Pure & Appl. Chem., Vol. 70, No. 1, pp. 117-142, 1998.
Printed in Great Britain
 1998 IUPAC

INTERNATIONAL UNION OF PURE
AND APPLIED CHEMISTRY
IUPAC-IUBMB-IUPAB INTER-UNION TASK GROUP ON THE
STANDARDIZATION OF DATA BASES OF PROTEIN AND NUCLEIC ACID
STRUCTURES DETERMINED BY NMR SPECTROSCOPY

RECOMMENDATIONS FOR THE
PRESENTATION OF NMR STRUCTURES OFPROTEINS AND NUCLEIC ACIDS(IUPAC Recommendations 1998)

Prepared for publication by

John L. Markley,a Ad Bax,b Yoji Arata,c C. W. Hilbers,d Robert Kaptein,e Brian D.
Sykes, f Peter E. Wright,g and Kurt Wüthrich haDepartment of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA;bLaboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda,

MD, USA; cWater Research Institute, Tsukuba, Japan; dLaboratory of Biophysical
Chemistry, University of Nijmegen, The Netherlands; eDepartment of Chemistry, University of Utrecht, The Netherlands; fDepartment of Biochemistry, University of Alberta, Edmonton, Alberta, Canada, gDepartment of Molecular Biology, The Scripps
Research Institute, La Jolla, CA, USA; hInstitut für Molekularbiologie und Biophysik,
ETH-Hönggerberg, Zürich, Switzerland (convenor of the task group to whom correspondence should be addressed).

Republication or reproduction of this report or its storage and/or dissemination by electronic means is permitted
without the need for formal IUPAC permission on condition that an acknowledgement, with full reference to thesource along with use of the copyright symbol , the name IUPAC and the year of publication are prominently
visible. Publication of a translation into another language is subject to the additional condition of prior approval
from the relevant IUPAC National Adhering Organization.

117

Recommendations for the Presentation of NMR
Structures of Proteins and Nucleic Acids(Recommendations 1998)
IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of
Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy
John L. Markley,a Ad Bax,b Yoji Arata,c C. W. Hilbers,d Robert Kaptein,e Brian D.
Sykes, f Peter E. Wright,g and Kurt Wüthrich haDepartment of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA;bLaboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda,

MD, USA; cWater Research Institute, Tsukuba, Japan; dLaboratory of Biophysical
Chemistry, University of Nijmegen, The Netherlands; eDepartment of Chemistry, University of Utrecht, The Netherlands; fDepartment of Biochemistry, University of Alberta, Edmonton, Alberta, Canada, gDepartment of Molecular Biology, The Scripps
Research Institute, La Jolla, CA, USA; hInstitut für Molekularbiologie und Biophysik,
ETH-Hönggerberg, Zürich, Switzerland (convenor of the task group to whom correspondence should be addressed).
Abstract: The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids throughunified nomenclature and reporting standards. Much of this document pertains to thereporting of data in journal articles; however, in the interest of the future developmentof structural biology, it is desirable that the bulk of the reported information be stored
in computer-accessible form and be freely accessible to the scientific community instandardized formats for data exchange. These recommendations stem from an
IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and
CODATA. The Task Group has reviewed previous formal recommendations and has
extended them in the light of more recent developments in the field of biomolecular
NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through tworounds of extensive modification to incorporate suggestions and criticisms.

TABLE OF CONTENTS
Page…………………………………………………………………………………. 119
Introduction
1. Definition of the system studied …………………………………………………………... 120
1.1.
Names of the molecules used ………………………………………………………. 120…………………………………………………………………………..120
1.2. Source……………………………. 120
1.3. Evidence for homogeneity and chemical identity…………………………………………………...
120
1.4. Sequence or chemical structure…………………………………………………………………..
120
1.5. Solution conditions…………………………………… 121
1.6. Isotope-labeled proteins and nucleic acids……………………………. 121
2. Atom identifiers for reporting chemical shift assignments…………………………………………………………...121
2.1. Proteins and peptides…………………………………………………... 121
2.1.1. Standard nomenclature…………………………………… 123
2.1.2. Notation for assignment ambiguities……………………………. 123
2.1.2.1.
Non-stereochemical ambiguity
2.1.2.2.
Stereochemical ambiguity involving prochiral centers ……...123
2.1.2.3.
Other stereochemical ambiguity ……………………………. 123…………………………………………………………………..
124
2.2. Nucleic acids…………………………………………………...
124
2.2.1. Standard nomenclature…………………………………… 124
2.2.2. Notation for assignment ambiguities

