Development of imaging scaffolds for cryo-electron microscopy

Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

Single Particle Kinetic and Electron Cryo-Microscopy Studies of Flavivirus Membrane Fusion

Recent technological breakthroughs in single-particle cryo-electron microscopy (cryo-EM) enable rapid atomic structure determination of biological macromolecules. A major bottleneck in the current single particle cryo-EM pipeline is the preparation of good quality frozen cryo-EM grids, which is mostly a trial-and-error process. Among many issues, preferred particle orientation and sample damage by air–water interface (AWI) are common practical problems. Here we report a method of applying metallo-supramolecular branched polymer (MSBP) in the cryo-sample preparation for high-resolution single-particle cryo-EM. Our data shows that MSBP keeps a majority of particles away from air–water interface and mitigates preferred orientation as verified by the analyses of apoferritin, hemagglutinin) trimer and various sample proteins. The use of MSBP is a simple method to improve particle distribution for high-resolution structure determination in single-particle cryo-EM.

Ryanodine-sensitive Ca2+ release channels, also known as the ryanodine receptors (RyRs), 2The abbreviations used are: RyR, ryanodine receptor; cryo-EM, cryoelectron microscopy; CY, cytoplasmic; TM, transmembrane; aa, amino acids; GFP, green fluorescent protein; IP3, inositol trisphosphate; CaM, calmodulin; FKBP, FK506-binding protein. are homotetramers of an ∼550-kDa subunit (Fig. 1) that are resident proteins of intracellular membranes such as the sarcoplasmic/endoplasmic reticulum. RyRs are responsible for the regulated release of Ca2+ from these lumenal stores. Over the last 10 years, single-particle cryoelectron microscopy (cryo-EM) has produced several low resolution (∼30 Å) structures of RyRs (for review, see Ref. 1Serysheva I.I. Chiu W. Ludtke S.J. Methods Cell Biol. 2007; 79: 407-435Crossref PubMed Scopus (8) Google Scholar). Recently, cryo-EM reconstructions of RyR1 have reached subnanometer resolution (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar), a breakthrough due to improvements in cryo-specimen preparation, instrumentation, image processing, and three-dimensional reconstruction techniques. RyRs have a mushroom shape with 4-fold symmetry (Fig. 1B) (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Most of the mass of RyR forms a large cytoplasmic (CY) assembly (280 × 280 × 120 Å) that is connected to the transmembrane (TM) region by a stalk-like structure. The CY region is strikingly empty with numerous distinctive structural domains and intervening cavities that appear suitable for interaction with modulators that bind within the N-terminal regions of RyR (Fig. 1). The clamp-shaped regions, located at the corners of the CY assembly, are likely regions for the interdigitation of neighboring RyRs seen in situ (5Block B.A. Franzini-Armstrong C. J. Cell Biol. 1988; 107: 1099-1112Crossref PubMed Scopus (47) Google Scholar) or for interaction with modulators. The clamp-shaped regions are interconnected to form a continuous network between the central rim and the CY stalk-like structure via several bridging densities. The TM region (120 × 120 × 60 Å) is rotated by ∼40° with respect to the CY region. The pore is thought to be composed of six to eight TM segments (RyR1 TM1–8, amino acids (aa) 4277–4323, 4343–4363, 4557–4576, 4637–4662, 4776–4800, 4803–4825, 4834–4854, and 4911–4935 with aa 4854–4911 forming the pore helix and selectivity filter) (Fig. 1A, line 1) (6Du G.G. Sandhu B. Khanna V.K. Guo X.H. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16725-16730Crossref PubMed Scopus (118) Google Scholar). This arrangement places both the N and C termini of RyR in the cytoplasm. Sequence numbers used in this review refer to rabbit RyR1 (Swiss-Prot accession number P11716) and, where indicated, RyR2 (Swiss-Prot accession number P30957). The best current maps of RyR1 have resolutions of ∼10 Å (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar), allowing delineation of secondary structure elements in the pore region (Fig. 2A): the helix running across the membrane (helix 1, possibly TM8), the short helix (helix 2, which is likely to be part of the pore helix) that forms the lumenal entrance of the channel, and helix 3 (located parallel to the membrane plane on the CY side of the TM region). Helices 1 and 2 lie near the 4-fold channel axis and resemble the inner (pore-lining) and pore helices in K+ channel structures (7Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Sciences (N. Y.). 