2004 NMR User Training Course High-Field Biomacromolecular Solution NMR Core Facility National Research Program for Genomic Medicine Date: September 06, 2004 (Monday) Place: B1C Lecture Room, IBMS, Academia Sinica, Taipei Title: Basic NMR Operation for Beginners 09:00-09:50 Lecture 1: Basic NMR Concept and Facility Overview (Dr. Chi-Fon Chang, Facility Manager) 10:00-10:50 Lecture 2: Steps for NMR Experiments small molecule (organics) (Dr. Casper Wu, Rezwave Application Scientist ) 11:00-11:50 Lecture 3: Steps for NMR Experiments larger molecule (biomolecules) (Dr. Wen-Jin Wu, Facility Staff Scientist) 13:00-13:30 Data Collection using Xwinmr (Dr. Chi-Fon Chang ) 13:30-14:00 Brief Introduction on Xwinplot (Dr. Casper Wu ) 14:00 --- Hands On -- B2, IBMS or B1, CHEM (Tsun-Ai Yu, Pei-Ju Fang, Wen-Jin Wu, Casper Wu, Chi-Fon Chang) 1
NMR Core Facility Overview Chi-Fon Chang 09/06/2004 2
Technician Director Operator Manager Operator Staff Scientist Secretary 3
NMR Room 4
500MHz with CryoProbe 600MHz with CryoProbe Computer Control 600MHz in 5
not available yet NMR System Location Remark 1 500 MHz 3 channels IBMS Upgraded since (Bruker AV) TXI probe Dec. 2002 Cryo probe Feb. 2004 2 600MHz 3 channels (Bruker DRX) TXI & other probes IBMS Available since Aug.2002 3 600MHz 3 channels CHEM Available since (Bruker AV) BBO & TXI probes Dec. 2002 TXI probe 4 600MHz 4 channels IBMS Available since (Bruker AV) QXI probe Jan. 2003 Cryo Probe March 2004 5 800MHz 4 channels IBMS Available since (Bruker AV) TXI Probe Cryo Probe July 2004 Dec. 2004 6
Service Items : : -- (Users Training Courses) --Advance NMR Workshop ( ) 7
NMR data processing software XWINNMR (process NMR data on IRIX 6.X & Linux ) nmrpipe (process NMR data on IRIX6.X & Linux) NMR data analysis software AURELIA (analyze NMR data on IRIX 6.X & Linux ) nmrdraw (analyze NMR data on IRIX 6.X & Linux ) nmrview (analyze NMR data on IRIX 6.X & Linux ) Sparky (analyze NMR data on Linux) CARA (analyze NMR data on PC) Structure Calculation program and software CSI Chemical Shift Index (making consensus plot on IRIX) TALOS (dihedral angles prediction on IRIX 6.X & Linux ) XPLOR or CNS (structure calculation on IRIX & Linux) ARIA (auto NOE assign and structure calculation on IRIX6.X & Linux) CYANA (auto NOE assign and structure calculation on IRIX6.5 & Linux) 8
NMR Core Facility ---User Information-- 9
2004.07.01 Bruker 500SB Bruker 500SB Cryo probe Bruker 600US Bruker 600UB Cryo probe Bruker 800US2 Bruker 800US2 Cryo probe 45 60 60 90 200 265 1000 500MHz ( 10 20 ) 600MHz 800MHz ( 10 30 ) ( 10 45 ) 30 250 30 300 30 450, 30, 10
( ) (user) (core) (user & core) (user) (user) (core) 11
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Operators Workstation Private Files Server ( 480G Hard Disk ) DNS/NAT/Firewall/FTP Server LAN Public DNS/WEB/MAIL Server ( NMR core facility ; nmr.sinica.edu.tw ) 15
http://www.nmr.sinica.edu.tw/ 16
Basic NMR Concepts Chi-Fon Chang 09/06/2004 17
The problem the we want to solve What we really see What we want to see How? 18
How about Spectroscopy? 19
Spectroscopy 1nm 10 10 2 10 3 10 4 10 5 10 6 10 7 (the wave) X-ray UV/VIS Infrared Microwave (the transition) Electronic Transition Vibration Rotation Radio Frequency Nuclear (spectrometer) X-ray UV/VIS Infrared/Raman CD Fluorescence NMR 20
Nuclear Magnetic Resonance Spectrometer B 0 : B 1 : ( ) 21
Before using NMR What s N, M, and R? Properties of the Nucleus Nuclear spin Nuclear magnetic moments The Nucleus in a Magnetic Field Precession and the Larmor frequency Nuclear Zeeman effect & Boltzmann distribution When the Nucleus Meet the right Magnet Nuclear Magnetic Resonance 22
