Abstract：
:As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30-200 microg in 8-35 microl volume. Extensive automation has been made possible by the combination of database tools, mechanization of key process steps, and the use of a micro-cryoprobe that gives excellent data while requiring little optimization and manual setup. In this perspective, we describe the overall process used by the NESG for screening NMR samples as part of a sample optimization process, assessing optimal construct design and solution conditions, as well as for determining protein rotational correlation times in order to assess protein oligomerization states. Database infrastructure has been developed to allow for flexible implementation of new screening protocols and harvesting of the resulting output. The NESG micro NMR screening pipeline has also been used for detergent screening of membrane proteins. Descriptions of the individual steps in the NESG NMR sample design, production, and screening pipeline are presented in the format of a standard operating procedure.
journal_name
J Biomol NMRjournal_title
Journal of biomolecular NMRauthors
Rossi P,Swapna GV,Huang YJ,Aramini JM,Anklin C,Conover K,Hamilton K,Xiao R,Acton TB,Ertekin A,Everett JK,Montelione GTdoi
10.1007/s10858-009-9386-zsubject
Has Abstractpub_date
2010-01-01 00:00:00pages
11-22issue
1eissn
0925-2738issn
1573-5001journal_volume
46pub_type
杂志文章abstract：:Solid-state NMR spectra of membrane proteins often show significant line broadening at cryogenic temperatures. Here we investigate the effects of several cryoprotectants to preserve the spectral resolution of lipid membranes and membrane peptides at temperatures down to ~200 K. Trehalose, glycerol, dimethylsulfoxide (...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-014-9845-z
更新日期：2014-08-01 00:00:00
abstract：:Simple pulse schemes are presented for the measurement of methyl (13)C and (1)H CSA values from (1)H-(13)C dipole/(13)C CSA and (1)H-(13)C dipole/(1)H CSA cross-correlated relaxation. The methodology is applied to protein L and malate synthase G. Average (13)C CSA values are considerably smaller for Ile than Leu/Val (...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-004-4349-x
更新日期：2004-12-01 00:00:00
abstract：:Recent efforts to reduce the measurement time for multidimensional NMR experiments have fostered the development of a variety of new procedures for sampling and data processing. We recently described concentric ring sampling for 3-D NMR experiments, which is superior to radial sampling as input for processing by a mul...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-008-9275-x
更新日期：2008-12-01 00:00:00
abstract：:Due to practical limitations in available 15N rf field strength, imperfections in 15N 180 degrees pulses arising from off-resonance effects can result in significant sensitivity loss, even if the chemical shift offset is relatively small. Indeed, in multi-dimensional NMR experiments optimized for protein backbone amid...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-006-9089-7
更新日期：2006-12-01 00:00:00
abstract：:While NMR studies of proteins typically aim at structure, dynamics or interactions, resonance assignments represent in almost all cases the initial step of the analysis. With increasing complexity of the NMR spectra, for example due to decreasing extent of ordered structure, this task often becomes both difficult and ...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-012-9652-3
更新日期：2012-09-01 00:00:00
abstract：:Protein expression in E. coli grown in shaker flasks is a routine and pivotal tool in many research laboratories. To maximize protein yields, cells are normally induced in the middle of the linear growth phase, typically at an OD600 of ≤ 1 for cells grown in Luria-Bertani (LB) medium at 37 °C. We recently showed that ...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-019-00285-x
更新日期：2019-12-01 00:00:00
abstract：:Although the order of the time steps in which the non-uniform sampling (NUS) schedule is implemented when acquiring multi-dimensional NMR spectra is of limited importance when sample conditions remain unchanged over the course of the experiment, it is shown to have major impact when samples are unstable. In the latter...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-019-00235-7
更新日期：2019-09-01 00:00:00
abstract：:The NMR derived translational diffusion coefficients were performed on unlabeled and uniformly labeled 13C,15N human insulin in water, both in neat, with zinc ions only, and in pharmaceutical formulation, containing only m-cresol as phenolic ligand, glycerol and zinc ions. The results show the dominant role of the pH ...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-018-0197-y