1998; 280: 69-77Crossref PubMed Scopus (5768) Google Scholar, 8Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Crossref PubMed Scopus (1209) Google Scholar). The orientation of helix 3 is similar to the slide helix seen in the KirBac1.1 structure (9Kuo A. Gulbis J.M. Antcliff J.F. Rahman T. Lowe E.D. Zimmer J. Cuthbertson J. Ashcroft F.M. Ezaki T. Doyle D.A. Sciences (N. Y.). 2003; 300: 1922-1926Crossref PubMed Scopus (738) Google Scholar). Two recent RyR1 structures (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar) were determined under conditions favoring the closed channel, the arrangement of the membrane-spanning helix that, in these structures, is quite different. Ludtke et al. (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) found a bent pore-lining helix (Fig. 2A) and suggested that the structure is similar to the open MthK channel (8Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Crossref PubMed Scopus (1209) Google Scholar). This raises questions as to whether "kinking" of the inner helix alone, as proposed for K+ channels, is adequate to open RyRs. However, the conformation of the pore-lining helix in the other high resolution RyR1 cryo-EM structure (3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar) is straight and resembles the closed KcsA channel structure (7Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Sciences (N. Y.). 1998; 280: 69-77Crossref PubMed Scopus (5768) Google Scholar). These differences may due to resolution or to the conformational state of the channel during the imaging. At the current level of resolution in the three-dimensional structure of RyR, it is not possible to elucidate the number of TM helices. Single-particle cryo-EM has been used to generate structures of RyR1 in different conformational states to explore the structural transitions associated with the RyR gating. RyR1 was frozen under conditions determined from electrophysiological and biochemical studies (10Orlova E.V. Serysheva I.I. van Heel M. Hamilton S.L. Chiu W. Nat. Struct. Biol. 1996; 3: 547-552Crossref PubMed Scopus (146) Google Scholar, 11Serysheva I.I. Schatz M. van Heel M. Chiu W. Hamilton S.L. Biophys. J. 1999; 77: 1936-1944Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Sharma M.R. Jeyakumar L.H. Fleischer S. Wagenknecht T. J. Biol. Chem. 2000; 275: 9485-9491Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) to drive the channel into either an opened or a closed state. Although the resolution of these structures was only 25–30 Å, global conformational changes associated with the closed-open transition of the RyR channel were detected in both the CY region (opening of the clamps) and the TM region (mass depletion from the center and a twisting motion of the TM region). Channel opening was proposed to be similar to the opening-closing of the iris in a camera diaphragm. Four approaches are used to identify the locations of specific sequences in the three-dimensional structures: 1) difference mapping with and without bound modulators; 2) difference mapping with and without sequence-specific antibodies; 3) insertion of tags such as green fluorescent protein (GFP) into defined positions in the primary sequence, followed by difference mapping; and 4) docking of molecular models of domains derived from crystal structures into the three-dimensional maps. There are three known RyR isoforms encoded by three distinct genes: RyR1 (expressed primarily in skeletal muscle), RyR2 (expressed in cardiac muscle and brain), and RyR3 (expressed in diaphragm, smooth muscle, and brain). These isoforms share ∼65% sequence identity. Regions of divergence (D1, aa 4254–4631 in RyR1 and aa 4210–4562 in RyR2; D2, aa 1342–1403 in RyR1 and aa 1353–1397 in RyR2; and D3, aa 1872–1923 in RyR1 and aa 1852–1890 in RyR2) are indicated in Fig. 1A (line 2). These divergent regions could be responsible