Biomedical subjects

S J Ludtke

Publications and source records attributed to S J Ludtke.

14 recordsLinked to original sources

PubMed2002

Structure of the voltage-gated L-type Ca2+ channel by electron cryomicroscopy.

Voltage-dependent L-type Ca(2+) channels play important functional roles in many excitable cells. We present a three-dimensional structure of an L-type Ca(2+) channel. Electron cryomicroscopy in conjunction with single-particle processing was used to determine a 30-A resolution structure of the channel protein. The asymmetrical channel structure consists of two major regions: a heart-shaped region connected at its widest end with a handle-shaped region. A molecular model is proposed for the arrangement of this skeletal muscle L-type Ca(2+) channel structure with respect to the sarcoplasmic reticulum Ca(2+)-release channel, the physical partner of the L-type channel for signal transduction during the excitation-contraction coupling in muscle.

Animals↗

PubMed2001

A 11.5 A single particle reconstruction of GroEL using EMAN.

Single-particle analysis has become an increasingly important method for structural determination of large macromolecular assemblies. GroEL is an 800 kDa molecular chaperone, which, along with its co-chaperonin GroES, promotes protein folding both in vitro and in the bacterial cell. EMAN is a single-particle analysis software package, which was first publicly distributed in 2000. We present a three-dimensional reconstruction of native naked GroEL to approximately 11.5 A performed entirely with EMAN. We demonstrate that the single-particle reconstruction, X-ray scattering data and X-ray crystal structure all agree well at this resolution. These results validate the specific methods of image restoration, reconstruction and evaluation techniques implemented in EMAN. It also demonstrates that the single-particle reconstruction technique and X-ray crystallography will yield consistent structure factors, even at low resolution, when image restoration is performed correctly. A detailed comparison of the single-particle and X-ray structures exhibits some small variations in the equatorial domain of the molecule, likely due to the absence of crystal packing forces in the single-particle reconstruction.

Chaperonin 60↗

PubMed2001

Bridging the information gap: computational tools for intermediate resolution structure interpretation.

Due to large sizes and complex nature, few large macromolecular complexes have been solved to atomic resolution. This has lead to an under-representation of these structures, which are composed of novel and/or homologous folds, in the library of known structures and folds. While it is often difficult to achieve a high-resolution model for these structures, X-ray crystallography and electron cryomicroscopy are capable of determining structures of large assemblies at low to intermediate resolutions. To aid in the interpretation and analysis of such structures, we have developed two programs: helixhunter and foldhunter. Helixhunter is capable of reliably identifying helix position, orientation and length using a five-dimensional cross-correlation search of a three-dimensional density map followed by feature extraction. Helixhunter's results can in turn be used to probe a library of secondary structure elements derived from the structures in the Protein Data Bank (PDB). From this analysis, it is then possible to identify potential homologous folds or suggest novel folds based on the arrangement of alpha helix elements, resulting in a structure-based recognition of folds containing alpha helices. Foldhunter uses a six-dimensional cross-correlation search allowing a probe structure to be fitted within a region or component of a target structure. The structural fitting therefore provides a quantitative means to further examine the architecture and organization of large, complex assemblies. These two methods have been successfully tested with simulated structures modeled from the PDB at resolutions between 6 and 12 A. With the integration of helixhunter and foldhunter into sequence and structural informatics techniques, we have the potential to deduce or confirm known or novel folds in domains or components within large complexes.

Algorithms↗

PubMed2001

Fourier amplitude decay of electron cryomicroscopic images of single particles and effects on structure determination.

Several factors, including spatial and temporal coherence of the electron microscope, specimen movement, recording medium, and scanner optics, contribute to the decay of the measured Fourier amplitude in electron image intensities. We approximate the combination of these factors as a single Gaussian envelope function, the width of which is described by a single experimental B-factor. We present an improved method for estimating this B-factor from individual micrographs by combining the use of X-ray solution scattering and numerical fitting to the average power spectrum of particle images. A statistical estimation from over 200 micrographs of herpes simplex virus type-1 capsids was used to estimate the spread in the experimental B-factor of the data set. The B-factor is experimentally shown to be dependent on the objective lens defocus setting of the microscope. The average B-factor, the X-ray scattering intensity of the specimen, and the number of particles required to determine the structure at a lower resolution can be used to estimate the minimum fold increase in the number of particles that would be required to extend a single particle reconstruction to a specified higher resolution. We conclude that microscope and imaging improvements to reduce the experimental B-factor will be critical for obtaining an atomic resolution structure.

Capsid↗

PubMed1999

EMAN: semiautomated software for high-resolution single-particle reconstructions.

