10 pages

English

tutorial

Navug

Le téléchargement nécessite un accès à la bibliothèque YouScribe
Tout savoir sur nos offres

10 pages

English

Le téléchargement nécessite un accès à la bibliothèque YouScribe
Tout savoir sur nos offres

A propos
Informations
Extrait

Description

SMART NOTEBOOK:A semi-automated approach for the rapid assignment of triple resonanceNMR spectra.Programmer: Robert BoykoConsultants: Carolyn SlupskyValerie BoothBrian SykesProtein Engineering Network Centres of Excellence, 713 HMRC,University of Alberta, Edmonton, Alberta, Canada. T6G 2S2e-mail: Carolyn.Slupsky@ualberta.ca, or Robert.Boyko@ualberta.ca orBrian.Sykes@ualberta.cahttp://canopus.pence.ualberta.ca/ftp/smartnotebook/Funded by:Smart Note Book Tutorial:---------------------------------------------------------------Summary of Features:1. Facilitates rapid backbone assignment using HSQC, HNCACB,and CBCA(CO)NNH. You may view other experiments at the sametime, such as the cc-TOCSY experiment.2. Uses NMRVIEW as the graphical interface.13 133. The assignment strategy uses both the Ca and Cb frequencymatch between neighboring residues and the BMRB database of15 13 13N, Ca, Cb values for particular amino-acid types.4. Selection of possible residue pairs is based on peak lists, howeverthe user views the actual spectra before determining the best fit.5. Uses a flexible programming strategy to allow the user to add orchange the functionality. For example, the user may add extraspectra, define new spectra and rules.The Basics:What you will need:1) A sequence file in nmrview format (see example file).2) A set of spectra: HSQC, HNCACB, CBCACONH, CCTOCSY(cctocsy is optional).3) NMRVIEW program4) Peak lists in nmrview .xpk format5) Sequence ...

Informations

Publié par	Navug
Nombre de lectures	31
Langue	English

Extrait

SMART NOTEBOOK:

A semi-automated approach for the rapid assignment of triple resonance

NMR spectra.

Programmer:

Robert Boyko

Consultants:

Carolyn Slupsky

Valerie Booth

Brian Sykes

Protein Engineering Network Centres of Excellence, 713 HMRC,

University of Alberta, Edmonton, Alberta, Canada. T6G 2S2

e-mail:

Carolyn.Slupsky@ualberta.ca

, or

Robert.Boyko@ualberta.ca

Brian.Sykes@ualberta.ca

http://canopus.pence.ualberta.ca/ftp/smartnotebook/

Funded by:

Smart Note Book Tutorial:

---------------------------------------------------------------

Summary of Features:

1. Facilitates rapid backbone assignment using HSQC, HNCACB,

and CBCA(CO)NNH. You may view other experiments at the same

time, such as the cc-TOCSY experiment.

2. Uses NMRVIEW as the graphical interface.

3. The assignment strategy uses both the

and

frequency

match between neighboring residues and the BMRB database of

values for particular amino-acid types.

4. Selection of possible residue pairs is based on peak lists, however

the user views the actual spectra before determining the best fit.

5. Uses a flexible programming strategy to allow the user to add or

change the functionality. For example, the user may add extra

spectra, define new spectra and rules.

The Basics:

What you will need:

1) A sequence file in nmrview format (see example file).

2) A set of spectra: HSQC, HNCACB, CBCACONH, CCTOCSY

(cctocsy is optional).

3) NMRVIEW program

4) Peak lists in nmrview .xpk format

5) Sequence file in nmrview sequence format

6) A set of initialization files to set up smartnotebook: init.spectra,

init.smartnotebook, and an nmrview startup script. (See: “Setting

up smartnotebook with your own data”.) We will provide default

versions of these.

Getting Started:

You will need to create the files necessary for assignment. Start by

processing with a package such as nmrpipe making sure that for

the 3D spectra x is HN, y is C and z is N. You will need to convert

the pipe format to nmrview format with the “-nv” option.

You will need to peak pick the spectra. You can do this

automatically with nmrview (see the appendix for instructions on

how to do this).

You should create a sequence file (see example directory).

After you have all of your files, quit nmrview. You will need to

check, and perhaps manually alter the following files that are

included in the SMARTNOTEBOOK package:

i) init.spectra to include the correct file names of your spectra (see

“custom configuration with init.spectra” section).

ii) init.smartnotebook to include the correct name of your sequence

file (see “custom configuration with init.smartnotebook” section).

Create your own nmrview script, and start up nmrview. Then in the

message window type “snb” or “smartnotebook”. At this point in

time, you should have three windows come up: A window

containing the 3D spectra in the hncacb(i-1) – cbcaconh(i) –

cctocsy(i) – hncacb(i) format; another window with your 2D HSQC;

and the “smartnotebook” window which should look like the

following:

HSQC

File

Spectral

Labelling

Connection

Editor

Chains

Pattern

Button

Protein

Sequence

Protein Sequence

is simply your protein sequence in a 1-letter code

format.

HSQC ID Menu

allows you to place cursors on a particular peak and

“set from spectrum” or look at all HSQC peaks.

File Menu

allows you to quit your session.

Spectral Labeling Menu

allows you to toggle on/off the peak labels

on each spectrum.

Connection Editor

allows you to query and modify particular

connectivities in the HSQC dataset. This feature is most likely

to be used in crowded regions of the spectrum where the

program has trouble picking out peaks that fit together.

Chains Menu

allows you to see and load in chains that you have

chained together or assigned.

Pattern Menu

allows you to select the type of pattern that you are

looking for. You can choose between: All, dxx (residue –

residue), dgx (glycine – residue), dxg (residue – glycine) and

dgg (glycine – glycine). (Because of the lack of a C

for

glycine, glycine has a special rules set.)