118

Additional atom identifiers for reporting conformational constraints and structures…….. 124…………………………………………………... 124
3.1. Pseudoatom nomenclature……………………..124
3.2. NOE constraints that can be identified by chemical shifts……………………………………………
125
4. Nomenclature for specifying conformation…………………………………………………………………..
126
4.1. Polypeptides……………………………. 126
4.1.1. Backbone torsion angles: φi , ψi , and ωi……………………………………………
126
j
4.1.2. Side-chain torsion angles: χ i…………….. 126
4.1.3. Specification of turns, disulfide bonds, and proline rings…………………………………………………………...127
4.2. Nucleic acids
4.2.1. Backbone and glycosidic torsion angles …………………………………… 127…………….. 127
4.2.2. Sugar pucker: torsion angles and angle of pseudorotation
4.2.3. Value ranges for torsion angles specifying commonly observed conformations …128…………………………………… 129
5. NMR data acquisition, processing, and referencing…………………………………………………………………..
129
5.1. Data acquisition…………………………………………………………………..
130
5.2. Data processing…………………………………………………...
130
5.3. Referencing of chemical shifts1…………………………………………………………...130
5.3.1. H chemical shifts…………….. 130
5.3.2. Chemical shifts of other nuclei such as 13C, 15N, and 31P…………………………………………………………………..
131
6. Resonance assignments…………………………….
131
6.1. Sequence-specific assignment of polypeptide chains……………………………………
131
6.1.1. Sequential backbone assignment…………………………………… 131
6.1.2. Amino acid side-chain assignment
6.2. Sequence-specific assignment of nucleic acids …………………………………… 132……………………. 132
6.3. Stereospecific assignment of diastereotopic substituents
6.4. Conformational equilibria ………………………………………………………….. 132
7. Conformational features derived from diagnostic NMR parameters ……………………. 132
7.1. Polypeptide secondary structure …………………………………………………... 132………………………………………………………………….. 134
7.2. Polynucleotides
7.2.1. Mononucleosides ………………………………………………………….. 134
7.2.2. Backbone torsion angles ………………………………………………….. 134
7.2.3. Secondary structure ………………………………………………………….. 134…………………………… 135
8. Collection of input constraints and structure calculation…………………………………………..
135
8.1. Conformational constraints…………………………………..
135
8.1.1. Distance constraints from NOE data……………………………
135
8.1.2. Torsion angle constraints from J-coupling data……………………………
135
8.1.3. Torsion angle constraints from chemical shifts
8.1.4. Constraints representing disulfide bonds, hydrogen bonding,…………………………………………………………. 135
and metal coordination…………………... 135
8.1.5. Supplementary constraints for structure refinement
8.1.6. Input for the final structure calculation ………………………………….. 136
8.2. Methods used for structure calculation and refinement …………………………… 136………………………………………………….. 136
8.2.1. Structure calculation
8.2.2. Preliminary structures to assign additional constraints …………………… 136…………………………………………………. 136
8.2.3. Structure refinement
9. Reporting three-dimensional structures …………………………………………………. 137
9.1. Presentation of structures ………………………………………………………… 137
9.2. Agreement of structures with constraints …………………………………………. 137…………………………………………………………. 137
9.3. Precision of structures…………………………………………………………. 137
9.4. Validation of structures
10. Data bank deposition of NMR structures and supporting data …………………………… 138………………………………………………………………………………… 139
Conclusion………………………………………………………………………… 139
References and notes
3.

INTRODUCTION
Solution-state NMR spectroscopy is used widely to determine the structures of peptides, proteins, andprotein-ligand complexes, as well as those of nucleic acids and their complexes with proteins, drugs, andother molecules. As the field has developed, a certain consensus has evolved on the presentation of NMR
solution structures. This has been helped indirectly by guidelines established for depositing primary experimental data and resulting structures in data banks such as the Protein Data Bank (ref. 1), BioMagResBank(ref. 2), Nucleic Acid Database (ref. 3), and by conventions used for inclusion in abstracting services, forexample, Macromolecular Structures (ref. 4). In consideration of the accumulated experience over the past
few years in presenting NMR structures, the time appeared to be appropriate for a formal examination of119

reporting conventions used in the past and for the development of a set of generally accepted guidelines forthe future. With these goals in mind, the present Task Group was convened as an IUPAC/IUBMB/IUPAB
Inter-Union venture with financial support from ICSU and CODATA. The present recommendations build
upon earlier recommendations for biochemical nomenclature (ref. 5), the presentation of proton (ref. 6) andnon-proton (ref. 7) NMR data for publication, and parameters and symbols for use in NMR (ref. 8).
1.

Definition of the system studied
1.1.

Names of the molecules used

It is helpful to use a notation that specifies the molecule type, natural species of origin, and fragment (if applicable): for example, “fragment consisting of residues 10218 of murine Blk, a B-cell specific protein
tyrosine kinase (PTC).” Note that recommendations for the naming of a variety of biological macromolecules have been published (ref. 914). Authors are encouraged to include identifiers used by publicly accessible databases (ref. 15), as these may be helpful for cross-referencing purposes. Such identifiers include
the accession codes used by the Protein Identification Resource (ref. 16), SWISS-PROT (ref. 17), the Enzyme Commission of IUBMB (ref. 18), and the Chemical Abstracts Service (ref. 19).
1.2. Source•••

Genus, species, strain or variant of gene (cloned or synthetic).
Expression vector and host.
If chemically synthesized, a description of the methods used.
1.3. Evidence for homogeneity and chemical identity••

Confirmation of chemical identity (e.g., from mass spectrometry or chemical sequencing).
Analytical method used to establish chemical homogeneity.
1.4. Sequence or chemical structure

•
•
•
•
•
•

Full sequence of the molecule (or reference to it).
Unambiguous definition of the sequence numeration of the molecule studied.
Description of additional covalent linkages and their locations: e.g., disulfide bridges, covalently attached cofactors, or metal ions.
Description of any differences between the system studied and the naturally-occurring biomolecule (e.g.,fragment with additional or fewer residues at either end of the biopolymer, post-transcriptional modifications in nucleic acids or post-translational modifications in proteins, or lack thereof).
Specification of noncovalent cofactors or prosthetic groups.
Quaternary structure (number and kind of subunits; symmetry, if known).
1.5. Solution conditions

•
•
•
•
•
•

Specification of the solvent constituents and their isotopic compositions.
Concentration of each solute component, including buffers, salts, antibacterial agents, etc.
Temperature and pressure along with methods used for their measurement.
Value of the pH, or pH* for uncorrected pH meter readings in 2H2O. Note that p2H (or “pD”) applies
only to measurements made with electrodes filled with 2H2O.
Comment on special measures taken to minimize self-aggregation (if applicable).
Type of sample cell used.

120

1.6. Isotope-labeled proteins and nucleic acids
Essential information would include descriptions of the methods used to prepare the labeled molecules andto determine the positions and levels of isotopic labeling in the system used. Percent incorporation should
be indicated when known, as illustrated in the following examples.
•
•
•
•
•

2.