Properties of the Nucleus Nuclear spin Nuclear spin is the total nuclear angular momentum quantum number. This is characterized by a quantum number I, which may be integral, half-integral or 0. Only nuclei with spin number I 0 can absorb/emit electromagnetic radiation. The magnetic quantum number m I has values of I, -I+1,..+I. ( e.g. for I=3/2, m I =-3/2, -1/2, 1/2, 3/2 ) 1. A nucleus with an even mass A and even charge Z nuclear spin I is zero Example: 12 C, 16 O, 32 S No NMR signal 2. A nucleus with an even mass A and odd charge Z integer value I Example: 2 H, 10 B, 14 N NMR detectable 3. A nucleus with odd mass A I=n/2, where n is an odd integer Example: 1 H, 13 C, 15 N, 31 P NMR detectable 23
Nuclear magnetic moments Magnetic moment µ is another important parameter for a nuclei µ = γ I (h/2π) I: spin number h: Plank constant 6.626*10-34 joul-sec γ: gyromagnetic ratio (property of a nuclei) 1H: I=1/2, γ = 267.512 *10 6 rad T -1 sec -1 13C: I=1/2, γ = 67.264*10 6 15N: I=1/2, γ = 27.107*10 6 Precession and the Larmor frequency The magnetic moment of a spinning nucleus processes with a characteristic angular frequency called the Larmor frequency ω, which is a function of r and B 0 Larmor frequency ω=rb 0 Linear precession frequency υ =ω/2π= rb 0 /2π Example: At what field strength do 1 H process at a frequency of 600.13MHz? What would be the process frequency for 13 C at the same field? 24
The Nucleus in a Magnetic Field B 0 B 0 ( the magnet of machine) (1) Provide energy for the nuclei to spin E i =-m i B 0 (rh/2π) Larmor precession υ =ω/2π= rb 0 /2π (2) Induce energy level separation (Zeeman effect & Boltzmann distribution) The stronger the magnetic field B 0, the greater separation P m=-1/2 / P m=+1/2 = e - E/kT m=-1/2 m=1/2 (3) The nuclei in both spin states are randomly oriented around the z axis. M z =M 0, M xy =0 ( where M 0 is the net nuclear magnetization) 25
When the Nucleus Meet the right Magnet: N. M. Resonance B 1 (the irradiation magnet, current induced) (1) Induce energy for nuclei to absorb, but still spin at ω or υ precession E induced = E=rhB 0 /2π=hυ precession E =E induced And now, the spin jump to the higher energy ( from m=1/2 m= 1/2) m= 1/2 (2) All of the individual nuclear magnetic moments become phase coherent, and the net M process around the z axis at a angel α M z =Mcosα M xy =Msinα. m= 1/2 26
What happen during irradiation When irradiation begins, all of the individual nuclear magnetic moments become phase coherent, and this phase coherence forces the net magnetization vector M 0 to process around the z axis. As such, M has a component in the x, y plan, M xy =Msinα. α is the tip angle which is determined by the power and duration of the electromagnetic irradiation. z α M o x x ω o B 1 y y M xy α deg pulse 90 deg pulse Hint: that s why we need to calibrate 90 o pulse!! 27
What happen after irradiation ceases After irradiation ceases, not only do the population of the states revert to a Boltzmann distribution, but also the individual nuclear magnetic moments begin to lose their phase coherence and return to a random arrangement around the z axis. (NMR!!) This process is called relaxation process ( ) There are two types of relaxation process : - T1(spin-lattice relaxation) -T2(spin-spin relaxation) 28
T1 (the spin lattice relaxation) How long after immersion in a external field does it take for a collection of nuclei to reach Boltzmann distribution is controlled by T1, the spin lattice relaxation time. ( ) Lost of energy in system to surrounding (lattice) as heat ( ) It s a time dependence exponential decay process of Mz components dm z /dt=-(m z -M z,eq )/T1 29
T2 (the spin spin relaxation) This process for nuclei begin to lose their phase coherence and return to a random arrangement around the z axis is called spin-spin relaxation. ( random ) The decay of M xy is at a rate controlled by the spin-spin relaxation time T2. dm x /dt=-m x /T2 dm y /dt=-m y /T2 de-phasing 30
