Biomedical subjects

Steven J Ludtke

Publications and source records attributed to Steven J Ludtke.

At least 19 recordsLinked to original sources

PubMed2006

Cryoelectron microscopy structures of rotavirus NSP2-NSP5 and NSP2-RNA complexes: implications for genome replication.

The replication and packaging of the rotavirus genome, comprising 11 segments of double-stranded RNA, take place in specialized compartments called viroplasms, which are formed during infection and involve a coordinated interplay of multiple components. Two rotavirus nonstructural proteins, NSP2 (with nucleoside triphosphatase, single-stranded RNA [ssRNA] binding and helix-destabilizing activities) and NSP5, are essential in these events. Previous structural analysis of NSP2 showed that it is an octamer in crystals, obeying 4-2-2 crystal symmetry, with a large 35-A central hole along the fourfold axis and deep grooves at one of the twofold axes. To ascertain that the solution structure of NSP2 is the same as that in the crystals and investigate how NSP2 interacts with NSP5 and RNA, we carried out single-particle cryoelectron microscopy (cryo-EM) analysis of NSP2 alone and in complexes with NSP5 and ssRNA at subnanometer resolution. Because full-length NSP5 caused severe aggregation upon mixing with NSP2, the deletion construct NSP566-188 was used in these studies. Our studies show that the solution structure of NSP2 is same as the crystallographic octamer and that both NSP566-188 and ssRNA bind to the grooves in the octamer, which are lined by positively charged residues. The fitting of the NSP2 crystal structure to cryo-EM reconstructions of the complexes indicates that, in contrast to the binding of NSP566-188, the binding of RNA induces noticeable conformational changes in the NSP2 octamer. Consistent with the observation that both NSP5 and RNA share the same binding site on the NSP2 octamer, filter binding assays showed that NSP5 competes with ssRNA binding, indicating that one of the functions of NSP5 is to regulate NSP2-RNA interactions during genome replication.

Binding Sites↗

PubMed2006

SPARX, a new environment for Cryo-EM image processing.

SPARX (single particle analysis for resolution extension) is a new image processing environment with a particular emphasis on transmission electron microscopy (TEM) structure determination. It includes a graphical user interface that provides a complete graphical programming environment with a novel data/process-flow infrastructure, an extensive library of Python scripts that perform specific TEM-related computational tasks, and a core library of fundamental C++ image processing functions. In addition, SPARX relies on the EMAN2 library and cctbx, the open-source computational crystallography library from PHENIX. The design of the system is such that future inclusion of other image processing libraries is a straightforward task. The SPARX infrastructure intelligently handles retention of intermediate values, even those inside programming structures such as loops and function calls. SPARX and all dependencies are free for academic use and available with complete source.

Computational Biology↗

PubMed2006

EMAN2: an extensible image processing suite for electron microscopy.

EMAN is a scientific image processing package with a particular focus on single particle reconstruction from transmission electron microscopy (TEM) images. It was first released in 1999, and new versions have been released typically 2-3 times each year since that time. EMAN2 has been under development for the last two years, with a completely refactored image processing library, and a wide range of features to make it much more flexible and extensible than EMAN1. The user-level programs are better documented, more straightforward to use, and written in the Python scripting language, so advanced users can modify the programs' behavior without any recompilation. A completely rewritten 3D transformation class simplifies translation between Euler angle standards and symmetry conventions. The core C++ library has over 500 functions for image processing and associated tasks, and it is modular with introspection capabilities, so programmers can add new algorithms with minimal effort and programs can incorporate new capabilities automatically. Finally, a flexible new parallelism system has been designed to address the shortcomings in the rigid system in EMAN1.

Algorithms↗

PubMed2006

Close membrane-membrane proximity induced by Ca(2+)-dependent multivalent binding of synaptotagmin-1 to phospholipids.

Synaptotagmin acts as a Ca(2+) sensor in neurotransmitter release through its two C(2) domains. Ca(2+)-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca(2+) binding to the C(2)B domain is more crucial for release than Ca(2+) binding to the C(2)A domain. Here we show that Ca(2+) induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C(2)B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C(2)B domain.

Animals↗

PubMed2006

An expanded conformation of single-ring GroEL-GroES complex encapsulates an 86 kDa substrate.