Uniform (random) labeling with 15N (extent of incorporation unknown): [U-15N]-ribonuclease.
Uniform (random) labeling with 13C, 15N, and 2H (labeling levels known): [U-98% 13C; U-90% 15N;
U-65% 2H]-d(GCGCAATTGCGC).
Residue-selective labeling: [98% 15N]-Cys rubredoxin.
Site specific labeling: [95% 13Cα]-Trp28 lysozyme.
Insertion of residues with natural isotopic composition into otherwise uniformly labeled proteins or nucleic acids are designated by NA for natural abundance: [U-98% 13C; NA-F,Y,W]-lysozyme.
Atom identifiers for reporting chemical shift assignments

The 1983 IUPAC-IUB recommendations for peptides and proteins (ref. 21) were never widely adopted by
the protein science community. Thus the present recommendations for peptides and proteins follow theearlier IUPAC-IUB “tentative rules” of 1969 (ref. 20) currently employed by biomolecular databases, withextensions and clarifications as required for NMR data. These rules designate all atoms by Greek letters (orRoman counterparts) and employ the main chain precedence rule for numbering prochiral sites. The present
recommendations for nucleic acids follow the IUPAC-IUB nomenclature (ref. 22), with three exceptions.
First, notation for the hydrogens at the C5′ position in ribose and deoxyribose follows the widespread use of
H5′ (for pro-S) and H5″ (for pro-R), rather than H5′1 and H5′2 (the latter notation (ref. 22) is inconsistent
with the numbering convention used with amino acids, in which such atoms would be designated as 2 and
3). Second, H2′ (for pro-S) and H2″ (for pro-R) are used to designate the hydrogens at the C2′-position in
deoxyribose. Third, the notation proposed for hydroxyl groups of ribose and deoxyribose rings in reference
22 is adapted from that used with amino acids, so that the hydrogen atoms are denoted by HO2′, HO3′, andHO5′, as appropriate, whereas the hydroxyl oxygens are denoted by O5′, etc.
In cases not covered by the present recommendations (e.g., unusual or modified amino acid residues or nucleic acid bases), authors are encouraged to refer to notation in use by the databases for such groups. If
new atom notation needs to be introduced, it will be helpful if authors define this by providing stereospecificdiagrams as appropriate.
2.1. Proteins and peptides
2.1.1. Standard nomenclature
Figure 1 defines the atom-naming conventions recommended here for proteins. Coordinate files should
contain all atoms, including hydrogen atoms. The 1969 recommendations (ref. 20) did not deal explicitlywith nomenclature for hydrogen atoms, and the rules are subject to different interpretations. The interpretation recommended here (Fig. 1) is that at any position of the side chain, the attached atom leading to themain chain always has the highest priority. For cyclic amino acids such as proline, the priority leads out
from the main chain Cα atom rather than from the N atom (i.e., the priority is given by Cα > Cβ > Cγ > ...
> N). In the peptide backbone, the nitrogen is denoted by N, its attached hydrogen by H, and the carbonyl
carbon and oxygen by C and O, respectively (ref. 20). In cases where the single letter designations are ambiguous, they may be primed (e.g., N′, C′, O′) (ref. 20); C′ is used quite widely to denote the peptide backbone carbonyl carbon. Although H alone is used by the biomolecular data banks (PDB and BMRB), it isrecommended here that the symbol HN (in widespread use in the NMR community) be used as the unambiguous designator for the backbone amide hydrogen. The hydrogens of an N-terminal amine are designatedas H1, H2, and H3 (when protonated) or H1 and H2 (when unprotonated). The oxygens of a C-terminal
carboxyl or carboxylate are O′ and O′′, and the hydrogen of the carboxyl is H′′. IUPAC rules (ref. 18) label
the hydrogens on methyl groups and protonated amines by “1”, “2”, and “3”, in the conventional way.

121

Thus, for example, the atom designations in amino acid side chains would be: Hβ1, Hβ2, and Hβ3 for thethree methyl hydrogens of alanine; Hζ1, Hζ2, and Hζ3 for the three amine hydrogens of a protonated lysine;and Hζ1 and Hζ2 for the two amine hydrogens of a neutral lysine.
In situations where Greek letters are unavailable (for example, on some computer displays), they may bereplaced by upper case Roman letters (α = A, β = B, γ = G, δ = D, ε = E, ζ = Z, η = H). When superscripts are not feasible, the full descriptor can be placed on a single line. When a full descriptor is used in a
subscript, its components are displayed on a single line: e.g., 3JH′Hα for a coupling constant or dHαHβ(commonly abbreviated dαβ) for a distance detected by a nuclear Overhauser effect (NOE). The computer
representation for ′is a single quote and for ′′ is two single quote symbols.
Hδ2

–

––

—

Nδ2 – Hδ22(Z)
Hδ21
(E)

Cβ

Hζ2

–

Hδ1

Nε1
Hε1

Hβ3
Cβ
Hβ2
(R)

Hε1

–

η2

Hδ1
Cγ

(R)

C–
– Cε2

Hδ2

Hε2

–

–

–

Cβ

Cζ – Oη – Hη

δ2

–

δ2

C–
– Cε2

Hδ2

Hε2

Val
Cγ2(Hγ2)3Cδ1– Cε1

––

–

–

––

–
–

(R)

ζ2

C –C

–

Hβ2 Cδ1

η2

C – H

ε2

Tyr

Cζ – Hζ

Cγ

–

Cγ

(R)

–

Cβ

Hβ2

(R)

–– –

C –
Cδ2

Cζ3

Cβ

–

ε3–

Sδ – Cε(Hε)3Hβ2 Hγ 3

(R)

Hζ3

Hε3

Cγ

––

Nζ(Hζ)3

Hε1

Cδ1– Cε1

–

+

Hδ1

–

Cε

Hβ3

–

(R)

–
– –

Cγ 2(Hγ 2)3

Cδ

Hε2

Hδ2

Phe

Hβ3 Hγ 2Hβ2 Hγ 3 Hδ2 Hε3

Hβ3

Hβ2

(R)

