Carlos A. Amezcua

NMRVIEW: AdvancedTopics in Protein NMRAnalysis

Carlos AmezcuaUniversity of Texas

Southwestern Medical Center at Dallas

Carlos A. Amezcua

Presentation Outline

n Introduction to STAR Files

n Backbone Assignments: CBCA Window

n Chemical Shift Index Analysis

n Titration and Screening Analysis

n Rate Analysis

Carlos A. Amezcua

STAR FILES

Carlos A. Amezcua

STAR Files: NMRView’s “Notebook”

n STAR (Self-defining Text Archive and Retrieval) files:universal archive files suitable for archiving all types oftext and numerical data in any order.

n The ASCII format allows them to be easily modified byany text editor.

n Currently used by BioMagResBank to store data frombiomolecular NMR spectroscopy (chemical shifts,coupling constants, dihedral angles, etc.).

Carlos A. Amezcua

Common Information Stored byNMRView’s STAR Files

n Molecule’s Data

n Sequence

n Chemical ShiftAssignments

n Peak lists

n 3D Structures

•NMRView is able to keepchemical shifts tables andpeak lists as individual files.

•Keeping a STAR file makesit easy to deposit data intoBMRB. NMRView can alsoread BMRB STAR files.

Carlos A. Amezcua

Format of NMRView’s STAR Files

data_defaultsave_hpask_Saveframe_category monomeric_polymer_Mol_type polymer_Mol_polymer_class protein_Name_common @_Name_variant @_Abbreviation_common @_Name_CAS @_CAS_registry_number @_Enzyme_commission_number @_Molecular_mass 12646.7_Details @_Sequence_citation_label @_Ambiguous_sequence_features @_Residue_count 114_Mol_residue_sequence @_Template_source seq_Template_filename hpask.seq

Carlos A. Amezcua

loop__Residue_seq_code_Residue_author_seq_code_Insertion_code_Residue_label_Segment_definition_code

1 1 . GLY .2 2 . ALA .3 3 . MET ....111 111 . PRO .112 112 . VAL .113 113 . GLU .114 114 . ARG .

stop_

save_save_Peak_Lists_Saveframe_category peak_lists

loop__Peaklist_name_Peaklist_label hnco 'H1 C13 N15 ' hncacb 'H1 C13 N15 ' cbcaconh 'H1 C13 N15 ' cconnh 'H1 C13 N15 ' hcconnh 'H1 H1b N15 ' hsqc 'H1 N15 ' hcchtocsy 'H1 H1b C13 ' noesy-hsqc15N 'H1 H1b N15 ’

stop_

Repetitive information is stored inthe form of loops

Carlos A. Amezcua

save_save_spectral_peak_list_hnco_Saveframe_category spectral_peak_list_Details ' @'_Software_label @_Experiment_label @_Number_of_spectral_dimensions @_Sample_label @_Sample_conditions_label @_Text_data_format @_Text_data ' @ '_Peaklist_dataset simnoe_Peaklist_scale 1.000000loop_ _Peaklist_label _Peaklist_dim _Peaklist_sw _Peaklist_sf _Peaklist_pattern _Peaklist_relation _Peaklist_idtol

H1 0 4738.21 599.6700 i.hn . 0.0500C13 1 1210.00 150.8120 i-1.c . 0.3000N15 2 1600.00 60.7710 i.n . 0.3000stop_

loop_ _Peak_list_number _Intensity_height _Intensity_volume _Intensity_volume2 _Peak_status _flag _comment _Dim_1_label _Dim_1_chem_shift _Dim_1_line_width _Dim_1_bounds _Dim_1_error _Dim_1_j _Dim_1_link0 _Dim_1_link1 _Dim_1_thread _Dim_1_user _Dim_2_label . . _Dim_3_label . . 0 22.102 0.000 0.000 0 2 {?}{22.HN} 9.402 0.135 0.104 {++} 0.000 0 0 0 {?}{21.C} 175.703 0.198 0.178 {++} 0.000 0 0 0 {?}{22.N} 130.096 0.288 1.031 {++} 0.000 0 0 0 {?} . . .stop_

Peak lists are conveniently storedin the STAR file

Carlos A. Amezcua

How to start a STAR File?

n Load your sequence:

n Save the STAR File

Carlos A. Amezcua

The CBCA Window

A semi-automated tool forbackbone assignments

Carlos A. Amezcua

Triple Resonance Experiments forBackbone Assignment

CBCA(CO)NH HNCACB

i-1 i

i-1 i 1H (ppm)

13C

(ppm

)

Res i-1, Cb

Res i-1, Ca

Res i, Cb

Res i, Ca

Carlos A. Amezcua

Assignment overview

n Match “clusters”: Linkclusters with common 13Cchemical shifts.

… Clust 4 --- Clust 22 --- Clust 50 …

n Match fragments intosequence:Search for unique chemicalshift patterns (e.g. 13Ca/13Cb)

K T TClust 4 Clust 22 Clust 50

Cluster Peaks 1 A1,A2,B40,B41,B42,B43 2 A8,A9,B25,B26,B27,B28 3 . 4 .

n Generate “clusters”:Cluster peaks from multipleexperiments using common15N/1H shifts.

