Sparky NMR Assignment and Integration Software NMR software •Paid –TopSpin •Developed and mainly used for Bruker spectrometers controlling •Many functions for processing, view and analysis of one- and multi-dimensional spectra •Basically impossible analysis of multi-dimensional –Mnova •Processing, analysis and visualization of 1D and 2D spectra •Support of different formats of spectra (Bruker, Jeol, Agilent...) •Free trial version –ACD/NMR processor •see Mnova •Free version for academic purposes with noncomplete functionalities NMR software •Freeware –NMRPipe •Multiplatform •Processing and analysis of any spectra •Based on UNIX systems → knowledge of UNIX environment is advantage –SpinWorks •For the Windows systems (emulator of Windows are needed for UNIX a Mac) •Processing and analysis of 1D and 2D spectra •User very unfriendly –Sparky Sparky NMR Assignment and Integration Software Originally developed on University of California, San Francisco, currently upgraded on University of Wisconsin–Madison (as NMRFAM-Sparky) •Multiplatform (Windows and Linux OS) •Free for download: https://nmrfam.wisc.edu/nmrfam-sparky-distribution/ •Written in Python language, open source, it is not developed anymore (last version is from the 2008) •Fast and undemanding for hardware •It can handle only multi-dimensional spectra (2D, 3D) Sparky - spectra •It can work with different formats of spectra, but to work with them is hard and slow (computationaly) –Conversion of spectra into *.ucsf format •Unix: bruk2ucsf 2rr name_of_the_spectrum.ucsf •Windows: on the command line bruk2ucsf.exe 2rr name_of_the_spectrum.ucsf –Spectrum does not contain any information about acquisition, temperature and so on → it is necessary to give the *.uscf file proper name (colchicine_noesy_800_D2O_25C.ucsf) •It has its own structure of folders –Lists, Projects, Save •Sparky start –Windows: /sparky/bin/sparky.bat Prikaz konverzie: bruk2ucsf -> nazov programu, 2rr -> subor s 2D spektrom (obe dimenzie su realne) Sparky - start •Windows: batch file – sparky.bat • main window changing the mode of the left mouse button by using F1-F12 keys File open/save spectrum or project main window changing the mode of the left mouse button by using F1-F12 keys Sparky - control •Control by using –Two letter shortcuts (case-sensitive) –Menu in the main window ·*.ucsf file - spectral data from a NMR exp ·*.save file – Sparky format (plain text) contains all settings, assignment ·*.proj file – Sparky project file contains link to multiple *save files •Open spectrum/project: fo/jo •Save spectrum/project as: fa/ja •Save spectrum/project: fs/js •Backup files*.bak are created automatically during the saving of the spectrum Takto to vyzera, ked sa otvori nove spektrum, neulozene zf – full view zf – odzoomovanie na full view vC – contour scale ct – contour levels Uprava kontur vC alebo ct adjustment of the contours: manually overwrite the values change by dragging the left mouse button Lowest positive Levels = 1 Lowest positive Levels = 3 Lowest positive Levels = 2 adjustment of the contours: 1D 2D zooming the spectrum: keyboard shortcut: zi (zoom in) preset zoom in alternatively use of middle mouse bottom pointer mode– zoom: key F11 selected region is zoomed by clicking and dragging of the mouse PEAK PICKING: pointer mode – find/add peak: key F8 one peak is added by one left click, peaks in every local maximum/minimum are added by selecting the rectangle region peak picking: changing the size of ornaments (peaks, labels...): ot → sizes → change the size as desirable (proper value 0.05) → apply PEAK SELECTION selection: pointer mode – select: key F1 one object is selected by clicking on it, more object are selected by dragging the mouse (or Shift+click) peak picking: adding of central peak from the selected group of peaks – pg (useful for multiplets in COSY spectra) Pooznačovať všetky relevantné píky (krospíky len na jednej polovici a diagonálne píky dokopy 16ks) peak picking: list of the peaks – lt Pooznačovať všetky relevantné píky (krospíky len na jednej polovici a diagonálne píky dokopy 16ks) identification of the spin systems: add vertical and horizontal line: key F3 add horizontal line: key F4 add vertical line: key F5 add a vector: key F7 F3 F5 F7 F4 identification of the spin systems: changing the color of ornaments (lines, labels...): ot → color → select object/s→ choose the color → apply identification of the spin systems: COSY – correlation through bonds, isolated spin systems Nájsť si spinový systém, odhadnút počet očakávaných signálov a skorelovať to so spektrom identification of the spin systems: COSY – correlation through bonds, isolated spin systems expected number of signals: Gly – 2: Hα, NH Cys – 4: Hα, NH, Hβ‘, Hβ‘‘ Glu – 3: Hα, Hβ, Hγ Nájsť si spinový systém, odhadnút počet očakávaných signálov a skorelovať to so spektrom (PREMYSLIEŤ POČET OČAKÁVANÝCH SIGNÁLOV) identification of the spin systems: COSY – correlation through bonds, isolated spin systems expected number of signals: Gly – 2: Hα, NH Cys – 4: Hα, NH, Hβ‘, Hβ‘‘ Glu – 3: Hα, Hβ, Hγ Priradiť nájdené spinové systémy k reziduám podľa odhadu počtu signálov Signal assignment: select the peak: pointer mode – select: key F1 → shortcut at – assignment → fill in the name of the group (e.g. abbreviation of residuum/molecule) and the name of the atom (eg. NH) according to the resonance in w1 and w2 axis → type apply Signal assignment: the assignment with the closest resonance to the current selected resonance is highlighted when clicking on the name of the axis. (not necessarily correct one!!), double click on the highlighted line adds the name of the group and atom label to currently selected peak. Use unique naming, otherwise resonances will be averaged. Display position of resonance by vR command (view Resonances). Dopriradzovať samostatne!!! signal assignment: shortcut tb – assignment table residues vs. atoms, statistical data can be shown as well shortcut lt – list of the peaks already with assignments, or with other information (→ Options) ! Always check SDev column for consistency of the assignment both can be exported as a plain text file (→ Save) Working with more spectra: Assignment made in on spectrum is available/shared also for other spectra, peak assignment can be completed using different experiments on condition that experiments were recorded at the same temperature, identical sample etc. During work you may save separately the spectra (file save – experiments of active window) and subsequently save the entire project working with more spectra: view assigned (averaged) resonances on the edge of the spectra - vR working with more spectra: assign ROESY spectrum by using at: Opíkovat ROESY a popriradzovať (ideálne pomocou at) working with more spectra: synchronize views of spectra: shortcut yt – it allows simultaniously move synchronized axes of spectra when moving one of them, click on the axes in spectra we want synchronize → Synchronize