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Protein sample production

NESG target selection DNA cloning protocols Protein expression and purification protocols  Sample Optimization Construct optimization Buffer optimization Cofactor optimization

SDS page gel Protein concentration measurements Assessment of Oligomerization Status gel-filtration and light scattering NMR determined Rotational correlation time Mass spectrum NMR screening 1H 1D screening Initial [15N,1H] HSQC

NMR Data Acquisition

Routine operation NMR sample tubes NMR Sample Preparation Inserting NMR Sample Tuning and matching Deuterium Lock Shimming Pulse width calibration Temperature calibration Chemical shift referencing Advanced operation Deuterium pulse width calibration and decoupling

NMR data acquisition for protein structure determination Common NMR experiment sets Custom NMR experiment setup scripts for VNMRJ Estimation of rotational correlation time Estimation of measurement time Simultaneous 13C,15N-resolved NOESY 2D [13C, 1H]-HSQC for fractionally 13C-labeled samples Long-range 15N-1H correlation experiments for histidine rings Non-uniform sampling (NUS) NUS - Varian NUS - Bruker

Maintenance (VARIAN) Installing and updating BioPack Full probefile calibration Rebooting spectrometer console Conditioning procedure for cryogenic probes

NMR Resonance Assignment

Principles and concepts Semi-automated protocols CARA Sparky XEASY

Automated resonance assignment AutoAssign AutoAssign server ABACUS PINE server

Validation and deposition Assignment validation suite (AVS) Linear analysis of chemical shift (LACS) Depositing chemical shifts

Principles and concepts Practical aspects Semi-automated protocols CARA Spin System Identification in 2D 15N-HSQC and 3D HNNCO Backbone Resonance Assignment Assignment of HA and HB Resonances with (4,3)D GFT HABCAB(CO)NHN Side Chain Assignment Aliphatic side-chain assignment Aromatic side-chain assignment Amide side-chain assignment Sparky XEASY Spin system identification Backbone resonance assignment GFT-based spectra HNCACAB/CABCA(CO)NH Conventional spectra HNCACB/CBCA(CO)NH HNCA/HN(CO)CA HNCO/HN(CA)CO NOESY/TOCSY Side chain resonance assignment Aliphatic GFT NMR spectra (4,3)D GFT HABCAB(CO)NHN (4,3)D GFT HCCH Conventional spectra HAHB(CO)NH HCCH-COSY HCCH-TOCSY Simultaneous NOESY (H)CC-TOCSY-(CO)NH H(CC-TOCSY-CO)NH Aromatic GFT-based spectra Conventional spectra Other Trp e1 NH and d1 CH Met e CH3 Asn d2 and Gln e2 NH2 NOESY peak integration Automated protocols AutoAssign AutoAssign server ABACUS PINE server Validation of resonance assignment Assignment validation suite (AVS) Linear analysis of chemical shift (LACS) Depositing chemical shifts

Structure Calculation and Validation Principles and concepts Practical aspects Structure calculation CYANA Getting started FOUND TALOS GLOMSA NOE calibration Manual structure calculation Automated NOESY assignment and structure calculation Structure calculation with residual dipolar couplings (link to REDCAT/PALES,FINDTENSOR, .rdc file, adding ORI to PDB file) Homodimer structure calculations AutoStructure Theory Getting started CYANA run XPLOR run Analyzing the output RPF/DP scores Structure calculation using AS-DP "Consensus" Approaches Overview of Consensus runs Finding Consensus NOE assignments Validation of Consensus runs CS-ROSETTA CS-DP ROSETTA CS-RDC-ROSETTA RDC-assisted dimer structure calculation Special topics Protein-Ligand complexes Metal ions Dimers Residual Dipolar Couplings REDCAT and REDCRAFT Paramagnetic constraints Structure Refinement CNS refinement XPLOR-NIH refinement ROSETTA refinement Validation and deposition PdbStat PSVS RPF analysis MolProbity server PDB and BMRB deposition ADIT-NMR HarvestDB SPINS

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