Hε1
δ1
N Cε1
Cγ +
δ2 ε2
C –N

––

Cγ

–

Hβ

Oε2 – Hε2

Hδ1

(R)

Cβ

Trp

Cβ

Cδ

Met

Cβ

Hγ

Hβ3

–

Hγ 3

(R)

O

– –

Hβ2

(R)

Hβ3 Hγ 2 Hδ3 Hε2Oγ1 – Hγ1Cγ(R)

Hε21(E)(R)

Hβ2 Cδ2(Hδ2)3

Cβ

(Z)

Cβ

His

ε1

–

(R)

H

Hγ 2

(E)

– ––

––

(R)

N

ε22

Hβ3 Cδ1(Hδ1)3Thr
Oγ – Hγ

ε2 –

Hβ3

Lys
Cγ

(Z)

–

– ––

Hγ 3

(R)

Hβ3

H

–

Cδ1(Hδ1)3

ε1

Cδ

Hβ2

(R)

Ser

C

β2

–

Hγ13

O

(R)

H

Hβ

Cγ

Leu

γ12

Cγ1

Cβ

S γ – Hγ

Hβ2

(R)

Cβ

C

Nη1 (Z)(R)Glu

Hβ3 Hγ 2–(H )3 C

N (E)Hη22 (E)

Oδ1
γ

–

(R)

–

Cβ

Oδ2 – Hδ2

γ2

Cζ

β

–

O

IIe

Hδ2

C

η2

Nε +

–

O

Gln

H

δ1

Hβ2

C

Hβ2 Hγ 3Hβ3Hη11 Hη12

β3

Cγ

δ

γ

H

–

O

(R)

Hβ3

β

C

(R)

Cys

Hβ2

C (H )3

––

––

H

(R)

Cβ

β

C

––

β2

γ2

C

C

Asp

Cβ

H

β

–

H

α

(R)

Hη21 (Z)

ε

–

C

α

Hδ3

–

α3

Hβ3 Hγ 2

–

Cβ
O

α

(R) H

C

–

α2

N

Asn
(R)

–

C

N

Hγ 3(R)

—

H

Cγ

Arg

—

α

α

H

–

Hβ3

Ala

H

–

(R) Hδ3
Hγ 2

Gly
—

–

Cδ
N

–

Pro

Hβ

Cγ1(Hγ1)3

Fig. 1. Recommended atom identifiers for the twenty common amino acids follow the 1969 IUPAC-IUB guidelines (ref 20).
Backbone atoms are shown for Pro, Gly, and Ala but not for the other L-amino acids (where they correspond to those boundedby the dashed line in the Ala structure). Greek letters are used as atom identifiers. The Cα or the substituent closer to Cα (inthe order Cα, Cβ, Cγ, . . . ) takes precedence over atoms in branches in defining stereochemical relationships. For example, if
tetrahedral carbon C has four substituents X, Y, Z, and Z′ (with priority X > Y > Z = Z′; i.e., Z and Z′ are diastereotopic substituents designated provisionally as unprimed and primed), their numbering is derived as follows: if one sights down theXC axis (with the X atom toward the viewer), the equivalent atoms, Z and Z′, are designated Z2 and Z3, such that Y, Z2, andZ3 follow a clockwise orientation. The side-chain -NH2 nitrogens of Arg are designated as Nη1 and Nη2 by their relationshipδ(cis or trans, respectively) to C . The hydrogen atoms of the side-chain -NH2 groups of Asn, Gln, and Arg are distinguishedby numbers (1βor 2) on theγ basis of theirrelationship (cis or trans, respectively) to the heavy atom three bonds closer to theεmain chain (C for Asn, C for Gln, N for Arg). Thus, each -NH2 hydrogen of Arg is distinguished by two numbers, the firstindicating the nitrogen to which it is attached and the second indicating the stereochemistry of the hydrogen itself. Numberingof Phe and Tyr rings gives higher priority to the atom with the smaller absolute value of the χ2 torsion angle (ref. 20). For
example, the ring carbons of Phe and Tyr lying in the plane with the smaller χ2 torsion angle are designated as Cδ1 and Cε1.
Indicated for reference in parentheses are the pro-R/pro-S designations for prochiral tetrahedral groups (with only the pro-R
indicated as "R") (ref. 23, 24) and the E/Z designations for planar groups (ref. 27, 28).

122

2.1.2. Notation for assignment ambiguities
It is important to distinguish clearly between resonances that have complete stereochemical assignments andthose that do not. Resonances with complete stereospecific assignments are denoted by the symbols shownin Fig. 1. Resonances having ambiguous assignments are denoted by special symbols, as defined below.
Knowledge about particular kinds of assignment ambiguity (stereospecific and non-stereospecific) can beimportant in structure determinations. Such information defines, for example, what kind of pseudoatoms(see 3.1. below) need to be invoked in a structure determination.
2.1.2.1.

Non-stereochemical ambiguity

A slash (/) should be used to indicate ambiguity in assignments. The slash is used to join the ambiguous
atom designators and chemical shifts. For example, four chemical shifts (6.84, 6.90, 7.35, and 7.42 ppm)
associated ambiguously with four hydrogens of a given tryptophan ring (Hε3, Hζ2, Hζ3, Hη2) are represented as: Hε3/ζ2/ζ3/η2 = 6.84/6.90/7.35/7.42 ppm. A single resonance at 4.30 ppm that is known to arise
from Hα of either Thr 10 or Thr 28 would be indicated as T10 Hα/T28 Hα = 4.30 ppm. Because signals
from individual hydrogens on methyl groups and amines are ordinarily not resolved by NMR spectroscopy,
the signals could be represented by the slash rule (for example, Hβ1/Hβ2/Hβ3, for the hydrogens of an alanine methyl group), but it is simpler when designating spectral features just to omit the numeration (Hβ, forthe alanine methyl).
2.1.2.2.