for important isoform-specific functions, or they could represent regions that are not evolutionarily conserved. Three-dimensional structures of all three RyR isoforms determined at ∼30 Å using single-particle cryo-EM are virtually identical (10Orlova E.V. Serysheva I.I. van Heel M. Hamilton S.L. Chiu W. Nat. Struct. Biol. 1996; 3: 547-552Crossref PubMed Scopus (146) Google Scholar, 11Serysheva I.I. Schatz M. van Heel M. Chiu W. Hamilton S.L. Biophys. J. 1999; 77: 1936-1944Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Sharma M.R. Jeyakumar L.H. Fleischer S. Wagenknecht T. J. Biol. Chem. 2000; 275: 9485-9491Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Using GFP insertions, D1 was mapped to subregion 3, whereas both D2 and D3 were mapped to the clamp in subregions 6 and 9, respectively (Fig. 1B). The three divergent regions all appear to be located in the CY domains close to the top of the structure, as would be expected for regions of RyR1 involved in interactions with CaV1.1. However, whether they are involved remains to be determined. Analysis of the primary sequence of RyR1 has revealed several motifs found in other proteins, but the functional roles of these motifs in RyRs are unknown. These motifs include 1) three SPRY domains (aa 582–798, 1014–1209, and 1358–1571) (Fig. 1A, line 3), which are domains found in members of the immunoglobulin superfamily; 2) a sequence that is repeated four times in two tandem domains (aa 842–955, 956–1069, 2726–2845, and 2846–2959) (Fig. 1A, line 4) with two additional truncated repeats located between the two domains (aa 1345–1360 and 1373–1388); 3) five MIR domains (aa 98–153, 160–205, 211–265, 271–334, and 336–394) (Fig. 1A, line 5), which are sequences found in RyRs, inositol trisphosphate receptors, and protein O-mannosyltransferases; and 4) two or more leucine zippers (aa 554–575 and 3039–3071 in RyR1 and aa 565–594, 1604–1632, and 3005–3036 in RyR2) (Fig. 1A, line 6), which are protein-protein interaction motifs and have been suggested to anchor PP1/spinophilin, PP2A/PR130, and protein kinase A/RII (protein kinase A regulatory subunit II)/mAKAP (muscle A-kinase anchoring protein) to RyR2 (13Marks A.R. Marx S.O. Reiken S. Trends Cardiovasc. Med. 2002; 12: 166-170Crossref PubMed Scopus (79) Google Scholar). The MIR domains are all located close to the N terminus, which is part of the clamp domain, suggesting that these domains may contribute to the structure/function of the clamps (4Serysheva I.I. Ludtke S.J. Baker M.L. Cong Y. Topf M. Eramian D. Sali A. Hamilton S.L. Chiu W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9610-9615Crossref PubMed Scopus (95) Google Scholar). Computational placement of a homology model based on the crystal structure of the inositol trisphosphate (IP3)-binding and suppressor domains of the IP3 receptor (4Serysheva I.I. Ludtke S.J. Baker M.L. Cong Y. Topf M. Eramian D. Sali A. Hamilton S.L. Chiu W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9610-9615Crossref PubMed Scopus (95) Google Scholar) places the first leucine zipper (aa 554–574) in the clamp domain in subregion 7 (Figs. 1B and 2B). If this is indeed the site that anchors kinases or phosphatases to RyR1, the targets of the enzymes may be sequences in close proximity to this site. Stewart et al. (15Stewart R. Zissimopoulos S. Lai F.A. Biochem. J. 2003; 376: 795-799Crossref PubMed Scopus (36) Google Scholar) proposed that the last 15 amino acids in the C-terminal tail are required for tetramer formation. Galvan and Mignery (16Galvan D.L. Mignery G.A. J. Biol. Chem. 2002; 277: 48248-48260Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) also suggested a C-terminal tetramerization domain but suggested that this was between aa 4869 and 5019 in RyR1. These regions are indicated in Fig. 1A (line 7). Because the C termini of RyRs are cytoplasmic, the region of tetramerization must also be in CY domains. The activities of all three RyR isoforms are modulated by a large number of agents, including ATP, Ca2+, Mg2+, calmodulin, FKBP12, junctin, triadin, calsequestrin, S100A, sorcin, and many others. Most of these modulators bind to the CY domains of the RyRs and allosterically regulate the opening of the Ca2+ conduction pathway in the TM