Electron cryomicroscopy reveals an unprecedented conformation of the single-ring mutant of GroEL (SR398) bound to GroES in the presence of Mg-ATP. This conformation exhibits a considerable expansion of the folding cavity, with approximately 80% more volume than the X-ray structure of the equivalent cis cavity in the GroEL-GroES-(ADP)(7) complex. This expanded conformation can encapsulate an 86 kDa heterodimeric (alphabeta) assembly intermediate of mitochondrial branched-chain alpha-ketoacid dehydrogenase, the largest substrate ever observed to be cis encapsulated. The SR398-GroES-Mg-ATP complex is found to exist as a mixture of standard and expanded conformations, regardless of the absence or presence of the substrate. However, the presence of even a small substrate causes a pronounced bias toward the expanded conformation. Encapsulation of the large assembly intermediate is supported by a series of electron cryomicroscopy studies as well as the protection of both alpha and beta subunits of the substrate from tryptic digestion.

Adenosine Triphosphate↗

PubMed2005

Structural analysis of the anaphase-promoting complex reveals multiple active sites and insights into polyubiquitylation.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase composed of approximately 13 distinct subunits required for progression through meiosis, mitosis, and the G1 phase of the cell cycle. Despite its central role in these processes, information concerning its composition and structure is limited. Here, we determined the structure of yeast APC/C by cryo-electron microscopy (cryo-EM). Docking of tetratricopeptide repeat (TPR)-containing subunits indicates that they likely form a scaffold-like outer shell, mediating assembly of the complex and providing potential binding sites for regulators and substrates. Quantitative determination of subunit stoichiometry indicates multiple copies of specific subunits, consistent with a total APC/C mass of approximately 1.7 MDa. Moreover, yeast APC/C forms both monomeric and dimeric species. Dimeric APC/C is a more active E3 ligase than the monomer, with greatly enhanced processivity. Our data suggest that multimerisation and/or the presence of multiple active sites facilitates the APC/C's ability to elongate polyubiquitin chains.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2005

Architecture of the ribosome-channel complex derived from native membranes.

The mammalian Sec61 complex forms a protein translocation channel whose function depends upon its interaction with the ribosome and with membrane proteins of the endoplasmic reticulum (ER). To study these interactions, we determined structures of "native" ribosome-channel complexes derived from ER membranes. We find that the ribosome is linked to the channel by seven connections, but the junction may still provide a path for domains of nascent membrane proteins to move into the cytoplasm. In addition, the native channel is significantly larger than a channel formed by the Sec61 complex, due to the presence of a second membrane protein. We identified this component as TRAP, the translocon-associated protein complex. TRAP interacts with Sec61 through its transmembrane domain and has a prominent lumenal domain. The presence of TRAP in the native channel indicates that it may play a general role in translocation. Crystal structures of two Sec61 homologues were used to model the channel. This analysis indicates that there are four Sec61 complexes and two TRAP molecules in each native channel. Thus, we suggest that a single Sec61 complex may form a conduit for translocating polypeptides, while three copies of Sec61 play a structural role or recruit accessory factors such as TRAP.

Animals↗

PubMed2005

Structure of Ca2+ release channel at 14 A resolution.

The 14 A resolution structure of the 2.3 MDa Ca2+ release channel (also known as RyR1) was determined by electron cryomicroscopy and single particle reconstruction. This structure was produced using collected data used for our previous published structures at 22-30 A resolution, but now taking advantage of recent algorithmic improvements in the EMAN software suite. This improved map clearly exhibits more structural detail and allows better defined docking of computationally predicted structural domain folds. Using sequence-based fold recognition, the N-terminal region of RyR1, residues 216-572, was predicted to have significant structural similarity with the IP3-binding core region of the type 1 IP3R. This putative structure was computationally localized to the clamp-shaped region of RyR1, which has been implicated to have a regulatory role in the channel activity.

Algorithms↗

PubMed2005

Electron cryomicroscopy of single particles at subnanometer resolution.

Electron cryomicroscopy and single-particle reconstruction have advanced substantially over the past two decades. There are now numerous examples of structures that have been solved using this technique to better than 10 A resolution. At such resolutions, direct identification of alpha helices is possible and, often, beta-sheet-containing regions can be identified. The most numerous subnanometer resolution structures are the icosahedral viruses, as higher resolution is easier to achieve with higher symmetry. Important non-icosahedral structures solved to subnanometer resolution include several ribosome structures, clathrin assemblies and, most recently, the Ca2+ release channel. There is now hope that, in the next few years, this technique will achieve resolutions approaching 4 A, permitting a complete trace of the protein backbone without reference to a crystal structure.

