minor capsid proteins. None of the proteins extracted by segmentation have known atomic structures. We generated models for the major coat protein using three comparative modeling tools, and evaluated each model using the cryo-EM map. Our analysis reveals a new architecture in the Iridoviridae family of viruses. It shares similarities with others in the same family, e.g., Chilo iridescent virus, but also shows new features of the major and minor capsid proteins.
View details for DOI 10.1016/j.str.2019.08.002
View details for Web of Science ID 000488523700009
View details for PubMedID 31447288
View details for PubMedCentralID PMC6853598
Rapid RNA structure determination through cryo-EM, high-throughput biochemistry, and computational modeling
AMER CHEMICAL SOC. 2019
View details for Web of Science ID 000525055504576
- The Chaperonin TRiC/CCT Associates with Prefoldin through a Conserved Electrostatic Interface Essential for Cellular Proteostasis CELL 2019; 177 (3): 751-+
- Cryo-EM structures of Helicobacter pylori vacuolating cytotoxin A oligomeric assemblies at near-atomic resolution PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2019; 116 (14): 6800–6805
- Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR-Csm revealed by cryo-EM and biochemistry CELL RESEARCH 2019; 29 (4): 305–12
The Chaperonin TRiC/CCT Associates with Prefoldin through a Conserved Electrostatic Interface Essential for Cellular Proteostasis.
Maintaining proteostasis in eukaryotic protein folding involves cooperation of distinct chaperone systems. To understand how the essential ring-shaped chaperonin TRiC/CCT cooperates with the chaperone prefoldin/GIMc (PFD), we integrate cryoelectron microscopy (cryo-EM), crosslinking-mass-spectrometry and biochemical and cellular approaches to elucidate the structural and functional interplay between TRiC/CCT and PFD. We find these hetero-oligomeric chaperones associate in a defined architecture, through a conserved interface of electrostatic contacts that serves as a pivot point for a TRiC-PFD conformational cycle. PFD alternates between an open "latched" conformation and a closed "engaged" conformation that aligns the PFD-TRiC substrate binding chambers. PFD can act after TRiC bound its substrates to enhance the rate and yield of the folding reaction, suppressing non-productive reaction cycles. Disrupting the TRiC-PFD interaction invivo is strongly deleterious, leading to accumulation of amyloid aggregates. The supra-chaperone assembly formed by PFD and TRiC is essential to prevent toxic conformations and ensure effective cellular proteostasis.
View details for PubMedID 30955883
Cryo-EM structures of Helicobacter pylori vacuolating cytotoxin A oligomeric assemblies at near-atomic resolution.
Proceedings of the National Academy of Sciences of the United States of America
Human gastric pathogen Helicobacter pylori (H. pylori) is the primary risk factor for gastric cancer and is one of the most prevalent carcinogenic infectious agents. Vacuolating cytotoxin A (VacA) is a key virulence factor secreted by H. pylori and induces multiple cellular responses. Although structural and functional studies of VacA have been extensively performed, the high-resolution structure of a full-length VacA protomer and the molecular basis of its oligomerization are still unknown. Here, we use cryoelectron microscopy to resolve 10 structures of VacA assemblies, including monolayer (hexamer and heptamer) and bilayer (dodecamer, tridecamer, and tetradecamer) oligomers. The models of the 88-kDa full-length VacA protomer derived from the near-atomic resolution maps are highly conserved among different oligomers and show a continuous right-handed beta-helix made up of two domains with extensive domain-domain interactions. The specific interactions between adjacent protomers in the same layer stabilizing the oligomers are well resolved. For double-layer oligomers, we found short- and/or long-range hydrophobic interactions between protomers across the two layers. Our structures and other previous observations lead to a mechanistic model wherein VacA hexamer would correspond to the prepore-forming state, and the N-terminal region of VacA responsible for the membrane insertion would undergo a large conformational change to bring the hydrophobic transmembrane region to the center of the oligomer for the membrane channel formation.
View details for PubMedID 30894496
- Structure of Calcarisporiella thermophila Hsp104 Disaggregase that Antagonizes Diverse Proteotoxic Misfolding Events STRUCTURE 2019; 27 (3): 449-+
Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR-Csm revealed by cryo-EM and biochemistry.