更新日期：2018-06-01 00:00:00
abstract：:The ability to simultaneously measure many long-range distances is critical to efficient and accurate determination of protein structures by solid-state NMR (SSNMR). So far, the most common distance constraints for proteins are 13C-15N distances, which are usually measured using the rotational-echo double-resonance (R...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-018-0187-0
更新日期：2018-05-01 00:00:00
abstract：:An approach to the determination of the 2-(13)C' chemical shift (CS) tensor orientation in pyrimidine bases via heteronuclear MAS NMR spectroscopy is presented. Considering a dipolar coupled spin 1/2 network of the type S1-I-S2 consisting of directly bonded heteronuclear spins, we have carried out numerical simulation...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1023/a:1008318530946
更新日期：2000-10-01 00:00:00
abstract：:NMR relaxometry plays crucial role in studies of protein dynamics. The measurement of longitudinal and transverse relaxation rates of [Formula: see text]N is the main source of information on backbone motions. However, even the most basic approach exploiting a series of [Formula: see text]N HSQC spectra can require se...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-017-0115-8
更新日期：2017-06-01 00:00:00
abstract：:We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273-6279 (1982)), types of amino acids are labeled with (1...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-010-9462-4
更新日期：2011-02-01 00:00:00
abstract：:Characterization of the structure and dynamics of nucleic acids by NMR benefits significantly from position specifically labeled nucleotides. Here an E. coli strain deficient in the transketolase gene (tktA) and grown on glucose that is labeled at different carbon sites is shown to facilitate cost-effective and large ...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-011-9586-1
更新日期：2012-02-01 00:00:00
abstract：:We perform a detailed comparison of fast backbone dynamics probed at amide nitrogen versus carbonyl carbon sites for dematin headpiece C-terminal domain (DHP) and its S74E mutant (DHPS74E). Carbonyl dynamics is probed via auto-correlated longitudinal rates and transverse C'/C'-C(alpha) CSA/dipolar and C'/C'-N CSA/dipo...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-010-9417-9
更新日期：2010-06-01 00:00:00
abstract：:One of the major bottlenecks in the determination of proteinstructures by NMR is in the evaluation of the data produced by theexperiments. An important step in this process is assignment, where thepeaks in the spectra are assigned to specific spins within specificresidues. In this paper, we discuss a spin system assig...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1023/A:1018329420659
更新日期：1997-10-01 00:00:00
abstract：:NMR View is a computer program designed for the visualization and analysis of NMR data. It allows the user to interact with a practically unlimited number of 2D, 3D and 4D NMR data files. Any number of spectral windows can be displayed on the screen in any size and location. Automatic peak picking and facilitated peak...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/BF00404272
更新日期：1994-09-01 00:00:00
abstract：:TROSY-based triple resonance experiments are essential for protein backbone assignment of large biomolecular systems by solution NMR spectroscopy. In a survey of the current Bruker pulse sequence library for TROSY-based experiments we found that several sequences were plagued by artifacts that affect spectral quality ...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-017-0133-6
更新日期：2017-09-01 00:00:00
abstract：:Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing with such complicated systems, given that the target molecules can be isotopically labeled. Methods of metabolic isotope la...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-018-0169-2
更新日期：2018-07-01 00:00:00
abstract：:An approach for the efficient implementation of RN(n)(nu) symmetry-based pulse schemes that are often employed for recoupling and decoupling of nuclear spin interactions in biological solid state NMR investigations is demonstrated at high magic-angle spinning frequencies. RF pulse sequences belonging to the RN(n)(nu) ...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-009-9335-x
更新日期：2009-08-01 00:00:00