Carlos A. Amezcua

The CBCA Analysis Step by Stepn Setup CBCA analysis preferencesn Clean and verify peaksn Filter peaks to eliminate artifactsn Cluster peaks based on common HN and N frequenciesn Verify and adjust peak clustersn Link clusters based on common CB and CA frequenciesn Confirm best cluster linksn Transfer chemical shifts to database

Under Developmentn Form fragments which are a series of overlapping clustersn Assign fragments to specific ranges of residues in sequence

Carlos A. Amezcua

Starting a CBCA Analysis

CBCA Window

Main Menu

Carlos A. Amezcua

Setting up Preferences

Carlos A. Amezcua

Examining the peaks13

C (p

pm)

15N (ppm) 15N (ppm)1H (ppm) 1H (ppm)

CBCA(CO)NH HNCACB HNCACBCBCA(CO)NH

Peak No.

Carlos A. Amezcua

Example of Multiple Rows

HNCA HNCAHN(CO)CA HN(CO)CA

CBCA(CO)NH CBCA(CO)NH HNCACB HNCACB

15N (ppm) 15N (ppm)1H (ppm) 1H (ppm)

13C

(ppm

)13

C (p

pm)

Carlos A. Amezcua

Filtering and Clustering Peaks

•Peaks are clustered based oncommon HN and N frequencies.

•This algorithm creates a Tcl arraycalled “Clust” that isn’t saved intothe STAR file database. Important:Save as text file using File --> Save

•It is possible to filter peaks bycomparing shifts using referencespectra like 15N/1H-HSQC orHNCO. This process eliminatesartifacts.

Carlos A. Amezcua

Editing Clusters

•Peaks can be shifted from one cluster to another by superimposing the cursor on the desiredpeak and typing the hot key “s”.

•Remember to “Relink” after modifying the cluster and to SAVE OFTEN.

CBCA(CO)NHCBCA(CO)NH HNCACB HNCACB

13C

(ppm

)

15N (ppm) 15N (ppm)1H (ppm) 1H (ppm)

Cluster No.

Carlos A. Amezcua

Matching Clusters•First setup the appropriate “MatchScript”(MatchCA, MatchCBCA2 orMatchCBCA4) and “MatchLists” in thecbcaPrefs window. It is easy to writescripts for other experiments.

•Next, link the clusters based on commonCa and Cb frequencies.

Carlos A. Amezcua

Editing Matching Clusters

Ca/Cb (i-1)N/H (i)

13C

(ppm

)

1H (ppm) 1H (ppm)1H (ppm)1H (ppm)

Ca/Cb (i-1, i)N/H (i)

Ca/Cb (i-2,i-1)N/H (i-1)

Ca/Cb (i)N/H (i+1)

Carlos A. Amezcua

The cbca Control Panel

MatchingCluster

MatchingScore (0-3)

Confirmed: 1Not confirmed: 0

No. of MatchingCarbons

Best match formatching cluster

ConfirmedCluster No.

(not confirmed: -1)

Carlos A. Amezcua

Matching Clusters to Sequence

Sequence: . . . A139 K140 T141 T142 E143 . . .Cluster No.: …, 121, 104, 38, …

T142T142T141 E143 AA CA CBALA 53.0 18.8SER 58.5 63.8THR 61.9 69.5LYS 56.8 32.8GLU 57.4 30.0GLY 45.2 ----

Chemical ShiftTables (BMRB)

Carlos A. Amezcua

Transfer of Chemical Shifts tothe Assign Panel

•Using the identified cluster/residue pairs,transfer the chemical shift information to theassign panel.

•This process can be done manually orautomatically through home made macros.

Carlos A. Amezcua

Secondary StructureInformation from BackboneAssignments

The Chemical Shift Index

Carlos A. Amezcua

Sequence Display

Sequence Display Window

Main Menu

Carlos A. Amezcua

Secondary Structure Informationfrom Backbone Chemical Shifts

Carlos A. Amezcua

Graphical Display of SecondaryStructure Elements on Sequence

Carlos A. Amezcua

Titration and ScreeningWindows

For these and other Tcl scripts check:

http://freedom7.swmed.edu/NMRview/index.html

Carlos A. Amezcua

Titration Experiments

• This 15N/1H-HSQC experimentshows a titration of a smallmolecule into a protein solution.

• A computer assisted method isneeded to follow the movingpeaks, record their chemicalshifts and obtain an average KD.

1H (ppm)

15N

(ppm

)

Carlos A. Amezcua

The Titration AnalysisWindow

n Automatically identifies and links shifting peaksof an HSQC titration series.

n Permits manual editing of linked peaks.

n Calculates equilibrium dissociation constants (KD)through an interface with GRACE.

n Written by Brad Holmes and Dr. Kevin Gardner.

Carlos A. Amezcua

A Typical Titration Session

• Load datasets and reference peak list

• Open the titration window

Carlos A. Amezcua

•Select the reference dataset: “Dataset for enumeration”

•Click on “Draw” and adjust the contour level

•Place a cursor box around the desired spectral region and clickon “PickAll”

Carlos A. Amezcua

• NMRView automatically pick peaks and generates peak lists forevery dataset.

