NMR paper Rapid screening of E. coli extracts by heteronuclear NMR.

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[NMR paper] Rapid screening of E. coli extracts by heteronuclear NMR.

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NMR processing:

MDD

NMR assignment:

Backbone:

Side-chains:

NOEs:

Structure from NMR restraints:

Ab initio:

Fragment-based:

Rosetta-NMR (Robetta)

Template-based:

GeNMR

I-TASSER

Refinement:

Amber

Structure from chemical shifts:

Fragment-based:

Homology-based:

Torsion angles from chemical shifts:

Preditor

TALOS

Promega- Proline

Secondary structure from chemical shifts:

CSI (via RCI server)

TALOS

MICS caps, β-turns

d2D

PECAN

Flexibility from chemical shifts:

RCI

Interactions from chemical shifts:

HADDOCK

Chemical shifts re-referencing:

NMR model quality:

NOEs, other restraints:

Chemical shifts:

RDCs:

Pseudocontact shifts:

Protein geomtery:

SAVES2 or SAVES4

Quality Control Check

NMR spectrum prediction:

FANDAS

MestReS

V-NMR

Flexibility from structure:

Backbone S2

Methyl S2

B-factor

Molecular dynamics:

Gromacs

Amber

Antechamber

Chemical shifts prediction:

From structure:

Shiftx2

Sparta+

Camshift

CH3shift- Methyl

ArShift- Aromatic

ShiftS

Proshift

PPM

CheShift-2- Cα

From sequence:

Shifty

Camcoil

Poulsen_rc_CS

Disordered proteins:

MAXOCC

Format conversion & validation:

CCPN

From NMR-STAR 3.1

Validate NMR-STAR 3.1

NMR sample preparation:

Protein disorder:

DisMeta

Protein solubility:

Isotope labeling:

Solid-state NMR:

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11-24-2010, 09:01 PM

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Rapid screening of E. coli extracts by heteronuclear NMR.

Rapid screening of E. coli extracts by heteronuclear NMR.

Related Articles Rapid screening of E. coli extracts by heteronuclear NMR.

Curr Protoc Protein Sci. 2003 May;Chapter 7:Unit 7.11

Authors: Gronenborn AM

Assessing whether a protein or protein complex is amenable to structural analysis is an important component in the structural genomics effort. In particular, if complete sets of structures for entire genomes are to be obtained within a reasonable time frame, high throughput methodologies for all steps along the way have to be developed. These days, cloning and expression systems are highly optimized and a variety of commercially available vectors can be used. However, heterologous proteins or protein domains expressed in bacteria may not be soluble or correctly folded, necessitating intricate solubilization and refolding schemes prior to structural or functional studies. NMR spectroscopy is an important tool for assessing the solubility, stability, and structural integrity of a gene product. It allows efficient evaluation of many variations of polypeptide length and sequence (without time- and labor-intensive purification/refolding procedures) using 1H-15N-HSQC spectroscopy of samples of 15N-labeled proteins directly from crude E. coli extracts. In addition to screening for particular properties of the expressed protein alone, it is also possible to map intermolecular interactions such as ligand binding. In this unit, the basic methodology for bacterial growth, isotope labeling, and spectroscopic evaluation of the protein structure is provided.

PMID: 18429247 [PubMed - indexed for MEDLINE]

Source: PubMed

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