abstract：:Defining the self-association state of a molecule in solution can be an important step in NMR-based structure determination. This is particularly true of peptides, where there can be a relatively small number of long-range interactions and misinterpretation of an intermolecular NOE as an intramolecular contact can hav...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1023/a:1008382624724
更新日期：2000-02-01 00:00:00
abstract：:The solvent-exposed regions of (U-13C)ascomycin when bound to its putative target protein, FKBP, have been identified based on the different proton longitudinal relaxation rates (R1 = 1/T1) measured in the absence and presence of the paramagnetic relaxation reagent, 4-hydroxy-2,2,6,6-tetramethyl-piperidinyl-1-oxy (HyT...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/BF02192797
更新日期：1992-01-01 00:00:00
abstract：:The first reconstitution of an Fe2S2 ferredoxin with a diamagnetic prosthetic group was recently described [Kazanis et al. (1995) J. Am. Chem. Soc., 117, 6625-6626]. The replacement of the iron-sulfur cluster of the bacterial ferredoxin putidaredoxin (Pdx) by gallium (Ga3+) renders the protein diamagnetic and permits ...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1023/a:1018369721091
更新日期：1997-06-01 00:00:00
abstract：:A simple spectroscopic filtering technique is presented that may aid the assignment of (13)C and (15)N resonances of methyl-containing amino-acids in solid-state magic-angle spinning (MAS) NMR. A filtering block that selects methyl resonances is introduced in two-dimensional (2D) (13)C-homonuclear and (15)N-(13)C hete...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-006-9078-x
更新日期：2006-11-01 00:00:00
abstract：:One-dimensional transient NOE build-up curves were measured for the synthetic disaccharide alpha-D-Fuc-(1-->4)-beta-D-GlcNAc 1 utilizing Gaussian shaped pulses. Simulated build-up curves from Metropolis Monte Carlo simulations were compared to the experimental data. Disaccharide 1 is structurally related to methyl bet...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/BF00176007
更新日期：1993-07-01 00:00:00
abstract：:NMR of macromolecules is limited by large transverse relaxation rates. In practice, this results in low efficiency of coherence transfer steps in multidimensional NMR experiments, leading to poor sensitivity and long acquisition times. The efficiency of coherence transfer can be maximized by design of relaxation optim...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-005-3592-0
更新日期：2005-05-01 00:00:00
abstract：:Simultaneous recording of different NMR parameters is an efficient way to reduce the overall experimental time and speed up structural studies of biological macromolecules. This can especially be beneficial in the case of fast NMR-based drug screening applications or for collecting NOE restraints, where prohibitively ...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-007-9178-2
更新日期：2007-10-01 00:00:00
abstract：:Accessible surface area (ASA) is the surface area of an atom, amino acid or biomolecule that is exposed to solvent. The calculation of a molecule's ASA requires three-dimensional coordinate data and the use of a "rolling ball" algorithm to both define and calculate the ASA. For polymers such as proteins, the ASA for i...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-015-9957-0
更新日期：2015-07-01 00:00:00
abstract：:Adiabatic pulses have been widely used for broadband decoupling and spin inversion at high magnetic fields. In this paper we propose adiabatic pulses and supercycles that can be used at high magnetic fields like 800 or 900 MHz to obtain broadband TOCSY sequences with C,C or H,H J-transfer. The new mixing sequences are...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1023/a:1026785725363
更新日期：2000-11-01 00:00:00
abstract：:Detection of (15)N in multidimensional NMR experiments of proteins has sparsely been utilized because of the low gyromagnetic ratio (γ) of nitrogen and the presumed low sensitivity of such experiments. Here we show that selecting the TROSY components of proton-attached (15)N nuclei (TROSY (15)NH) yields high quality s...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-015-9991-y
更新日期：2015-12-01 00:00:00
abstract：:In this work, we show how the water flip-back approach that is widely employed in solution-state NMR can be adapted to proton-detected MAS solid-state NMR of highly deuterated proteins. The scheme allows to enhance the sensitivity of the experiment by decreasing the recovery time of the proton longitudinal magnetizati...
journal_title：Journal of biomolecular NMR
pub_type： 杂志文章
doi：10.1007/s10858-015-9902-2
更新日期：2015-02-01 00:00:00