Carlos A. Amezcua

• To link the shifting peaks click on the “Link Peaks” button

Carlos A. Amezcua

•A very useful tool to keep track of where the peaks are movingto can be accessed with the “DrawBoxes’ button.

Carlos A. Amezcua

Modifying Linked Peaks

• Position the cursor on top of the desired peak and click the“Find” button under the corresponding peak list.

Carlos A. Amezcua

• After selecting peaks for the new vector, click on “DrawBoxes”to see the new direction of the moving peak.

Carlos A. Amezcua

KD Determination

• After inspecting the peaks, fill in the ligand concentration fora given dataset, the protein concentration and click on“Calculate KDs”.

Carlos A. Amezcua

Interface to Grace for Data Fitting•The interface with GRACE calculates a fit for every shiftingpeak above the threshold level and outputs an averaged KD inthe NMRView Tk Console.

Fitting Equation:

Dd = Ddmax *{(L+PT+Kd)- [(L+PT+Kd)2-(4*L*PT)]1/2}

[2*PT]L = Ligand concentrationPT = Total ligand concentrationKd = Equilibrium dissociation constantDd = Chemical shift change at ligand concentration LDdmax = Chemical shift change at saturation

Carlos A. Amezcua

Tools for Ligand Discovery

Carlos A. Amezcua

Screening Generates Lots of Data

Carlos A. Amezcua

Dataset Sum Average Max Peaks Gamma ratio: 0.101example-103 5.458 0.012 0.139 445example-140 5.157 0.013 0.128 396example-54 5.009 0.005 0.146 951example-113 4.857 0.010 0.120 488example-26 4.770 0.010 0.134 477example-129 4.757 0.009 0.123 521example-148 4.620 0.008 0.130 561example-119 4.593 0.009 0.106 512example-135 4.422 0.008 0.142 533example-23 4.404 0.011 0.135 383example-61 4.402 0.005 0.118 826example-99 4.330 0.005 0.112 956

Output File: Ranking of spectra by sum of minimum chemical shifts

The Screening Window

•This new tool allows to rapidlypeak pick many spectra andcalculates minimum chemicalshifts (mcs) with respect to areference.

•mcs = sqrt{(dref-d)2H + [(dref-d)

*(gHET/gH)]2HET}

Carlos A. Amezcua

Rate Analysis

Carlos A. Amezcua

Typical Experiments

n Relaxation experiments: T1, T2

n Chemical exchange: N-H ---> N-D

n Slow conformational changes

Carlos A. Amezcua

The Rate Analysis Window•Open datasets and read the peak list

•Type the “Matrix Root”, “Matrix Extension”, Noise “SDev”, and select the control “Peaklist”

•Click on “Setup” and select the file with the “Extensions and Delays”

Carlos A. Amezcua

Selecting Fitting Parameters

Carlos A. Amezcua

Fitting Results

Carlos A. Amezcua

Output files

filename.txtPeak No. Residue Average Value Maximum Minimum Std. Dev.relax.0 213.HN 628.234152 640.609113 615.592839 0.322123relax.1 230.HN 627.522038 640.356408 616.455145 0.480259relax.2 137.HN 640.591471 651.978668 629.362775 0.437270relax.3 145.HN 639.115880 657.254495 624.447290 0.233065relax.4 135.HN 657.214250 675.046681 641.816510 0.310350relax.5 211.HN 699.213025 711.432576 689.888055 0.492043

filename.datRes T1(s) Err(max) Err(min)213 0.628234152 0.012641313 0.012374961230 0.627522038 0.011066893 0.01283437137 0.640591471 0.011228696 0.011387197145 0.63911588 0.01466859 0.018138615135 0.65721425 0.01539774 0.017832431211 0.699213025 0.00932497 0.012219551

• File in GRACE format

• GRACE plot

Carlos A. Amezcua

HetNOE Analysis andWrite Modelfree

Carlos A. Amezcua

Heteronuclear NOE Analysis

Output FilePeak Res NOE Errorhsqc.0 90.HN 0.844579727153 0.0220038139944hsqc.1 107.HN 0.765441512887 0.0228763153881hsqc.2 14.HN 0.65637966805 0.0194502074689hsqc.3 22.HN 0.757088658147 0.0299520766773hsqc.4 12.HN 0.749387683695 0.0262421273618hsqc.5 88.HN 0.720154882832 0.0150859901438

Carlos A. Amezcua

MODELFREEInterface

•This window helps to generate inputfiles to be used with Art Palmer’sModelfree4 program (for 15N-relaxationanalysis).(http://cpmcnet.columbia.edu/dept/gsas/biochem/labs/palmer/software.html)

•Requires T1and T2 files generatedthrough the Rate Analysis window andNOE files generated with the HetNOEAnalysis window.

Carlos A. Amezcua

Good Luck andHappy Analysis

Acknowledgements:ÿKevin Gardner

ÿBrad Holmes

ÿJohn Shriver

ÿRenee Valentine