Animals↗

PubMed2005

The pore structure of the closed RyR1 channel.

Using single particle electron cryomicroscopy, several helices in the membrane-spanning region of RyR1, including an inner transmembrane helix, a short pore helix, and a helix parallel to the membrane on the cytoplasmic side, have been clearly resolved. Our model places a highly conserved glycine (G4934) at the hinge position of the bent inner helix and two rings of negative charges at the luminal and cytoplasmic mouths of the pore. The kinked inner helix closely resembles the inner helix of the open MthK channel, suggesting that kinking alone does not open RyR1, as proposed for K+ channels.

Amino Acid Sequence↗

PubMed2005

A structure of the human apoptosome at 12.8 A resolution provides insights into this cell death platform.

Apaf-1 and cytochrome c coassemble in the presence of dATP to form the apoptosome. We have determined a structure of the apoptosome at 12.8 A resolution by using electron cryomicroscopy and single-particle methods. We then docked appropriate crystal structures into the map to create an accurate domain model. Thus, we found that seven caspase recruitment domains (CARDs) form a central ring within the apoptosome. At a larger radius, seven copies of the nucleotide binding and oligomerization domain (NOD) associate laterally to form the hub, which encircles the CARD ring. Finally, an arm-like helical domain (HD2) links each NOD to a pair of beta propellers, which bind a single cytochrome c. This model provides insights into the roles of dATP and cytochrome c in assembly. Our structure also reveals how a CARD ring and the central hub combine to create a platform for procaspase-9 activation.

Apoptosis↗

PubMed2004

Structure of the bifunctional and Golgi-associated formiminotransferase cyclodeaminase octamer.

Mammalian formiminotransferase cyclodeaminase (FTCD), a 0.5 million Dalton homo-octameric enzyme, plays important roles in coupling histidine catabolism with folate metabolism and integrating the Golgi complex with the vimentin intermediate filament cytoskeleton. It is also linked to two human diseases, autoimmune hepatitis and glutamate formiminotransferase deficiency. Determination of the FTCD structure by X-ray crystallography and electron cryomicroscopy revealed that the eight subunits, each composed of distinct FT and CD domains, are arranged like a square doughnut. A key finding indicates that coupling of three subunits governs the octamer-dependent sequential enzyme activities, including channeling of intermediate and conformational change. The structure further shed light on the molecular nature of two strong antigenic determinants of FTCD recognized by autoantibodies from patients with autoimmune hepatitis and on the binding of thin vimentin filaments to the FTCD octamer.

Amino Acid Sequence↗

PubMed2004

Mitochondrial ATP synthasome: three-dimensional structure by electron microscopy of the ATP synthase in complex formation with carriers for Pi and ADP/ATP.

The terminal steps involved in making ATP in mitochondria require an ATP synthase (F(0)F(1)) comprised of two motors, a phosphate carrier (PIC), and an adenine nucleotide carrier (ANC). Under mild conditions, these entities sub-fractionate as an ATP synthase/PIC/ANC complex or "ATP synthasome" (Ko, Y.H., Delannoy, M, Hullihen, J., Chiu, W., and Pedersen, P.L. (2003) J. Biol. Chem. 278, 12305-12309). As a first step toward obtaining three-dimensional information about this large complex or "metabolon" and the locations of PIC and ANC therein, we dispersed ATP synthasomes into single complexes and visualized negatively stained images by electron microscopy (EM) that showed clearly the classical headpiece, central stalk, and basepiece. Parallel immuno-EM studies revealed the presence of PIC and ANC located non-centrally in the basepiece, and other studies implicated an ATP synthase/PIC/ANC stoichiometry near 1:1:1. Single ATP synthasome images (7506) were boxed, and, using EMAN software, a three-dimensional model was obtained at a resolution of 23 A. Significantly, the basepiece is oblong and contains two domains, the larger of which connects to the central stalk, whereas the smaller appears as an extension. Docking studies with known structures together with the immuno-EM studies suggest that PIC or ANC may be located in the smaller domain, whereas the other transporter resides nearby in the larger domain. Collectively, these finding support a mechanism in which the entry of the substrates ADP and P(i) into mitochondria, the synthesis of ATP on F(1), and the release and exit of ATP are very localized and highly coordinated events.