Stereochemical ambiguity involving prochiral centers

The nomenclature described in 2.1.2.1. also applies to situations in which two diastereotopic substituents
have not been assigned stereospecifically. For example, resonances at 2.44 ppm and 3.12 ppm that were
ambiguously assigned to the Hβ2 and Hβ3 atoms of a given residue are reported as Hβ2/β3 = 3.12/2.44 ppm.
A single peak at 2.44 ppm that could be assigned to Hβ2, Hβ3, or both is reported as Hβ2/β3 = 2.44 ppm. If
it has been established that both have the same chemical shift, they are reported as: Hβ2 = 2.44 ppm, Hβ3 =2.44 ppm.
2.1.2.3.

Other stereochemical ambiguity

When flips of the symmetrical ring of a phenylalanine or tyrosine are slow on the chemical shift time scale,signals from the δ- and ε-atoms can have non-equivalent shifts. In such cases, information about assignments relative to the χ2 torsion angle is needed to define the specific atom designators 1 and 2. This information often is not known in advance of a full structure determination, so it is useful to specify this level ofambiguity again with the slash rule. For example, if proton signals at 6.60, 6.95, 7.12, and 7.22 ppm havebeen identified with a particular, slowly-rotating, tyrosine ring at position 12, with no further information,
they are reported as Y12 Hδ1/ε1/δ2/ε2 = 7.12/6.95/7.22/6.60 ppm. If, in addition to the above, it is known
that the signals at 7.12 and 7.22 ppm arise from δ-hydrogens, then the signals are reported as Y12 Hδ1/δ2 =7.12/7.22 ppm and Y12 Hε1/ε2 = 6.60/6.95 ppm. If it has been further determined that the signals at 6.95
and 7.12 ppm correspond to hydrogens on the same side of the ring, they are reported as Hδx = 7.22 ppm,
Hδy = 7.12 ppm, Hεx = 6.60 ppm, Hεy = 6.95 ppm.
In cases where the signals from the side-chain amide hydrogens of Asn or Gln, or the guanidino NH2 nitrogens or hydrogens of Arg are known to be coincident, the same chemical shift is assigned to both atom
designators. In cases where information for unambiguous, individual assignments is lacking, the ambiguity
is represented by the “slash rule”. For example, ambiguous assignments would be indicated by Gln Hε21/ε22,
Arg Hη11/η12, or Arg Hη11/η12/η21/η22.

123

2.2. Nucleic acids
2.2.1. Standard nomenclature
Figure 2 shows the recommended designators for the atoms of ribose, deoxyribose, and the common bases(ref. 22). The IUPAC-IUB notation for the methyl group of T is C7(H7)3. The phosphorus-bound oxygens
are named O3′, O5′, OP1, and OP2. In the CIP rules (Cahn-Ingold-Prelog, ref. 25, 26), OP1 corresponds
to pro-R and OP2 to pro-S.
2.2.2. Notation for assignment ambiguities
The slash rule is used to designate diastereotopic substituents of prochiral centers that have not been assigned stereospecifically. For example, if H5′ and H5″ of a given ribose are identified at 4.12 ppm and 4.53
ppm but have not been assigned individually, they are designated as H5′/H5″ = 4.53/4.12 ppm.
3.

Additional atom identifiers for reporting conformational constraints and structures

Structural constraints involving atoms for which the NMR signals have not been assigned individually are
frequently employed in the determination of macromolecular structures from NMR data. In such cases,
special symbols are used to identify the structural constraints. These may take the form of pseudoatoms
that represent groups of atoms, or ofsymbols that link specific constraints with
NMR chemical shifts.
ribose3.1. Pseudoatom nomenclature

base
H1'

C1'

H1'

O2'-R

C2'

C2'

H3'

C3'

O4'

H4'

H5''
(R)

H5'

H5''
(R)

H4'

A

H3'
O3'-R

O3'-R
H5'

(R)
H2''

R-O5'
C3'

O4'

H2'

C1'

R-O5'

G
–
–

N6

–

–

–

–

–

–

H22

U
–

N3

C4
– –

C5

–

C7(H7)3

C2
C6
– – –
O2 –
N1
H6

C2
– C6
– –
O2 –
N1
H6

–

–

–

H3–

–
–

C5

H5

–
–

–
–

C2
– C6
–
O2 –
N1
H6

N3

C4
– –

–

–

H3–

H5
––C4–
–
N3
C5
–

O4

–
–

N4

–
–

O4

–

–

–

–

–

T

H42

–

H41

–– –

–– –

C

–
–

C6
– – – N7
N1
C5
C8 H8
H21–
– C2–– – C4–
N9
N2
N3

–

H1 –

––C6– – N7
N1
C5
C8 H8
– C2–– – C4– N9
N3
H2

–

–

O6

H62

–

H61

–
–

3.2. NOE constraints that canbe identified by chemical
shifts

base

H2'

–

NOE constraints to groups of atoms forwhich the resonances have not been assigned individually are commonly specified by pseudo-structures in which certain
atoms have been joined together into
pseudoatoms. Relevant pseudoatoms forthe standard amino acid residues havebeen defined (ref. 30) and are in common
use in the NMR literature. An NOE to
one or all protons in the group is measured relative to a location central to thegroup (ref. 31). All pseudoatoms needed
to describe proteins and nucleic acids canbe specified in terms of two letters, Q and
M, in conjunction with the usual atom
identifiers (Roman letters are used insteadof Greek letters for atom positions withinamino acids, and R is used to denote aring). M describes the location of methylgroups, and Q is used in all other situations. Table 1 lists all pseudoatoms in thecommon amino acids and nucleotides.