The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading nucleic acids. They accomplish this task through the coordinated cleavage of invading substrates of single-stranded RNA and DNA (ssDNA and ssRNA) by the Csm (type III-A) or Cmr (type III-B) effector complexes. The ssRNA is complementarily bound to the CRISPR RNA (crRNA). However, the structural basis for the DNase and RNase activation of the Csm nucleoprotein complex is largely unknown. Here we report cryo-EM structures of the Csm-crRNA complex, with or without target ssRNA, at near-atomic resolution. Our cryo-EM maps allow us to build atomic models of the key macromolecular components, including Cas10, Csm2, Csm3, Csm4, crRNA and the invading ssRNA. Our structure resolves unambiguously the stoichiometry and tertiary structures of the Csm protein complex and the interactions between protein components and the crRNA/ssRNA. Interestingly, the new atomic structures of the Csm proteins presented here are similar to those of previously known Csm proteins in other species despite their low sequence similarity. Our combined structural and biochemical data suggest that ssRNA cleavage is preferentially carried out near its 5'-end, that the extent of interactions among the ssRNA, crRNA and the protein components regulates the DNase activity of the Csm complex, and that the 3' flanking sequence of ssRNA activates the Cas10 DNase activity allosterically.
View details for PubMedID 30814678
- Outcomes of the Cryo-EM Map and Model Challenges CELL PRESS. 2019: 160A
Cryo-EM structure of a 40 kDa SAM-IV riboswitch RNA at 3.7 Å resolution.
2019; 10 (1): 5511
Specimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The high flexibility of pure RNAs makes it difficult to obtain high-resolution structures by cryo-EM. In bacteria, riboswitches regulate sulfur metabolism through binding to the S-adenosylmethionine (SAM) ligand and offer compelling targets for new antibiotics. SAM-I, SAM-I/IV, and SAM-IV are the three most commonly found SAM riboswitches, but the structure of SAM-IV is still unknown. Here, we report the structures of apo and SAM-bound SAM-IV riboswitches (119-nt, ~40 kDa) to 3.7 Å and 4.1 Å resolution, respectively, using cryo-EM. The structures illustrate homologies in the ligand-binding core but distinct peripheral tertiary contacts in SAM-IV compared to SAM-I and SAM-I/IV. Our results demonstrate the feasibility of resolving small RNAs with enough detail to enable detection of their ligand-binding pockets and suggest that cryo-EM could play a role in structure-assisted drug design for RNA.
View details for DOI 10.1038/s41467-019-13494-7
View details for PubMedID 31796736
Assessment of structural features in Cryo-EM density maps using SSE and side chain Z-scores
JOURNAL OF STRUCTURAL BIOLOGY
2018; 204 (3): 564–71
We introduce a new method for assessing resolvability of structural features in density maps from Cryo-Electron Microscopy (Cryo-EM) using fitted or derived models. It calculates Z-scores for secondary structure elements (SSEs) and side chains. Z-scores capture how much larger the cross-correlation score (CCS) is for atoms in such features at their placed locations compared to the CCS at displaced positions. Z-scores are larger when the structural features are well-resolved, as confirmed by visual analysis. This method was applied to all 66 maps submitted to the 2015/2016 EMDB map challenge. For each map, the fitted model provided by the map committee was used in this assessment. The average Z-scores for each map and fitted model correlate moderately well with reported map resolutions (r2 = 0.45 for SSE Z-scores and r2 = 0.56 for side chain Z-scores). Rankings of the submitted maps based on average Z-scores seem to more closely agree with visual analysis. Z-scores can also be used to pinpoint which parts of a model are well-resolved in a map, and which parts of the model may need further fitting or refinement to make the model better match the density.
View details for DOI 10.1016/j.jsb.2018.08.015
View details for Web of Science ID 000454373000022
View details for PubMedID 30144506
View details for PubMedCentralID PMC6525962
Structure of Calcarisporiella thermophila Hsp104 Disaggregase that Antagonizes Diverse Proteotoxic Misfolding Events.
Structure (London, England : 1993)
Hsp104 is an AAA+ protein disaggregase with powerful amyloid-remodeling activity. All nonmetazoan eukaryotes express Hsp104 while eubacteria express an Hsp104 ortholog, ClpB. However, most studies have focused on Hsp104 from Saccharomyces cerevisiae and ClpB orthologs from two eubacterial species. Thus, the natural spectrum of Hsp104/ClpB molecular architectures and protein-remodeling activities remains largely unexplored. Here, we report two structures of Hsp104 from the thermophilic fungus Calcarisporiella thermophila (CtHsp104), a 2.70A crystal structure and 4.0A cryo-electron microscopystructure. Both structures reveal left-handed, helical assemblies with all domains clearly resolved. We thus provide the highest resolution and most complete view of Hsp104 hexamers to date. We also establish that CtHsp104 antagonizes several toxic protein-misfolding events invivo where S. cerevisiae Hsp104 is ineffective, including rescue of TDP-43, polyglutamine, and alpha-synuclein toxicity. We suggest that natural Hsp104 variation is an invaluable, untapped resource for illuminating therapeutic disaggregases for fatal neurodegenerative diseases.