region. RyR activities are also modulated by post-translation modifications such as phosphorylation, oxidation, and S-nitrosylation. Prerequisites for the delineation of the mechanisms of channel regulation by individual modulators and modifications are the identity of the sites of modulator interaction or modification in the primary sequence and the location of these sites in the three-dimensional structure. Single-particle cryo-EM has been used to analyze macromolecular interactions of RyR with the modulatory proteins calmodulin and FKBP12 (for original references on these studies, see reviews in Refs. 1Serysheva I.I. Chiu W. Ludtke S.J. Methods Cell Biol. 2007; 79: 407-435Crossref PubMed Scopus (8) Google Scholar and 17Durham W.J. Wehrens X.H.T. Sood S. Hamilton S.L. Carafoli E. Brini M. Calcium Signaling and Disease. Springer-Verlag New York Inc., New York2007: 273-321Google Scholar). Because most channel modulators bind in the CY assembly, there must be mechanisms whereby changes in the conformation of specific domains in the CY assembly are transduced, first into changes in the column-like structure and then into changes in the TM helices of the pore. CaV1.1—In skeletal muscle, the interaction between CaV1.1 in the transverse tubules and RyR1 in the sarcoplasmic reticulum has been shown to involve an orthograde signal, whereby CaV1.1 signals RyR1 to open, and a retrograde signal, through which RyR1 increases the Ca2+ current through CaV1.1 (18Dirksen R.T. Front. Biosci. 2002; 7: d659-d670Crossref PubMed Google Scholar). Multiple regions of RyR1 are likely to be involved in the coupling to CaV1.1 (Fig. 1A, line 8) (reviewed in Ref. 18Dirksen R.T. Front. Biosci. 2002; 7: d659-d670Crossref PubMed Google Scholar), including aa 1635–2636, 2659–3720, and 1837–2154 and multiple regions between aa 1 and 1680 (19Sheridan D.C. Takekura H. Franzini-Armstrong C. Beam K.G. Allen P.D. Perez C.F. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 19760-19765Crossref PubMed Scopus (62) Google Scholar). The sites of interaction appear to be distributed over a rather large part of the CY domain of RyR1. Based on freeze-fracture studies (20Paolini C. Fessenden J.D. Pessah I.N. Franzini-Armstrong C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12748-12752Crossref PubMed Scopus (63) Google Scholar), the most likely regions of RyR1 to be in contact with CaV1.1 include subregions 4 and 6 (Fig. 1B). Several of the sequences suggested to contribute to mechanical coupling have been found within these two subdomains. Ser437 of RyR2 (Ser422 in RyR1) maps to a location between subregions 5 and 9. Ser2368 in RyR2 (Arg2400 in RyR1) maps to a location between subregions 5 and 6. An interaction of CaV1.1 with these regions of RyR1 could directly control the opening of the clamp and allosterically modulate the channel gating. Ca2+—Regulation of RyRs by Ca2+ varies with the RyR isoform. The most important modulator of RyR2 activity in cardiac muscle is Ca2+, where Ca2+ enters through CaV1.2 and activates RyR2. Ca2+ also regulates RyR1 activity, but depending on the concentration, it can either activate or inhibit its activity. These findings suggest that RyR1 has at least two Ca2+-binding sites. Both RyR1 and RyR2 are activated at 0.1–10 μm Ca2+ (21Chen S.R. MacLennan D.H. J. Biol. Chem. 1994; 269: 22698-22704Abstract Full Text PDF PubMed Google Scholar), but RyR1 is more sensitive to inhibition by higher concentrations of Ca2+. Lumenal Ca2+ also regulates the activity of RyRs (22Jiang D. Xiao B. Yang D. Wang R. Choi P. Zhang L. Cheng H. Chen S.R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13062-13067Crossref PubMed Scopus (361) Google Scholar). Possible binding sites for Ca2+ (Fig. 1A, line 9) are located between aa 1861 and 2094 (21Chen S.R. MacLennan D.H. J. Biol. Chem. 1994; 269: 22698-22704Abstract Full Text PDF PubMed Google Scholar), 3657 and 3776 (21Chen S.R. MacLennan D.H. J. Biol. Chem. 1994; 269: 22698-22704Abstract Full Text PDF PubMed Google Scholar), and 4381 and 4626 (23Treves S. Chiozzi P. Zorzato F. Biochem. J. 1993; 291: 757-763Crossref PubMed Scopus (26) Google Scholar) and two putative EF-hand motifs located at aa 4079–4092 (EF1) and 4115–4126 (EF2) (24Xiong H. Feng X. Gao L. Xu L. Pasek D.A. Seok J.H. Meissner G. Biochemistry. 