Animals↗

PubMed2004

A 9 angstroms single particle reconstruction from CCD captured images on a 200 kV electron cryomicroscope.

Sub-nanometer resolution structure determination is becoming a common practice in electron cryomicroscopy of macromolecular assemblies. The data for these studies have until now been collected on photographic film. Using cytoplasmic polyhedrosis virus (CPV), a previously determined structure, as a test specimen, we show the feasibility of obtaining a 9 angstroms structure from images acquired from a 4 k x 4 k Gatan CCD on a 200 kV electron cryomicroscope. The match of the alpha-helices in the protein components of the CPV with the previous structure of the same virus validates the suitability of this type of camera as the recording media targeted for single particle reconstructions at sub-nanometer resolution.

Cryoelectron Microscopy↗

PubMed2004

Experimental verification of conformational variation of human fatty acid synthase as predicted by normal mode analysis.

Fatty acid synthase (FAS) is a 550 kDa homodimeric enzyme with multiple functional and structural domains. Normal mode analysis of a previously determined 19 A structure of FAS suggested that this enzyme might assume different conformational states with several distinct hinge movements. We have used a simultaneous multiple-model refinement method to search for the presence of the structural conformers from the electron images of FAS. We have demonstrated that the resulting models observed in the electron images are consistent with the predicted conformational changes. This technique demonstrates the potential of the combination of normal mode analysis with multiple model refinement to elucidate the multiple conformations of flexible proteins. Since each of these structures is based on a more homogeneous particle set, this technique has the potential, provided that sufficient references are used, to improve the resolution of the final reconstructions of single particles from electron cryomicroscopy.

Cryoelectron Microscopy↗

PubMed2004

Seeing GroEL at 6 A resolution by single particle electron cryomicroscopy.

We present a reconstruction of native GroEL by electron cryomicroscopy (cryo-EM) and single particle analysis at 6 A resolution. alpha helices are clearly visible and beta sheet density is also visible at this resolution. While the overall conformation of this structure is quite consistent with the published X-ray data, a measurable shift in the positions of three alpha helices in the intermediate domain is observed, not consistent with any of the 7 monomeric structures in the Protein Data Bank model (1OEL). In addition, there is evidence for slight rearrangement or flexibility in parts of the apical domain. The 6 A resolution cryo-EM GroEL structure clearly demonstrates the veracity and expanding scope of cryo-EM and the single particle reconstruction technique for macromolecular machines.

Chaperonin 60↗

PubMed2003

Structure of the type 1 inositol 1,4,5-trisphosphate receptor revealed by electron cryomicroscopy.

The three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) has been determined by electron cryomicroscopy and single-particle reconstruction. The receptor was immunoaffinity-purified and formed functional InsP3- and heparin-sensitive channels with a unitary conductance similar to native InsP3Rs. The channel structure exhibits the expected 4-fold symmetry and comprises two morphologically distinct regions: a large pinwheel and a smaller square. The pinwheel region has four radial curved spokes interconnected by a central core. The InsP3-binding core domain has been localized within each spoke of the pinwheel region by fitting its x-ray structure into our reconstruction. A structural mapping of the amino acid sequences to several functional domains is deduced within the structure of the InsP3R1 tetramer.

Animals↗

PubMed2003

Object oriented database and electronic notebook for transmission electron microscopy.

As high-resolution biological transmission electron microscopy (TEM) has increased in popularity over recent years, the volume of data and number of projects underway has risen dramatically. A robust tool for effective data management is essential to efficiently process large data sets and extract maximum information from the available data. We present the Electron Microscopy Electronic Notebook (EMEN), a portable, object-oriented, web-based tool for TEM data archival and project management. EMEN has several unique features. First, the database is logically organized and annotated so multiple collaborators at different geographical locations can easily access and interpret the data without assistance. Second, the database was designed to provide flexibility to the user, so it can be used much as a lab notebook would be, while maintaining a structure suitable for data mining and direct interaction with data-processing software. Finally, as an object-oriented database, the database structure is dynamic and can be easily extended to incorporate information not defined in the original database specification.

Databases, Factual↗