2' deoxyribose

Fig. 2. Nomenclature, structures, and atom numbering for the sugars andNOEs used for structural constraints that the bases contained in common nucleotides (ref. 22). The identifiers
shown here for the hydrogen atoms largely follow previous recommendaare attributed to pairs of diastereotopic or tions as discussed in section 2. of the text. “R” indicates pro-R.

124

TABLE 1. Pseudoatoms for the Common Amino Acids and Nucleotides Used in the
Determination of Structures of Proteins and Nucleic Acids from NMR Data.a
1H atoms representedb
Residue
Pseudoatom
Gly
Ala
Val
Ile
Leu

Pro
Ser, Asp, Cys, His, Trp
Thr
Asn
Glu
Gln
Lys
Arg

Met
Phe, Tyrβ-D-ribose
2′-β -D-deoxyribose
ACGTa Ref. (30).

QA
MB
MG1, MG2
QG
MG, MD
QG
MD1, MD2QB
QD
QB, QG, QD
QB
MG
QB
QD
QB, QG
QB, QG
QE
QB, QG, QD, QE
QZ
QB, QG, QD
QH1, QH2
QH
QB, QGMEQB
QD, QE
QR
Q5′
Q2′
Q5′
Q6
Q4
Q2
M7

α-methylene
β-methyl
γ1-, γ2-methyl
all six γ-methyl
γ2-, δ1-methyl
γ1-methylene
δ1-, δ2-methyl
β-methylene
all six δ-methyl
β-, γ-, δ-methylene
β-methylene
γ2-methyl
β-methylene
δ2-amido
β-, γ-methylene
β-, γ-methylene
ε2-amido
β-, γ-, δ-, ε-methylene
ζ-aminoβ-, γ-, δ-methylene
η11 and η12, η21 and η22
all four η-guanidino
β-, γ-methylene
ε-methyl
β-methylene
δ1- and δ2-ring, ε1- and ε2-ring
all ring
5′-methylene
2′-methylene
5′-methylene
6-amino4-amino2-amino7-methylb See Figures 1 and 2.

symmetry-related protons which have non-degenerate chemical shifts and have not been assigned individually, can be distinguished unambiguously on the basis of the chemical shifts. For example, an NOE observed at 3.12 ppm to one of a pair of methylene proton resonances at 2.44 ppm and 3.12 ppm, which wereassigned ambiguously to the Hβ2/β3 atoms of Tyr57 is reported as an NOE to Tyr57 Hβ2/β3 (3.12 ppm). Alternatively, the symbols “L” and “H” can be used to represent the resonances at lower frequency (historically referred to as “upfield”) and higher frequency (historically “downfield”), respectively. In the above
example, the NOE would then be to Tyr57 H βL.
4.

Nomenclature for specifying conformation

Torsion angles are defined in terms of the substituent with highest preference on each of the two atoms thatspan the rotatable bond (ref. 20, 35). The IUPAC-IUB convention (ref. 20, 22) specifies that the main chain
atoms of a peptide or nucleic acid take precedence over others. For example, in a peptide, the backbone

125carbon and nitrogen atoms take precedence over other heavier atoms such as the carbonyl oxygen or cysteine Cβ (ref. 20). Otherwise atom preferences follow standard conventions (ref. 25, 26). A given torsion
angle θ about the BC bond of a molecule ABCD (where A is the atom with highest preference
attached to B, and D is the atom with highest preference attached to C) is the angle between the planes
containing ABC and BCD, or alternatively the angle between the projections of BA and CD
onto a plane normal to BC. The torsion angle is written in full as θ (A,B,C,D). When the projections of
the two bonds BA and CD coincide (eclipsed or cis conformation), θ = 0°; when the projections of thetwo bonds BA and CD are opposed (trans conformation), θ = 180°. If for a given torsion angle θ thatis neither 0° nor 180°, when looking (in either direction) along the central bond BC, the minimal rotationof the front bond that would be required to achieve the eclipsed conformation is clockwise, θ is considered
to be positive; if the required minimal rotation is counter-clockwise, θ is considered to be negative. As defined below, various abbreviations are used to denote specific torsion angles in proteins and nucleic acids; inother situations (or to avoid any ambiguity) the four atoms that determine the torsion angle should bespecified.
4.1. Polypeptides
4.1.1. Backbone torsion angles: φ i , ψ i , and ω i
The φi torsion angle describes rotations about the Ni  Cαi bond (relevant four atoms C′i-1  Ni  Cαi C′i), and the ψ i torsion angle describes rotations about the Cαi  C′i bond (relevant atoms Ni  Cαi  C′i
 Ni+1). The ω i torsion angle describes rotations about the peptide bond, C′i-1  Ni (relevant atoms Cαi-1
 C′i-1  Ni  Cαi).
4.1.2. Side-chain torsion angles: χ ij
Side-chain torsion angles for residue i are designated by χ ij , where j represents the rotatable bond: j = 1 for
the Cα  Cβ bond (where the relevant atoms are N  Cα  Cβ X) ; j = 2 for the Cβ  Cγ bond (where
the relevant atoms are Cα  Cβ  Cγ  X), etc. Figure 3 shows how the χ1 angle is related to the angle
between the projections of the bonds Cα  Hα and Cβ
residues withIle
 Hβ (or Cβ  Hβ2) for the common amino acids. In
β2
β3
and
H
H
amino acids with branched side chains, two superscripts
HαHα
are used, the first to indicate the position of the bond and
β3
γ2
H
R
C
Cγ 1
the second to indicate the branch (e.g, the Cα  Cβ 
1
χ = – 60°
χ1 = – 60°CαCαCγ1  Cδ1 torsion angle of isoleucine at residue position
CNNC
i is denoted by χ 2i ,1 ) (ref. 20).
β2
β4.1.3. Specification of turns,
bonds, and proline rings

disulfide

H

H

Thr

Val

Hα

Turns can be defined unambiguously by the torsion angles involved, i.e., φi+1, ψ i+1, φi+2, and ψ i+2 (ref. 36).
Disulfide bridges between residues i and j can be de2
1
scribed by the torsion angles χ 1i , χ i2 , χ i3 , χ j , and χ j ; inaddition, it is recommended that the handedness be indicated by R or S (ref. 37). Proline ring puckers can be
defined by the torsion angles χ 1i , χ i2 , χ i3 and χ i4 (seecaption to Fig. 4); alternatively, use can be made of thefact that the value of χ 1i alone is indicative of the two
major pucker forms (ref. 38) (Fig. 4).