1998; 37: 4804-4814Crossref PubMed Scopus (54) Google Scholar). Mutation E3986A (Glu4042 in rabbit RyR1) in mouse RyR2 reduced Ca2+ activation by >1000-fold (25Li P. Chen S.R. J. Gen. Physiol. 2001; 118: 33-44Crossref PubMed Scopus (113) Google Scholar). Fessenden et al. (26Fessenden J.D. Feng W. Pessah I.N. Allen P.D. J. Biol. Chem. 2004; 279: 53028-53035Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) scrambled the putative EF-hand motifs and found that these mutations did not alter the response to either depolarization or RyR agonists (caffeine, 4-chloro-m-cresol) in intact myotubes. Surprisingly, no high affinity [3H]ryanodine binding was observed in membranes expressing the EF2 mutation, but the channels in bilayers were activated by Ca2+ in the micromolar range. These findings suggest that the putative EF-hands contribute to Ca2+ modulation of the channel but are not sufficient for Ca2+ regulation of this close to these putative Ca2+-binding sites have not been mapped in the three-dimensional structures of RyRs. However, et al. L. Zhang R. Hamilton S.L. Biophys. J. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) suggested an interaction of the regions putative and EF2 with the calmodulin region (aa suggesting that these two EF-hands are close to 3, where the site is located also bind CaM, which is a Ca2+-binding protein. RyR1 is the protein of sarcoplasmic reticulum membranes A. Xu L. G. Meissner G. Biophys. J. Full Text PDF PubMed Scopus Google Scholar). At micromolar Ca2+ both RyR1 and RyR2. However, at activates RyR1 but RyR2 D. and A. in Scholar). RyR3 also CaM, and the of on the channel are Xu L. Pasek D.A. Chen S.R. Meissner G. Biochemistry. 2005; PubMed Scopus Google Scholar). The site on RyR1 aa (Fig. 1A, line G. Zhang L. G. Hamilton S.L. Biochemistry. 1999; PubMed Scopus Google Scholar, Xu L. Pasek D.A. Meissner G. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) and at an close to aa H. Zhang Hamilton S.L. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). difference the site was found to be located in CY subregion 3 (Fig. 1B). the location of the site is to Å in the of Ca2+ with respect to its for M. Wagenknecht T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). This of could be due to a of binding Ca2+ a of the site RyR1 Ca2+. These findings suggest that subregion 3 a regulatory for channel activation and but additional studies are required to elucidate the molecular of regulation of RyR1. FKBP12 and bind to RyRs and regulate activities A.R. Physiol. 1996; PubMed Scopus Google Scholar). Although FKBP12 and are to bind to all RyR FKBP12 with RyR1, whereas with RyR2. The bound to RyR3 is not bind to RyR1 and RyR2 on site RyR1 subunit and a closed state of the channel A.R. Physiol. 1996; PubMed Scopus Google Scholar). The binding site has been suggested to be between aa and in RyR1 (Fig. 1A, line Mutation of this site FKBP12 binding M. J. Reiken S. F. Marx S.O. A.R. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), but this may be due to an regulation of the binding site S. Lai F.A. Cell Biochem. Biophys. 2005; PubMed Scopus Google Scholar). Using difference mapping of three-dimensional reconstructions of RyR1 with and without FKBP12, the binding site for FKBP12 is found at the between clamp subregions 5 and and subregion 3, neighboring clamps in the RyR CY region (Fig. 1B). of the location of FKBP12 by difference mapping M. X. Allen P.D. J. Mol. Biol. 2006; PubMed Scopus Google Scholar) with the docking of the IP3 homology model (4Serysheva I.I. Ludtke S.J. Baker M.L. Cong Y. Topf M. Eramian D. Sali A. Hamilton S.L. Chiu W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9610-9615Crossref PubMed Scopus (95) Google Scholar) that there is a binding for FKBP12 by and (Fig. 2B). or not FKBP12 at this remains to be protein has been shown to with RyR1 and to regulate its response to both and W. J. Yang T. Allen P.D. Pessah I.N. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (125) Google Scholar). A possible sequence is found between aa and has been mapped between domains 5 and 6 Wang R. Zhang J. Chen S.R. Wagenknecht T. