Recent Technical Advances in Sample Preparation for Single-Particle Cryo-EM.

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

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.

OVERVIEW OF STANDARDS FOR COAL AND COKE SAMPLING © N.M. Нolik (State Enterprise “Ukrainian State Research Institute for Carbochemistry (UKHIN)”, 7 Vesnina str., Kharkiv, 61023, Ukraine), D.V. Miroshnichenko, Doctor of Technical Sciences (National Technical University "Kharkiv Polytechnic Institute", 2, Kyrpychova str., Kharkiv, 61002, Ukraine (NTU “KhPI”) The article discusses the main stages of testing solid fuels, in particular hard coal and coke, including sample collection, preparation and examination. The importance of obtaining a representative sample that corresponds to the average composition of the entire fuel and is sufficient in weight for accurate measurements is emphasised. It is determined that the heterogeneity of the composition of solid fuels complicates the process of taking a representative sample, which is critical for the accuracy of research. The main stages of obtaining an analytical sample used to determine the chemical and physical characteristics of fuel are described. Special attention is paid to the importance of compliance with the requirements of standards during sample collection and preparation. An overview of the main national and international regulations on sampling and preparation of solid fuels is provided, as well as the scope and key provisions of each standard. The aspects of the importance of the stage of sampling and preparation of solid fuel samples that affect the accuracy of the results of studies of its properties are considered. The main types of samples are listed; requirements for samples, covering the mass and number of spot samples; sampling methods; general requirements for the design of mechanical samplers and sample preparation equipment. The author analyses a number of current Ukrainian regulatory documents and points out the need for their harmonisation with international standards to improve the accuracy of the sampling process. The aspects of the importance of the stage of sampling and preparation of solid fuel samples that affect the accuracy of the results of studies of its properties are considered. The article considers the requirements for determining the systematic error of testing hard coal, anthracite or coke, the sequence of testing and calculation and the criteria for evaluating the test results are presented. The necessity of checking mechanical samplers to determine the presence or absence of a systematic sampling error is noted. The importance of participating in international standardisation is highlighted, which allows for continuous improvement of sampling and sample preparation methods. Keywords: sampling, sample preparation, solid fuel, coal, coal coke, point sample, mechanical sampler, systematic error. Corresponding author N.M. Golik, e-mail: aisim@ukhin.org.ua

Development of imaging scaffolds for cryo-electron microscopy

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

The Thermoplasma VCP-like ATPase from Thermoplasma acidophilum (VAT) ATPase is a member of the two-domain AAA ATPases and homologous to the mammalian p97/VCP and NSF proteins. We show here that the VAT ATPase complex unfolds green fluorescent protein (GFP) labeled with the ssrA-degradation tag. Increasing the Mg2+ concentration derepresses the ATPase activity and concomitantly stimulates the unfolding activity of VAT. Similarly, the VATDeltaN complex, a mutant of VAT deleted for the N domain, displays up to 24-fold enhanced ATP hydrolysis and 250-fold enhanced GFP unfolding activity when compared with wild-type VAT. To determine the individual contribution of the two AAA domains to ATP hydrolysis and GFP unfolding we performed extensive site-directed mutagenesis of the Walker A, Walker B, sensor-1, and pore residues in both AAA domains. Analysis of the VAT mutant proteins, where ATP hydrolysis was confined to a single AAA domain, revealed that the first domain (D1) is sufficient to exert GFP unfolding indistinguishable from wild-type VAT, while the second AAA domain (D2), although active, is significantly less efficient than wild-type VAT. A single conserved aromatic residue in the D1 section of the pore was found to be essential for GFP unfolding. In contrast, two neighboring residues in the D2 section of the pore had to be exchanged simultaneously, to achieve a drastic inhibition of GFP unfolding.

In this paper the advantages of application of the nanocomposite track membranes (NCTM) with TiN coating as the substrates for sample preparation for their investigation by methods of electron microscopy are discussed. This membrane material was obtained by the ion-plasma sputtering of TiN coating on the polymer track-etched membrane. The physicochemical properties of NCTM were researched. Preparation technique allows to separate nanoand microdimensional samples from dry, liquid and gas mixtures for their investigation by methods of scanning and transmission electron microscopy.