We present EMAN (Electron Micrograph ANalysis), a software package for performing semiautomated single-particle reconstructions from transmission electron micrographs. The goal of this project is to provide software capable of performing single-particle reconstructions beyond 10 A as such high-resolution data become available. A complete single-particle reconstruction algorithm is implemented. Options are available to generate an initial model for particles with no symmetry, a single axis of rotational symmetry, or icosahedral symmetry. Model refinement is an iterative process, which utilizes classification by model-based projection matching. CTF (contrast transfer function) parameters are determined using a new paradigm in which data from multiple micrographs are fit simultaneously. Amplitude and phase CTF correction is then performed automatically as part of the refinement loop. A graphical user interface is provided, so even those with little image processing experience will be able to begin performing reconstructions. Advanced users can directly use the lower level shell commands and even expand the package utilizing EMAN's extensive image-processing library. The package was written from scratch in C++ and is provided free of charge on our Web site. We present an overview of the package as well as several conformance tests with simulated data.

Algorithms↗

PubMed1997

Effect of changing the size of lipid headgroup on peptide insertion into membranes.

Adsorption of amphiphilic peptides to the headgroup region of a lipid bilayer is a common mode of protein-membrane interactions. Previous studies have shown that adsorption causes membrane thinning. The degree of the thinning depends on the degree of the lateral expansion caused by the peptide adsorption. If this simple molecular mechanism is correct, the degree of lateral expansion and consequently the membrane thinning should depend on the size of the headgroup relative to the cross section of the hydrocarbon chains. Previously we have established the connection between the alamethicin insertion transition and the membrane thinning effect. In this paper we use oriented circular dichroism to study the effect of varying the size of the headgroup, while maintaining a constant cross section of the lipid chains, on the insertion transition. A simple quantitative prediction agrees very well with the experiment.

Adsorption↗

PubMed1996

Membrane pores induced by magainin.

Magainin, found in the skin of Xenopus laevis, belongs to a broad class of antimicrobial peptides which kill bacteria by permeabilizing the cytoplasmic membrane but do not lyse eukaryotic cells. The 23-residue peptide has been shown to form an amphiphilic helix when associated with membranes. However, its molecular mechanism of action has been controversial. Oriented circular dichroism has detected helical magainin oriented perpendicular to the plane of the membrane at high peptide concentrations, but Raman, fluorescence, differential scanning calorimetry, and NMR all indicate that the peptide is associated with the head groups of the lipid bilayer. Here we show that neutron in-plane scattering detects pores formed by magainin 2 in membranes only when a substantial fraction of the peptide is oriented perpendicular to the membrane. The pores are almost twice as large as the alamethicin pores. On the basis of the in-plane scattering data, we propose a toroidal (or wormhole) model, which differs from the barrel-stave model of alamethicin in that the lipid bends back on itself like the inside of a torus. The bending requires a lateral expansion in the head group region of the bilayer. Magainin monomers play the role of fillers in the expansion region thereby stabilizing the pore. This molecular configuration is consistent with all published magainin data.

Alamethicin↗

PubMed1996

Mechanism of alamethicin insertion into lipid bilayers.

Alamethicin adsorbs on the membrane surface at low peptide concentrations. However, above a critical peptide-to-lipid ratio (P/L), a fraction of the peptide molecules insert in the membrane. This critical ratio is lipid dependent. For diphytanoyl phosphatidylcholine it is about 1/40. At even higher concentrations P/L > or = 1/15, all of the alamethicin inserts into the membrane and forms well-defined pores as detected by neutron in-plane scattering. A previous x-ray diffraction measurement showed that alamethicin adsorbed on the surface has the effect of thinning the bilayer in proportion to the peptide concentration. A theoretical study showed that the energy cost of membrane thinning can indeed lead to peptide insertion. This paper extends the previous studies to the high-concentration region P/L > 1/40. X-ray diffraction shows that the bilayer thickness increases with the peptide concentration for P/L > 1/23 as the insertion approaches 100%. The thickness change with the percentage of insertion is consistent with the assumption that the hydrocarbon region of the bilayer matches the hydrophobic region of the inserted peptide. The elastic energy of a lipid bilayer including both adsorption and insertion of peptide is discussed. The Gibbs free energy is calculated as a function of P/L and the percentage of insertion phi in a simplified one-dimensional model. The model exhibits an insertion phase transition in qualitative agreement with the data. We conclude that the membrane deformation energy is the major driving force for the alamethicin insertion transition.

Adsorption↗

PubMed1996

Neutron scattering in the plane of membranes: structure of alamethicin pores.

A technique of neutron in-plane scattering for studying the structures of peptide pores in membranes is described. Alamethicin in the inserted state was prepared and undeuterated and deuterated dilauroyl phosphatidylcholine (DLPC) hydrated with D2O or H2O. Neutron in-plane scattering showed a strong dependence on deuteration, clearly indicating that water is a part of the high-order structure of inserted alamethicin. The data are consistent with the simple barrel-stave model originally proposed by Baumann and Mueller. The theoretical curves computed with this model at four different deuteration conditions agree with the data in all cases. Both the diameter of the water pore and the effective outside diameter of the channel are determined accurately. Alamethicin forms pores in a narrow range of size. In a given sample condition, > 70% of the peptide forms pores of n and n +/- 1 monomers. The pore size varies with hydration and with lipid. In DLPC, the pores are made of n = 8-9 monomers, with a water pore approximately 18 A in diameter and with an effective outside diameter of approximately 40 A. In diphytanoyl phosphatidylcholine, the pores are made of n approximately 11 monomers, with a water pore approximately 26 A in diameter, with an effective outside diameter of approximately 50 A.