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar), suggesting that may also via the clamp domain of RyR1. and S. A. H. A. C. F. M. J. Wang J. D. G. A.R. J. Cell Biol. 2003; PubMed Scopus Google Scholar) have suggested that RyR (aa in RyR1 and aa in RyR2) from RyRs, to Ca2+ A number of other have to these findings 2007; 101: PubMed Scopus Google Scholar, S. J. R. 2006; PubMed Scopus Google Scholar, G. H. Chen X. H. S.R. 2008; PubMed Scopus Google Scholar). site of is aa (aa in Possible sites are indicated in Fig. 1A (line of GFP at in RyR2 in RyR1) this site to domain to the site X. Xiao B. S. X. F. J. R. J. Chen S.R. Wagenknecht T. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). The putative site was mapped by GFP at and was found to be located in to the site X. Xiao B. S. J. Wagenknecht T. Chen S.R. Biochem. J. 2008; PubMed Scopus Google Scholar). are in the is the primary of (Fig. 1, line in J. C. Meissner G. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). et al. W.J. P. C. S. Galvan D.L. Baker M.R. F. R. Hamilton S.L. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar) that RyR1 the found with RyR1 mutations associated with have been by et al. P. R.T. C. Hamilton S.L. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) under conditions in which the channel was primarily in a open conformation (Fig. 1A, line and by et al. J. M. D. Pessah I.N. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) under conditions in which the channel was in a primarily closed Although the differences may be due to differences in it is also possible that different are the channel is in the open closed are the in the three-dimensional Several can be in the docking model (Fig. is in the site and is located in are located in 5 and 6 (Fig. 1B) the region from aa to to these Wang R. Zhang J. Chen S.R. Wagenknecht T. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). in RyR1 central and of recent review by et al. W.J. Wehrens X.H.T. Sood S. Hamilton S.L. Carafoli E. Brini M. Calcium Signaling and Disease. Springer-Verlag New York Inc., New York2007: 273-321Google Although there is for the mutations to in three regions of RyR1, it that mutations can the primary sequence (Fig. 1A, line that the channel to be more sensitive to whereas central mutations either the channel or through the for the location of of the N-terminal mutations can be from the docking model (Fig. 2B). Channel opening is associated with structural transitions in the CY clamp subregions and (Fig. 2A) (10Orlova E.V. Serysheva I.I. van Heel M. Hamilton S.L. Chiu W. Nat. Struct. Biol. 1996; 3: 547-552Crossref PubMed Scopus (146) Google Scholar, 11Serysheva I.I. Schatz M. van Heel M. Chiu W. Hamilton S.L. Biophys. J. 1999; 77: 1936-1944Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). the three-dimensional locations of the and sites and other important functional the clamp subregions are likely to be involved in modulation of the channel activity. These findings suggest a between the conformation of the clamp and channel with many mutations associated with and central in mutations that the channel to open more are located in clamp subregions and (Fig. 2) (4Serysheva I.I. Ludtke S.J. Baker M.L. Cong Y. Topf M. Eramian D. Sali A. Hamilton S.L. Chiu W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9610-9615Crossref PubMed Scopus (95) Google Scholar). Although RyR1 was the first structures by single-particle cryo-EM at resolution M. R. J. Fleischer S. Wagenknecht T. J. Cell Biol. 1994; PubMed Scopus Google Scholar, I.I. E.V. Chiu W. Hamilton S.L. van Heel M. Nat. Struct. Biol. PubMed Scopus Google Scholar), the RyR assembly remains the most for structural due to its RyR1 reconstructions at ∼10 Å by two have important into its pore structure, and control by multiple intracellular (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar). Several higher resolution structures of RyR1, including from cryo-specimen of membrane proteins, of the protein from its with and of different conformational are to the channel in a functional state and either to all modulators or to all binding sites with modulators. New are to the number of has to be and the of are that of these to be are 1) crystal structures of individual or into the cryo-EM and 2) and the models into the cryo-EM the in the resolution of RyR structure are likely to in the with L. Serysheva