126

γ2

Hα
γ1

C

α

C

O
χ1 = – 60°
N

C
H

β

γ1

C

C

α

Cγ 2
χ1 = ±180°
N

C
H

β

Fig. 3. Definition of the χ1 angle for the common aminoacids. Note that although the Hα Cα Cβ Hβ(β2) torsion
angles are equivalent in all the examples shown, the valueof χ1 for Val differs from those of the other amino acids as aconsequence of the definition of this torsion angle.

4.2. Nucleic acids––
C4.2.1. Backbone and glycosidic
torsion angles

Hγ 2

O

H

Hβ2

O3′i-1 Pi O5′i C5′i
Pi O5′i C5′i C4′iO5′i C5′i C4′i C3′iC5′i C4′i C3′i O3′i
C4′i C3′i O3′i Pi+1
C3′i O3′i Pi+1 O5′i+1
O4′i C1′i N1i C2i
O4′i C1′i N9i C4i

Hγ 2

Hδ2

N
N

N

Hδ3

H

H

α
β
γ
δ
ε
ζ
χ(Py)
χ(Pu)

C

α
γ3

For residue i the backbone (α, β , γ, δ, ε, ζ)and glycosidic (χ) torsion angles for purine(Pu) and pyrimidine (Py) bases are defined asfollows (ref. 22) (see also Fig. 5):

Hβ2

O
δ2

–
–

Notation in current use in the nucleic acidfield is presented here. In line with current
usage, multiple notations are provided incertain instances. Older conventions for defining torsion angles can be found in reference 22.

δ3

H

α

H

β3

H

Hγ 3

Hβ3

DOWN

UP

Fig. 4. Ring pucker in the proline ring (ref. 38). In the DOWN conformation, the torsion angles χ1 and χ3 are positive and χ2 and χ4 arenegative; in the UP conformation, the torsion angles χ1 and χ3 arenegative and χ2 and χ4 are positive. Typical values for the torsion angles also depend on whether the XxxPro peptide bond is cis ortrans. Average values (in degrees) found from a number of X-ray
structures of proteins for χ1, χ2, χ3, χ4 were (ref. 38): trans DOWN,
18, −29, 17, −4; cis DOWN, 26, −38, 18, −7; trans UP, −27, 35, −34,
20; cis UP, −21, 36, −36, 17.

4.2.2. Sugar pucker: torsion angles and angle of pseudorotation

––

––

5'

––

––

––

Each of the sugar torsion angles ν 0, ν 1, ν 2, ν 3 and ν 4 is defined by four atoms as follows (ref. 22) (see also
Fig. 5):
ν 0 C4′ O4′ C1′ C2′
ν 1 O4′ C1′ C2′ C3′base
ν 2 C1′ C2′ C3′ C4′χ
ν 3 C2′ C3′ C4′ O4′
ν0 C1' ν1ν 4 C3′ C4′ O4′ C1′
O4'
C2'
ν4
ν22
Note that ν 3 and δ, which characterize torsion angles about the same bond (C3′ C4′), will have dif- – O3'(i –1) – P(i) – O5' – C5' – C4' –– C3' – O3' – P(i +1) –αβγ δ/ν33εζferent values for any given conformation.
3'

The sugar is generally non-planar, and it is recom- Fig. 5. Designation of the torsion angles in the sugarmended that the sugar ring conformation (pucker) be phosphate backbone (α, β , γ, δ, ε, ζ), the glycosidic bond (χ),
and the endocyclic torsion angles in the sugar ring (ν0  ν4)
described by specifying two parameters: the phase (ref. 22).

angle of pseudorotation (P) and the puckering amplitude ψ m. In practice, the pseudorotation parameters can be determined from measurements of the threebond J-couplings between the protons attached to C1′, C2′, C3′, and C4′. The following equations characterize relationships among the torsion angles defined by these protons and the pseudorotation parameters(ref. 39, 40):

φ1′2′ = 121.4 + 1.03 ψ m cos (P − 144°)
φ1′2′′ = 0.9 + 1.02 ψ m cos (P − 144°)
φ2′3′ = 2.4 + 1.06 ψ m cos (P)

φ2′′3′ = 122.9 + 1.06 ψ m cos (P)
φ3′4′ = − 124.0 + 1.09 ψ m cos (P + 144°)

127

ndo

exo
–

en

do

E

2

1
2

P=0°
3
2

T

36

E

T

T
E

d

4

r

T

0
4

T
0

E
0
1

T

d

r
4

E

E

1
4
3

T
E

3

44

T

E

±180°

°
44

1

'–

ex

o

o
end

C3'–

°

T

2

2
3

C1

2
1

C2'–

–1

°

T

exo

°

–108

o
nd

04'– endo

E

4
0

ex

72°

–72

°

E

1
0

0

–e

'
C4

'–
C4

3
4

1

04'– exo

o

°

3

108

'–

°
36

C3'–
e

C2'–

C1

Fig. 6. Pseudorotation cycle of the furanose ring showing the relationships among the phase angle of pseudorotation (P), theenvelope (E) and twist (T) notation, and the endo and exo notation. Conformations corresponding to the northern half aredesignated as N-type, and those corresponding to the southern half are designated as S-type. Ranges of the P values usuallyobserved experimentally for the N and S conformations are represented by ‘r’ for ribo- and ‘d’ for 2′-deoxyribofuranose ringsof β-D-nucleosides and nucleotides (ref. 22). Note that the symmetrical twist (T) conformations are found at even multiples of
18° and that the symmetrical envelope conformations are found at odd multiples of 18°.