Search Button

allows you to look for possible connections between

the queried HSQC ID number and all other HSQC ID

numbers.

Either type in a HSQC peak number or select an HSQC peak by

placing the black cursors in the HSQC on a particular peak, and

selecting under the HSQC ID menu “set from spectra”.

Begin to search for connectivities by clicking on the “Search”

Button. You should see something like the following:

Connectivities

Found

Button to

draw

spectra

Button

to add

current

selection to

chain

HSQC I.D. number

queried

Pattern

You may select a particular pattern to query using the “Pattern” menu.

For example, the above pattern is “all”

Once you have chosen a pattern and selected and HSQC I.D.

number, click on the “Search” button. The left hand box shows you

four things: Fit, Connection, Pattern, and Status. Fit is an

indication of how well the chemical shifts match between spectra.

The higher the number, the better the fit. The connection refers to

the HSQC peak I.D. numbers being connected. The first number

coincides to the HSQC I.D. number of residue i-1, and the second

number coincides to the HSQC I.D. number of residue i. The

connections are listed in order with the best matching pairs at the

top. The pattern is the particular pattern that the connectivity

matches (for example, dxx refers to generic residue – generic

residue … excluding glycine). The Status refers to the current

status of the two HSQC peak I.D.’s in the connection. The first

character refers to the first HSQC I.D, the second character to the

second HSQC I.D. number. If an HSQC peak I.D. number has

been chained, the status will report a “C”. If the status shows an

“A”, it means that HSQC I.D. peak number has been assigned. A

status of “-“ means that the peak is currently not placed in any

chain.

You can now click on “Draw Spectra”. This will present the hncacb

spectrum of residue i-1 in the left-most part of the panel

corresponding to the HSQC I.D. number on the left and the black

cursors centered on the peak in the HSQC spectrum. The

cbcaconh and hncacb (and cctocsy) of residue i are presented to

the right of the first hncacb spectrum and correspond to the HSQC

I.D. number on the right and the red cursors on the HSQC

spectrum. Note that on the 3D spectra, the connecting peaks have

cursors running through them.

Black cursors cross

at N,HN of residue i-1

Red cursors

cross at N,HN

of residue i.

Connection

between N,HN

of residue i to

Ca and Cb of

residue i-1

N,HN of

residue i

N,HN of

residue i-1

Check all of the connectivities to see which one fits best. If you

don’t like a particular connectivity, (eg. The Ca and Cb don’t match

properly), you may use the “delete” button under the “conection

editor” to put an “x” next to that connection. It will not be removed

from the data, but it is useful so you don’t have to re-query that

choice again. Under the ‘connections’ menu, the “edit” button

allows you to change the peaks in the hncacb that define a Ca and

Cb for a particular residue. This is important since the rules that

define connection patterns may be simple, and while

smartnotebook might recognize a pattern, it may have a difficult

time deciding where all the peaks in the pattern fit.

Arrow

tells

you which

connection

Peak I.D.

numbers

from hncacb

connected.

Quit window

button.

HSQC peak I.D. numbers

in connectivity with pattern.

Here, the user is presented with four possible pattern configurations. The

arrow on the left indicates the current configuration of peaks. The user

clicks on the line that correctly specifies the pattern of choice. Note that

when you select a different pattern from what the computer originally

chose, when you quit this window the “Fit” will change, and you will have

to re-select that peak from the connections window.

10)

Once you have made a good connectivity choice, click on the “Add

to chain” button. The HSQC I.D. numbers then appear in the right

hand box of the smart notebook panel with the HSQC I.D. number

representing residue i-1 first, followed by residue i (sequential

order). Once you have done this, residues in the sequence change

color to indicate possible assignments assessed using your

chemical shifts file (or the BMRB database by default). Any residue

colored blue (our default) indicates the starting place for a possible

assignment.

HSQC Peak

I.D. numbers

in Chain

Delete

Button

Best Fit

query

All plausible

assignments

in spectrum.

11)

Continue chaining HSQC I.D. peak numbers until you have a

single choice. If you incorrectly add a connectivity to the chain,

select the incorrect peak and press the “delete” button (must be at

either end of the chain). You may delete the entire chain by

pressing the “delete” button when no hsqc I.D. number has been

chosen.

12)

Once you have a chain, click on the “Best Fit” button. You will see

a box appear that shows the segment of the sequence that your

chain fits the best. There is a sliding standard deviation scale that

you may use to make the criteria stricter or looser. You may also

click on another residue in the protein sequence panel to see why

other options may not be optimal for assignment. If you like the

assignment, click on the “assign” button. An unassign button

allows you to change your mind about the assignment of a chain.

A chain that has been assigned is colored black.

Sliding Standard

Deviation Scale

Assign

Button

Unassign

Button

Quit

Window

button

Checking other

assignments

indicates where

discrepencies

exist to the BMRB

chemical shift

database.

This table shows how well each proposed chemical shift fits to the

sequence. Discrepencies to your chemical shifts file are indicated with an

“x”.

All errors and messages are noted in the nmrview console and are

prefixed by “snb:”

Now that you are familiar with smartnotebook, please follow the

instructions under “setting up smartnotebook with your own data”

to look at your own data. If you have any questions or comments,

feel free to e-mail or telephone Carolyn Slupsky or Robert Boyko.

Univers
Ebooks
Livres audio
Presse
Podcasts
BD
Documents

Livre audio en ligne - Développement personnel Livre en ligne Tout le catalogue Tous les Intérêts

tutorial

YouScribe

Le catalogue

Le service

Les conditions