Michael Levitt

KoBaMIN: a knowledge-based minimization web server for protein structure refinement

The KoBaMIN web server provides an online interface to a simple, consistent and computationally efficient protein structure refinement protocol based on minimization of a knowledge-based potential of mean force. The server can be used to refine either a single protein structure or an ensemble of proteins starting from their unrefined coordinates in PDB format. The refinement method is particularly fast and accurate due to the underlying knowledge-based potential derived from structures deposited in the PDB; as such, the energy function implicitly includes the effects of solvent and the crystal environment. Our server allows for an optional but recommended step that optimizes stereochemistry using the MESHI software. The KoBaMIN server also allows comparison of the refined structures with a provided reference structure to assess the changes brought about by the refinement protocol. The performance of KoBaMIN has been benchmarked widely on a large set of decoys, all models generated at the seventh worldwide experiments on critical assessment of techniques for protein structure prediction (CASP7) and it was also shown to produce top-ranking predictions in the refinement category at both CASP8 and CASP9, yielding consistently good results across a broad range of model quality values. The web server is fully functional and freely available at http://csb.stanford.edu/kobamin.

Structural patterns in globular proteins.

A simple diagrammatic representation has been used to show the arrangement of α helices and β sheets in 31 globular proteins, which are classified into four clearly separated classes. The observed arrangements are significantly non-random in that pieces of secondary structure adjacent in sequence along the polypeptide chain are also often in contact in three dimensions.

A simplified representation of protein conformations for rapid simulation of protein folding.

This report is one of a series of papers that introduce and use a new and highly simplified treatment of protein conformations. The first paper (Levitt & Warshel, 1975) outlined fhe approach and showed how it could be used to simulate the “renaturation” of a small protein. The present paper describes fhe representation in some detail and tests the methods extensively under a variety of different conditions. The third paper (Warshel & Levitt, 1976) is devoted to a study of the folding pathway and stability of a mainly a-helical protein. In this work, I show how the concept of time-averaged forces, introduced previously (Levitt & Warshel, 1975), can be used to simplify conformafional energy calculations on globular proteins. A detailed description is given of the simplified molecular geometry, the parameterization of suitable force fields, the best energy minimization procedure, and fhe techniques for escaping from local minima. Extensive tests of the method on the native conformation of pancreatic trypsin inhibitor show that the simplifications work well in representing the stable native conformation of this globular protein. Further tests show that simulated folding of pancreatic trypsin inhibitor from open chain conformations gives compact calculated conformations thaf have many features in common with the actual native conformafion. Folding simulafions are done under a variety of conditions, and fhe relevance of such calculations fo the actual in vitro folding process is discussed at some length. These same techniques have many potential applications including enzyme-substrate binding, changes in protein tertiary and quaternary structure, and protein-protein inferactiona.

Computer simulation of protein folding

A new and very simple representation of protein conformations has been used together with energy minimisation and thermalisation to simulate protein folding. Under certain conditions, the method succeeds in ‘renaturing’ bovine pancreatic trypsin inhibitor from an open-chain conformation into a folded conformation close to that of the native molecule.

Conformation of amino acid side-chains in proteins☆

We have analysed the side-chain dihedral angles in 2536 residues from 19 protein structures. The distributions of x1 and x2 are compared with predictions made on the basis of simple energy calculations. The x1 distribution is trimodal; the g− position of the side-chain (trans to Hα), which is rare except in serine, the t position (trans to the amino group), and the g+ position (trans to the carbonyl group), which is preferred in all residues. Characteristic x2 distributions are observed for residues with a tetrahedral γ-carbon, for aromatic residues, and for aspartic acid/asparagine. The number of configurations actually observed is small for all types of side-chains, with 60% or more of them in only one or two configurations. We give estimates of the experimental errors on x1 and x2 (3 ° to 16 °, depending on the type of the residue), and show that the dihedral angles remain within 15 ° to 18 ° (standard deviation) from the configurations with the lowest calculated energies. The distribution of the side-chains among the permitted configurations varies slightly with the conformation of the main chain, and with the position of the residue relative to the protein surface. Configurations that are rare for exposed residues are even rarer for buried residues, suggesting that, while the folded structure puts little strain on side-chain conformations, the side-chain positions with the lowest energy in the unfolded structure are chosen preferentially during folding.