Single-particle electron cryomicroscopy is an essential tool for high-resolution 3D reconstruction of proteins and other biological macromolecules. An important challenge in cryo-EM is the reconstruction of non-rigid molecules with parts that move and deform. Traditional reconstruction methods fail in these cases, resulting in smeared reconstructions of the moving parts. This poses a major obstacle for structural biologists, who need high-resolution reconstructions of entire macromolecules, moving parts included. To address this challenge, we present a new method for the reconstruction of macromolecules exhibiting continuous heterogeneity. The proposed method uses projection images from multiple viewing directions to construct a graph Laplacian through which the manifold of three-dimensional conformations is analyzed. The 3D molecular structures are then expanded in a basis of Laplacian eigenvectors, using a novel generalized tomographic reconstruction algorithm to compute the expansion coefficients. These coefficients, which we name spectral volumes, provide a high-resolution visualization of the molecular dynamics. We provide a theoretical analysis and evaluate the method empirically on several simulated data sets.

Single-particle cryo-electron microscopy (cryoEM) methods are powerful for solving high-resolution structures of biological macromolecules. Locating numerous particles from micrographs is essential for three-dimensional reconstruction but challenging due to the extremely low signal-to-noise ratio and various particle shapes in micrographs. In this study, we devise the TransPicker, a two-dimensional particle picking framework based on a novel end-to-end transformer-based detective method named crDETR (cryoEM DEtection TRansformer). crDETR applies an improved deformable Transformer to perform inference in parallel on particle relocation and global context, without hand-crafted components like anchors, non-maximum suppression procedure, or sliding windows. Also, it uses a combined loss function to guarantee fast convergence. It uses divide-and-conquer to overcome the limitations of the object query number. Moreover, we develop a series of optimizations, including denoising, enhancing, bad particle filtering, adding masks on carbon areas and ice contaminants to decrease the false-positive ratio and improve the accuracy of particle picking. Experimental results on various datasets demonstrate that TransPicker can select particles with more accuracy, especially in high noise compared with other methods. To our knowledge, TransPicker is the first application of the transformer technique in cryoEM particle picking.

The power spectrum of proteins at high frequencies is remarkably well described by the flat Wilson statistics. Wilson statistics therefore plays a significant role in X-ray crystallography and more recently in electron cryomicroscopy (cryo-EM). Specifically, modern computational methods for three-dimensional map sharpening and atomic modelling of macromolecules by single-particle cryo-EM are based on Wilson statistics. Here the first rigorous mathematical derivation of Wilson statistics is provided. The derivation pinpoints the regime of validity of Wilson statistics in terms of the size of the macromolecule. Moreover, the analysis naturally leads to generalizations of the statistics to covariance and higher-order spectra. These in turn provide a theoretical foundation for assumptions underlying the widespread Bayesian inference framework for three-dimensional refinement and for explaining the limitations of autocorrelation-based methods in cryo-EM.

<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.

Cryo-electron microscopy (cryo-EM) single particle method (SPM) reconstructs the three-dimensional (3D) density map of biological macromolecules using 2D particle images with estimated orientations. The estimated orientations have errors which result in the decrease in resolution of the reconstructed map. We propose a wavelet orthonormal bases based iteration method by refining alternatively the orientations and the map using Levenberg–Marquardt algorithm and soft-thresholding, respectively. The convergence analysis of the proposed algorithm is provided and numerical experiments for simulated particle images show its good performance.

Single particle cryo electron microscopy (cryo-EM) is now the major method for the determination of integral membrane protein structure. For the success of a given project the type of membrane mimetic used for extraction from the native cell membrane, purification to homogeneity and finally cryo-grid vitrification is crucial. Although small molecule amphiphiles - detergents - are the most widely used membrane mimetic, specific tailoring of detergent structure for single particle cryo-EM is rare and the demand for effective detergents not satisfied. Here, we compare the popular detergent lauryl maltose-neopentyl glycol (LMNG) with the novel detergent neopentyl glycol-derived triglucoside-C11 (NDT-C11) in its behavior as free detergent and when bound to two types of multisubunit membrane protein complexes - cyanobacterial photosystem I (PSI) and mammalian F-ATP synthase. We conclude that NDT-C11 has high potential to become a very useful detergent for single particle cryo-EM of integral membrane proteins.

Electron microscopy as a critical tool in the determination of pore forming mechanisms in proteins.

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