Structure determination of clathrin coats to subnanometer resolution by single particle cryo-electron microscopy

Cryo-sample preparation is a vital step in the process of obtaining high-resolution structures of macromolecules by using the single-particle cryo–electron microscopy (cryo-EM) method; however, cryo-sample preparation is commonly hampered by high uncertainty and low reproducibility. Specifically, the existence of air-water interfaces during the sample vitrification process could cause protein denaturation and aggregation, complex disassembly, adoption of preferred orientations, and other serious problems affecting the protein particles, thereby making it challenging to pursue high-resolution 3D reconstruction. Therefore, sample preparation has emerged as a critical research topic, and several new methods for application at various preparation stages have been proposed to overcome the aforementioned hurdles. Here, we summarize the methods developed for enhancing the quality of cryo-samples at distinct stages of sample preparation, and we offer insights for developing future strategies based on diverse viewpoints. We anticipate that cryo-sample preparation will no longer be a limiting step in the single-particle cryo-EM field as increasing numbers of methods are developed in the near future, which will ultimately benefit the entire research community.

<b>Abstract ID 24997</b> <b>Poster Board 83</b> Phospholipase C β (PLCβ) plays an important role in cardiovascular diseases and opioid analgesia. PLCβ catalyzes the hydrolysis of the inner membrane lipid into two crucial secondary messengers, phosphatidylinositol-4,5-bisphosphate (PIP₂) to inositol-1,4,5-triphosphate (IP₃) and diacylglycerol (DAG), which activate multiple signaling pathways to modulate cellular behavior. PLCβ is activated downstream of G-protein coupled receptors (GPCRs) by the heterotrimeric G protein subunits Gα_q and Gβγ. In small-angle X-ray scattering (SAXS) experiments, the solution structure of PLCβ had additional density unaccounted for in crystal structures, suggesting that its structure was more dynamic than previously thought. To test this, we used cryo-electron microscopy (cryo-EM) single particle analysis to determine the solution structure of full-length PLCβ3 to 4.1 Å resolution. PLCβ3 adopted a compact, autoinhibited conformation under these conditions. This structure confirmed that the proximal C-terminal domain interacts with the core of PLCβ, but the autoinhibitory X–Y linker does not, suggesting more complex regulation of the lipase by the membrane. We are currently working to determine the structure of PLCβ3 on a model membrane to reveal the basally active state of the lipase.

Electron cryomicroscopy (cryo-EM) is a powerful tool in structural biology for determining 3D structures of macromolecular complexes. In the previous decade, many technological advancements have been introduced to improve the methodology of single particle cryo-EM, and hence the resolution of its protein reconstructions. These advancements include developments in both software algorithms and microscope hardware. Despite all these developments, the resolution of single particle cryo-EM cannot surpass 1.5 Å, a barrier blocking direct visualization of atomic details of the protein structure. Here, we show that by improving the optical performance of the electron microscope with new hardware, such resolution barrier can be removed, and atomic resolution of 1.25 Å can be reached with an engineered protein apoferritin. The atomic resolution protein structure helped set benchmarks for validating data quality in high resolution cryo-EM. We found that the new microscope also benefited to other proteins with lower contrast and higher flexibility, such as proteasome, chaperonin and fatty acid synthetase. For these proteins, sample properties and behavior were the major limitations. We anticipate that this would be alleviated by introducing other new microscope hardware, so that in the future, time and effort of structural biologists can concentrate on biochemical preparation of the sample, and single particle cryo-EM can routinely yield high quality protein structures at atomic resolution.