Collecting NMR signals The detection of NMR signal is on the xy plane. The oscillation of Mxy generate a current in a coil, which is the NMR signal. m= 1/2 E =hv pression m= 1/2 Due to the relaxation process, the time dependent spectrum of nuclei can be obtained. This time dependent spectrum is called free induction decay (FID) M xy time ( relaxation ) ( T1 &T2) 31
NMR Data Processing The FID (free induction decay) is then Fourier transform to frequency domain to obtain υ pression ( chemical shift) for each different nuclei. m= 1/2 E =hv pression m= 1/2 0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 t1 sec Time (sec) Frequency (Hz) 32
Basic of NMR signal assignment It s easy to understand that different nucleus type will give different NMR signal. ( v =w/2π= γb0/2π, γ: gyromagnetic ratio is the property of a nuclei.) However, it is very important to know that for same nucleus type, but different nucleus could generate different signal. This is also what make NMR useful and interesting. < At 14.7 Tesla > 600MHz is Our spinning rate H H C like to process around 150MHz N is the slower dancer at 60MHZ H H H 13 CO NH 13 CH 4 N 33
Basic of NMR signal assignment Electron surrounding each nucleus in a molecule serves to shield that nucleus from the applied magnetic field. This shielding effect cause different v in the spectrum B eff =B 0 -B i B i = σb 0 where B i induced by cloud electron where σ is the shielding constant B eff =(1-σ) B 0 v precession = (rb 0 /2π) (1-σ) σ =0 σ >0 σ <0 naked nuclei nuclei is shielded by electron cloud electron around this nuclei is withdraw, i.e. deshielded 34
Chemical Shift The chemical shift of a nucleus is the difference between the resonance frequency of the nucleus and a standard, relative to the standard. This quantity is reported in ppm and given the symbol delta, δ = (ν - ν REF ) x10 6 / ν REF In 1H NMR spectroscopy, this standard is often tetramethylsilane, Si(CH 3 ) 4, abbreviated TMS, or 2,2-dimethyl-2-silapentane-5-sulfonate, DSS, in biomolecular NMR. The good thing is that since it is a relative scale, the δ for a sample in a 100 MHz magnet (2.35 T) is the same as that obtained in a 600 MHz magnet (14.1 T). Deshielded (low field) 15 Acids Aldehydes Aromatics Amides Olefins 10 7 5 Alcohols, protons α to ketones 2 Aliphatic 0 TMS ppm Shielded (up field) 35
J-coupling Nuclei which are close to one another could cause an influence on each other's effective magnetic field. If the distance between non-equivalent nuclei is less than or equal to three bond lengths, this effect is observable. This is called spin-spin coupling or J coupling. 1 H 1 H 1 H 13 C one-bond three-bond Each spin now seems to has two energy sub-levels depending on the state of the spin it is coupled to: αβ I S ββ S I αα βα J (Hz) I S The magnitude of the separation is called coupling constant (J) and has units of Hz. 36
Basic of Assignment v precession = (rb 0 /2π) (1-σ) = v 0, precession (1-σ) σ =0 σ >0 σ <0 naked nuclei nuclei is shielded by electron cloud electron around this nuclei is withdraw, i.e. deshielded HO-CH 2 -CH 3 low field ω o frequency high field ppm*10-6 = ν/ν0 = σ 37
Steps for NMR Experiment 38
Introduction of NMR Experiments Homo Nuclear 1D NMR 1D one pulse 1H Aromatic & Amide Aliphatic R1 R2 N Cα CΟ N Cα CΟ.. H H H H 39
Homo/Hetro Nuclear 2D NMR Basic 1D Experiment Basic 2D Experiment 40
2D Homo Nuclear 1H-1H Aliphatic R1 R2 N Cα CΟ N Cα CΟ.. H H H H 41
2D Hetero Nuclear 1H-15N 1H 15N Aromatic & Amide R1 R2 N Cα CΟ N Cα CΟ.. H H H H 42
Multi-Dimensional NMR Basic 3D Experiment 2D 2D 3D f2 t1 t2 t3 f2 f1 f3 f1 43
3D Hetero Nuclear 1H-15N-1H 1H 1H 1H 15N R1 R2 N Cα CΟ N Cα CΟ.. H H H H 44
15N 3D 15N-edit TOCSY-HSQC 1H-all 1H-NH 45
15N 3D CBCA(CO)NH 13Ca & Cb 1H-NH 46
Thank you!! cfchang@ibms.sinica.edu.tw Some figures are copy from : Joseph P. Hornak, Ph.D. http://www.cis.rit.edu/htbooks/nmr/ 47