Protein normal-mode dynamics: trypsin inhibitor, crambin, ribonuclease and lysozyme

We have developed a new method for modelling protein dynamics using normal-mode analysis in internal co-ordinates. This method, normal-mode dynamics, is particularly well suited for modelling collective motion, makes possible direct visualization of biologically interesting modes, and is complementary to the more time-consuming simulation of molecular dynamics trajectories. The essential assumption and limitation of normal-mode analysis is that the molecular potential energy varies quadratically. Our study starts with energy minimization of the X-ray co-ordinates with respect to the single-bond torsion angles. The main technical task is the calculation of second derivative matrices of kinetic and potential energy with respect to the torsion angle co-ordinates. These enter into a generalized eigenvalue problem, and the final eigenvalues and eigenvectors provide a complete description of the motion in the basic 0.1 to 10 picosecond range. Thermodynamic averages of amplitudes, fluctuations and correlations can be calculated efficiently using analytical formulae. The general method presented here is applied to four proteins, trypsin inhibitor, crambin, ribonuclease and lysozyme. When the resulting atomic motion is visualized by computer graphics, it is clear that the motion of each protein is collective with all atoms participating in each mode. The slow modes, with frequencies of below 10 cm-1 (a period of 3 ps), are the most interesting in that the motion in these modes is segmental. The root-mean-square atomic fluctuations, which are dominated by a few slow modes, agree well with experimental temperature factors (B values). The normal-mode dynamics of these four proteins have many features in common, although in the larger molecules, lysozyme and ribonuclease, there is low frequency domain motion about the active site.

Structure of nucleosome core particles of chromatin.

Crystals have been obtained of nucleosome cores and analysed by X-ray diffraction and electron microscopy. The core is a flat particle of dimensions about 110 × 110 × 57 Å, somewhat wedge shaped, and strongly divided into two ‘layers’, consistent with the DNA being wound into about 1¾ turns of a fiat superhelix of a pitch about 28 Å. The organisation of the DNA can be correlated with the results of enzyme digestion studies. A change in the screw of the DNA double helix on nucleosome formation can be deduced.

The ASTRAL Compendium in 2004

The ASTRAL Compendium provides several databases and tools to aid in the analysis of protein structures, particularly through the use of their sequences. Partially derived from the SCOP database of protein structure domains, it includes sequences for each domain and other resources useful for studying these sequences and domain structures. The current release of ASTRAL contains 54,745 domains, more than three times as many as the initial release 4 years ago. ASTRAL has undergone major transformations in the past 2 years. In addition to several complete updates each year, ASTRAL is now updated on a weekly basis with preliminary classifications of domains from newly released PDB structures. These classifications are available as a stand-alone database, as well as integrated into other ASTRAL databases such as representative subsets. To enhance the utility of ASTRAL to structural biologists, all SCOP domains are now made available as PDB-style coordinate files as well as sequences. In addition to sequences and representative subsets based on SCOP domains, sequences and subsets based on PDB chains are newly included in ASTRAL. Several search tools have been added to ASTRAL to facilitate retrieval of data by individual users and automated methods. ASTRAL may be accessed at http://astral.stanford. edu/.

Aromatic rings act as hydrogen bond acceptors

Simple energy calculations show that there is a significant interaction between a hydrogen bond donor (like the greater than NH group) and the centre of a benzene ring, which acts as a hydrogen bond acceptor. This interaction, which is about half as strong as a normal hydrogen bond, contributes approximately 3 kcal/mol (1 cal = 4.184 J) of stabilizing enthalpy and is expected to play a significant role in molecular associations. It is of interest that the aromatic hydrogen bond arises from small partial charges centred on the ring carbon and hydrogen atoms: there is no need to consider delocalized electrons. Although some energy calculations have included such partial charges, their role in forming such a strong interaction was not appreciated until after aromatic hydrogen bonds had been observed in protein-drug complexes.

Helix to helix packing in proteins

Abstract Analysis of the pattern of residue to residue contacts at the interface of 50 helix to helix packings observed in ten proteins of known structure supports a model for helix to helix packing in which the ridges and grooves on the helix surface intercalate. These ridges are formed by rows of residues whose separation in sequence is usually four, occasionally three and rarely one. The model explains the observed predominance of packings whose interhelical angle is ~ −50 °. Of the 50 packings, 38 agree with the model and the general features of another ten packings are described by an extension to the model in which ridges can pack across each other if a small side-chain occurs at the place where they cross.