Ligand-Induced Conformational Changes in Tetrameric IP3R1 Revealed by Single-Particle Cryo-EM

High-throughput cryo-electron tomography enables multiscale visualization of the inner life of microbes.

Bacteria downregulate their ribosomal activity through dimerization of 70S ribosomes, yielding inactive 100S complexes. In Escherichia coli, dimerization is mediated by the hibernation promotion factor (HPF) and ribosome modulation factor. Here we report the cryo-electron microscopy study on 100S ribosomes from Lactococcus lactis and a dimerization mechanism involving a single protein: HPFlong. The N-terminal domain of HPFlong binds at the same site as HPF in Escherichia coli 100S ribosomes. Contrary to ribosome modulation factor, the C-terminal domain of HPFlong binds exactly at the dimer interface. Furthermore, ribosomes from Lactococcus lactis do not undergo conformational changes in the 30S head domains upon binding of HPFlong, and the Shine–Dalgarno sequence and mRNA entrance tunnel remain accessible. Ribosome activity is blocked by HPFlong due to the inhibition of mRNA recognition by the platform binding center. Phylogenetic analysis of HPF proteins suggests that HPFlong-mediated dimerization is a widespread mechanism of ribosome hibernation in bacteria.

Single particle cryo-electron microscopy (cryo-EM) has matured into a robust method for the determination of biological macromolecule structures in the past decade, complementing X-ray crystallography and nuclear magnetic resonance. Constant methodological improvements in both cryo-EM hardware and image processing software continue to contribute to an exponential growth in the number of structures solved annually. In this review, we provide a historical view of the many steps that were required to make cryo-EM a successful method for the determination of high-resolution protein complex structures. We further discuss aspects of cryo-EM methodology that are the greatest pitfalls challenging successful structure determination to date. Lastly, we highlight and propose potential future developments that would improve the method even further in the near future.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

BackgroundProteins in their majority act rarely as single entities. Multisubunit macromolecular complexes are the actors in most of the cellular processes. These nanomachines are hold together by weak protein-protein interactions and undergo functionally important conformational changes. TFIID is such a multiprotein complex acting in eukaryotic transcription initiation. This complex is first to be recruited to the promoter of the genes and triggers the formation of the transcription preinitiation complex involving RNA polymerase II which leads to gene transcription. The exact role of TFIID in this process is not yet understood.MethodsLast generation electron microscopes, improved data collection and new image analysis tools made it possible to obtain structural information of biological molecules at atomic resolution. Cryo-electron microscopy of vitrified samples visualizes proteins in a fully hydrated, close to native state. Molecular images are recorded at liquid nitrogen temperature in low electron dose conditions to reduce radiation damage. Digital image analysis of these noisy images aims at improving the signal-to-noise ratio, at separating distinct molecular views and at reconstructing a three-dimensional model of the biological particle.ResultsUsing these methods we showed the early events of an activated transcription initiation process. We explored the interaction of the TFIID coactivator with the yeast Rap1 activator, the transcription factor TFIIA and the promoter DNA. We demonstrated that TFIID serves as an assembly platform for transient protein-protein interactions, which are essential for transcription initiation.ConclusionsRecent developments in electron microscopy have provided new insights into the structural organization and the dynamic reorganization of large macromolecular complexes. Examples of near-atomic resolutions exist but the molecular flexibility of macromolecular complexes remains the limiting factor in most case. Electron microscopy has the potential to provide both structural and dynamic information of biological assemblies in order to understand the molecular mechanisms of their functions.

Biochemical and structural characterization of the capsid-bound tegument proteins of human cytomegalovirus