View details for PubMedID 30595457
Comparison of Crystal and Cryoem Structures of Hsp104 and ClpB Disaggregases
WILEY. 2018: 32–33
View details for Web of Science ID 000450682700039
The first single particle analysis Map Challenge: A summary of the assessments
JOURNAL OF STRUCTURAL BIOLOGY
2018; 204 (2): 291–300
The recent successes of cryo-electron microscopy fostered great expectation of solving many new and previously recalcitrant biomolecular structures. However, it also brings with it the danger of compromising the validity of the outcomes if not done properly. The Map Challenge is a first step in assessing the state of the art and to shape future developments in data processing. The organizers presented seven cases for single particle reconstruction, and 27 members of the community responded with 66 submissions. Seven groups analyzed these submissions, resulting in several assessment reports, summarized here. We devised a range of analyses to evaluate the submitted maps, including visual impressions, Fourier shell correlation, pairwise similarity and interpretation through modeling. Unfortunately, we did not find strong trends. We ascribe this to the complexity of the challenge, dealing with multiple cases, software packages and processing approaches. This puts the user in the spotlight, where his/her choices becomes the determinant of map quality. The future focus should therefore be on promulgating best practices and encapsulating these in the software. Such practices include adherence to validation principles, most notably the processing of independent sets, proper resolution-limited alignment, appropriate masking and map sharpening. We consider the Map Challenge to be a highly valuable exercise that should be repeated frequently or on an ongoing basis.
View details for DOI 10.1016/j.jsb.2018.08.010
View details for Web of Science ID 000447226400018
View details for PubMedID 30114512
View details for PubMedCentralID PMC6205511
The 3.5-A CryoEM Structure of Nanodisc-Reconstituted Yeast Vacuolar ATPase Vo Proton Channel
The 3.5-A CryoEM Structure of Nanodisc-Reconstituted Yeast Vacuolar ATPase Vo Proton Channel
2018; 69 (6): 993-1004
The molecular mechanism of transmembrane proton translocation in rotary motor ATPases is not fully understood. Here, we report the 3.5-Å resolution cryoEM structure of the lipid nanodisc-reconstituted Vo proton channel of the yeast vacuolar H+-ATPase, captured in a physiologically relevant, autoinhibited state. The resulting atomic model provides structural detail for the amino acids that constitute the proton pathway at the interface of the proteolipid ring and subunit a. Based on the structure and previous mutagenesis studies, we propose the chemical basis of transmembrane proton transport. Moreover, we discovered that the C terminus of the assembly factor Voa1 is an integral component of mature Vo. Voa1's C-terminal transmembrane α helix is bound inside the proteolipid ring, where it contributes to the stability of the complex. Our structure rationalizes possible mechanisms by which mutations in human Vo can result in disease phenotypes and may thus provide new avenues for therapeutic interventions.
View details for DOI 10.1016/j.molcel.2018.02.006
View details for PubMedCentralID PMC5893162
Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly
2018; 172 (5): 966-978
Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22-α23. Biochemical, biophysical, and mutational analyses revealed that inter-eNP contacts within α22-α23 are critical for viral NC assembly and regulate viral RNA synthesis. These observations suggest that the N terminus and α22-α23 of eNP function as context-dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target.
View details for DOI 10.1016/j.cell.2018.02.009
View details for PubMedCentralID PMC5973842
Resolution and Probabilistic Models of Components in CryoEM Maps of Mature P22 Bacteriophage
2016; 110 (4): 827-839
CryoEM continues to produce density maps of larger and more complex assemblies with multiple protein components of mixed symmetries. Resolution is not always uniform throughout a cryoEM map, and it can be useful to estimate the resolution in specific molecular components of a large assembly. In this study, we present procedures to 1) estimate the resolution in subcomponents by gold-standard Fourier shell correlation (FSC); 2) validate modeling procedures, particularly at medium resolutions, which can include loop modeling and flexible fitting; and 3) build probabilistic models that combine high-accuracy priors (such as crystallographic structures) with medium-resolution cryoEM densities. As an example, we apply these methods to new cryoEM maps of the mature bacteriophage P22, reconstructed without imposing icosahedral symmetry. Resolution estimates based on gold-standard FSC show the highest resolution in the coat region (7.6 Å), whereas other components are at slightly lower resolutions: portal (9.2 Å), hub (8.5 Å), tailspike (10.9 Å), and needle (10.5 Å). These differences are indicative of inherent structural heterogeneity and/or reconstruction accuracy in different subcomponents of the map. Probabilistic models for these subcomponents provide new insights, to our knowledge, and structural information when taking into account uncertainty given the limitations of the observed density.
View details for DOI 10.1016/j.bpj.2015.11.3522
View details for Web of Science ID 000370763800011
View details for PubMedID 26743049
View details for PubMedCentralID PMC4775875