Completely automated, highly error-tolerant macromolecular structure determination from multidimensional nuclear overhauser enhancement spectra and chemical shift assignments

The major rate-limiting step in high-throughput NMR protein structure determination involves the calculation of a reliable initial fold, the elimination of incorrect nuclear Overhauser enhancement (NOE) assignments, and the resolution of NOE assignment ambiguities. We present a robust approach to automatically calculate structures with a backbone coordinate accuracy of 1.0-1.5 A from datasets in which as much as 80% of the long-range NOE information (i.e., between residues separated by more than five positions in the sequence) is incorrect. The current algorithm differs from previously published methods in that it has been expressly designed to ensure that the results from successive cycles are not biased by the global fold of structures generated in preceding cycles. Consequently, the method is highly error tolerant and is not easily funnelled down an incorrect path in either three-dimensional structure or NOE assignment space. The algorithm incorporates three main features: a linear energy function representation of the NOE restraints to allow maximization of the number of simultaneously satisfied restraints during the course of simulated annealing; a method for handling the presence of multiple possible assignments for each NOE cross-peak which avoids local minima by treating each possible assignment as if it were an independent restraint; and a probabilistic method to permit both inactivation and reactivation of all NOE restraints on the fly during the course of simulated annealing. NOE restraints are never removed permanently, thereby significantly reducing the likelihood of becoming trapped in a false minimum of NOE assignment space. The effectiveness of the algorithm is demonstrated using completely automatically peak-picked experimental NOE data from two proteins: interleukin-4 (136 residues) and cyanovirin-N (101 residues). The limits of the method are explored using simulated data on the 56-residue B1 domain of Streptococcal protein G.     

Measurement of small ion beams by thermal ionisation mass spectrometry using new 10 Ohm resistors

We tested 5 newly manufactured – prototype – 10¹³ Ohm resistors in the feedback loop of Faraday cup amplifiers to measure small ion beams by Thermal Ionisation Mass Spectrometry (TIMS). The high Ohmic resistors installed in the TRITON Plus at the VU University Amsterdam theoretically have 10 times lower noise levels relative to the default 10¹¹ Ohm resistors. To investigate the precision and accuracy of analyses using these new amplifiers we measured Sr and Nd isotopes of reference standards at a range of ion currents (3.2 × 10⁻¹⁶ to 1 × 10⁻¹² A, corresponding to intensities of 32 μV to 100 mV on a default 10¹¹ Ohm amplifier) and on small amounts of material (100 and 10 pg). Internal precision and external reproducibility for Sr and Nd isotope ratios are both better when collected on 10¹³ compared 10¹² Ohm resistors and to the default 10¹¹ Ohm resistors. At an ⁸⁷Sr ion current of 3 × 10⁻¹⁴ A (3 mV on a 10¹¹ Ohm amplifier) the internal precision (2 SE) of ⁸⁷Sr/⁸⁶Sr is 5 times better for 10¹³ Ohm resistors compared to 10¹¹ Ohm resistors. The external reproducibility (2 SD) at this beam intensity is 9 times better.     

AssignFit: a program for simultaneous assignment and structure refinement from solid-state NMR spectra

AssignFit is a computer program developed within the XPLOR-NIH package for the assignment of dipolar coupling (DC) and chemical shift anisotropy (CSA) restraints derived from the solid-state NMR spectra of protein samples with uniaxial order. The method is based on minimizing the difference between experimentally observed solid-state NMR spectra and the frequencies back calculated from a structural model. Starting with a structural model and a set of DC and CSA restraints grouped only by amino acid type, as would be obtained by selective isotopic labeling, AssignFit generates all of the possible assignment permutations and calculates the corresponding atomic coordinates oriented in the alignment frame, together with the associated set of NMR frequencies, which are then compared with the experimental data for best fit. Incorporation of AssignFit in a simulated annealing refinement cycle provides an approach for simultaneous assignment and structure refinement (SASR) of proteins from solid-state NMR orientation restraints. The methods are demonstrated with data from two integral membrane proteins, one α-helical and one β-barrel, embedded in phospholipid bilayer membranes.