Alamethicin↗

PubMed1995

Antimicrobial peptide pores in membranes detected by neutron in-plane scattering.

Antimicrobial peptides isolated from the host defense systems of animals have been shown to exert their activity directly on the lipid bilayer of cell membranes, but the antimicrobial mechanisms are not clear, due chiefly to the difficulty of discerning the high-order structures formed by these peptides in membranes. Previously we have shown that these peptides insert into the membrane when their concentrations exceed a lipid-dependent critical value. With neutron in-plane scattering we now show that inserted alamethicin creates aqueous pores approximately greater than 18 A in diameter. The density of pores is consistent with the assumption that all of the alamethicin is involved in pore formation. Pores were not detected below the critical concentration. Thus concentration-dependent pore formation appears to be the molecular mechanism of antimicrobial action.

Alamethicin↗

PubMed1995

X-ray diffraction study of lipid bilayer membranes interacting with amphiphilic helical peptides: diphytanoyl phosphatidylcholine with alamethicin at low concentrations.

A variety of amphiphilic helical peptides have been shown to exhibit a transition from adsorbing parallel to a membrane surface at low concentrations to inserting perpendicularly into the membrane at high concentrations. Furthermore, this transition has been correlated to the peptides' cytolytic activities. X-ray lamellar diffraction of diphytanoyl phosphatidylcholine-alamethicin mixtures revealed the changes of the bilayer structure with alamethicin concentration. In particular, the bilayer thickness decreases with increasing peptide concentration in proportion to the peptide-lipid molar ratio from as low as 1:150 to 1:47; the latter is near the threshold of the critical concentration for insertion. From the decreases of the bilayer thickness, one can calculate the cross sectional expansions of the lipid chains. For all of the peptide concentrations studied, the area expansion of the chain region for each adsorbed peptide is a constant 280 +/- 20 A2, which is approximately the cross sectional area of an adsorbed alamethicin. This implies that the peptide is adsorbed at the interface of the hydrocarbon region, separating the lipid headgroups laterally. Interestingly, the chain disorder caused by a peptide adsorption tends to spread over a large area, as much as 100 A in diameter. The theoretical basis of the long range nature of bilayer deformation is discussed.

Alamethicin↗

PubMed1994

Cooperative membrane insertion of magainin correlated with its cytolytic activity.

Using oriented circular dichroism, we have found that magainin adopts an alpha-helical conformation with two distinct orientations when interacting with a lipid bilayer. At low concentrations, magainin is absorbed parallel to the membrane surface. However, at high concentrations, magainin is inserted into the membrane. This transition occurs at roughly the same critical concentration required for cytolytic activity, implying that the membrane insertion is responsible for magainin's cell-lysing activity.

Anti-Infective Agents↗

PubMed1994

Closed state of gramicidin channel detected by X-ray in-plane scattering.

An analogue of gramicidin A (gA) was synthesized with the formyl group replaced by a BOC group. The analogue (BOC-gA) exhibited single channel conduction, but the channel is 5-order-of-magnitude destabilized relative to the gA channel. Hydrated mixtures of gramicidin and dilauroyl phosphatidylcholine in the molar ratio of 1:10 were prepared into uniformly aligned multiple bilayers, and X-ray scattering with the momentum transfer in the plane of the membrane was measured. Analysis with the help of computer simulations showed that 70% of BOC-gA are monomers. Thus for the first time it was shown that gramicidin monomers are stable inside the monolayers of a lipid membrane. Furthermore, the monomers have the same beta helical conformation as the dimeric channel. The result suggests the possibility that when a gramicidin channel is closed, it dissociates into two monomers floating in opposite monolayers.

Computer Simulation↗

PubMed1993

X-ray scattering with momentum transfer in the plane of membrane. Application to gramicidin organization.

We demonstrate a technique for measuring x-ray (or neutron) scattering with the momentum transfer confined in the plane of membrane, for the purpose of studying lateral organization of proteins and peptides in membrane. Unlike freeze-fracture electron microscopy or atomic force microscopy which requires the membrane to be frozen or fixed, in-plane x-ray scattering can be performed with the membrane maintained in the liquid crystalline state. As an example, the controversial question of whether gramicidin forms aggregates in membrane was investigated. We used dilauroylphosphatidylcholine (DLPC) bilayers containing gramicidin in the molar ratio of 10:1. Very clear scattering curves reflecting gramicidin channel-channel correlation were obtained, even for the sample containing no heavy atoms. Thallium ions bound to gramicidin channels merely increase the magnitude of the scattering curve. Analysis of the data shows that the channels were randomly distributed in the membrane, similar to a computer simulation of freely moving disks in a plane. We suggest that oriented proteins may provide substantial x-ray contrast against the lipid background without requiring heavy-atom labeling. This should open up many possible new experiments.

Biophysical Phenomena↗