Protein assignment

General introduction

Chris Spronk, January 2009.

This tutorial is divided in a number of topics (shown on the right) and is designed to achieve 2 main goals:

• 1. to familiarise users who are new to CcpNmr Analysis with an important part of the programs' functionality and core concepts.

  2. to teach the basic steps that are typically performed in the interpretation and assignment of NMR data of a double labeled protein.

The tutorial is set up such that the user can start from a variety of stages in the tutorial, by loading project files that have been prepared and are available for download.

Data

The data used in this tutorial come from the 95 residue protein NapD1. The protein consists of alpha-helices, beta-sheets, turns and a flexible tail, and the NMR data contain easy and more difficult parts to assign. As such, NapD provides a representative example for other real life cases. The tutorial data can be downloaded here, and contains ~1.5Gb of NMR data and associated files:

• • 15N-HSQC, 600MHz

  • CBCAcoNH, 600MHz

  • HNCACB, 600MHz

  • HNcaHA, 600MHz

  • HBHAcbcacoNH, 600MHz

  • hCCH-TOCSY, 600MHz

  • HcCH-TOCSY, 600MHz

  • 13C-NOESY-HSQC_aro, aromatic region, 800MHz

  • 13C-NOESY-HSQC, aliphatic region, 800MHz

  • 15N-NOESY-HSQC, 800MHz

The spectra, projects and related data can be downloaded from here in 6 tgz files:

proteinAssignmentTutorialFiles1.tgz

proteinAssignmentTutorialFiles2.tgz

proteinAssignmentTutorialFiles3.tgz

proteinAssignmentTutorialFiles4.tgz

proteinAssignmentTutorialFiles5.tgz

proteinAssignmentTutorialFiles6.tgz

Tutorial directory structure

The main data directory contains the sequence of the protein in 'fasta' format, NapD.fasta, and the following subdirectories:

• • Azara: Contains NMR spectra

  • RCSB_DEPOSITION: contains structures, constraints, chemical shifts etcetera that were deposited at the RCSB.

  • NapD

  • NapD_1

  • NapD_2

  • NapD_....(saved projects)

Saved projects' descriptions

The NapD_.... directories contain the saved intermediate project states:

• 1. NapD_1: Project set up; sequence loaded; Molecules defined

  2. NapD_2: Spectra loaded; spectra and windows customised

  3. NapD_3: 15N-HSQC peaks automatically picked; noise peaks removed; added manually picked peaks. A clean starting point for 3d peak picking.

  4. NapD_4: Initialised NH roots and spin systems; identified NH2's; Auto arranged peak labels

  5. NapD_5: CBCAcoNH/HNCACB/HNcaHA/HBHAcbcacoNH linked to 15N-HSQC, 3D peaks picked and assigned to root frequencies. Non root resonances created

  6. NapD_6: Some side chains assigned in the hCCH-TOCSY

  7. NapD_7: Short stretch of sequential amino acids assigned

Next: Setting up the tutorial project

Topics

• Setting up the tutorial project

• Loading the spectra

• Customising project settings

• Introduction to protein backbone assignment

  • 15N-HSQC peak picking

  • Assigning root resonances (1)

  • Assigning root resonances (2)

  • 3D peak picking (1)

  • 3D peak picking (2)

  • Assigning non-root resonances

  • Identifying spin system types

  • Assigning side chain resonances

  • Sequential assignment (1)

  • Sequential assignment (2)