In these equations the non-equilateral character of the sugar ring is accounted for. The φ’s can be derived
from the J-couplings by means of appropriate Karplus equasyn
tions.
–sp

Relationships among the phase angle of pseudorotation andthe alternative E/T and endo/exo notations are illustrated by
the pseudorotation wheel in Fig. 6. In the E/T nomenclature,
the puckered forms are designated by E (envelope form) andT (twist form) (ref. 22). For example, P = +18° is equivalent
to C3′-endo or 3 E , and P = −162° is equivalent to C3′-exo
or 3 E . Symmetrical twist conformations, with two atoms at
equal distance with respect to the plane defined by the threeremaining ring atoms, can also be represented; for example,
P = 0° is equivalent to C3′-endo/ C2′-exo, or to 32T .
4.2.3. Value ranges for torsion angles specifying commonly observed conformations
In practice, torsion angles are often not known exactly, but
can be assigned to particular conformational regions. The
notation used most frequently by spectroscopists and X-ray
crystallographers for ranges of torsion angles is cis , trans,−gauche, and +gauche as shown in Fig. 7. The IUPAC-IUB
Commission on Nucleotide Nomenclature recommended the
Klyne-Prelog notation (ref. 22), with ± synperiplanar (± sp),± synclinal (± sc), ± anticlinal (± ac), and ± antiperiplanar(± ap) as defined in Fig. 7. The terms syn (0° ± 90°) andanti (180° ± 90°) have special meanings in nucleotide chemistry in that they are used to define the orientation of the basewith respect to the sugar. Table 2 lists the predominant conformations of nucleic acids and the associated ranges of torsion angles.

128

+sp
(cis)

–30°

–sc

0°

+30°

+sc

Front
bond

–60°
(–gauche)+60°(+gauche)

–90°

+90°

–120°
–ac

+120°
+ac
–150°

–ap

±180°
(trans)

+150°

+ap

anti
Fig. 7. Relationships among different terminologies
used for the approximate description of a torsion
angle in terms of conformational regions. Torsion
angles are defined by holding the front bond at zero
position, as indicated in the figure, and by measuring
the angle between the front bond and the back bondwhile looking along the central bond. The KlynePrelog convention (whose ranges are abbreviated as:± sp, ± synperiplanar; ± sc, ± synclinal; ± ap, ± antiperiplanar; ± ac, ± anticlinal) has been recommended by the IUPAC-IUB Commission on Nucleotide Nomenclature (ref. 22). However, spectroscopists commonly use a different nomenclature
which specifies four 60° regions: cis (0° ± 30°),trans (180° ± 30°), +gauche (60° ± 30°) and −gauche(−60° ± 30). The terms syn (0° ± 90°) and anti
(180° ± 90°) are discussed in the text.

TABLE 2. Notation Commonly used to Describe the Major Conformational Forms of
Polynucleotides in Terms of Torsion Angle Ranges Obtained from Fiber Diffraction
Measurements.a

α

β

γ

δ

ε

ζ

χ

A-RNA

g−
- sc

t
+ ap

g+
+ sc

g+
+ sc

t
- ap

g−
− sc

anti
− ap

A-DNA

g−
− sc

t
+ap

g+
+sc

g+
+sc

[t]
− ac

g−
− sc

anti
− ap

B-DNA

g−
− sc

[t]
-ac

g+
+sc

t
+ap

t
+ap

[g−]
− ac

anti
− ac

Z-DNA Gb

g+
+sc

t
− ap

t
+ap

g+
+sc

g−
− sc

[[g+]]
+ac

syn
+sc

Z-DNA Cc[[g−]]
− ac

t
− ap

g+
+sc

[t]
+ac

[[g−]]
− ac

g−
− ac

anti
− ap

a For reference, the alternative Klyne-Prelog notation (ref. 22) is given below each recommended
symbol. The cis (c), trans (t), +gauche (g+), −gauche (g−) notation does not provide a definition for
the torsion angle ranges 90° to 150° and − 90° to − 150° (Fig. 7). Several torsion angles in standard
polynucleotide conformations fall outside the ranges defined by these symbols. This information is
also given in the table in that angle values outside the range by less than 10° are enclosed by brackets and those outside the range by more than 10° are enclosed by double brackets. In these cases,
the Klyne-Prelog notation provides a more accurate representation of the torsion angle range.
bGuanine residue in Z-DNA. cCytosine residue in Z-DNA.

5.

NMR data acquisition, processing, and referencing

This section gives recommendations on how to present information needed to provide a comprehensive description of the experiments used in a structure determination.
5.1. Data acquisition
The following information describes the data collection process. In particular, a complete account is neededfor experiments employed in the collection of conformational constraints.
•

NMR hardware used. Customarily the magnetic field is specified by the resonance frequency of a particular nucleus, usually 1H; other minimal information includes the type of console, type of probe head
and the size and nature of the sample tube/container. Additional details might be desirable for the description of novel experimental protocols or home-built systems.

•

Protocols for the acquisition of raw data. This normally includes a diagram of the pulse sequence
and/or an ASCII file with the code for the pulse program, or a reference if previously published routines
were used. It is important to include information on details of solvent suppression, timing parameters,
r.f. power levels (or equivalent 90° pulse duration), phase cycling, pulse shapes, any magnetic field gradients (magnitudes and durations), the modes of acquisition for each dimension (including method for
quadrature detection), the recycle delay, and the total time required for data acquisition.
Description of time domain data. This usually includes acquisition times, spectral widths, possible useof non-linear sampling, and the number of real or complex data points in each time domain.

•129

5.2. Data processing
A comprehensive description of data processing normally includes the following information:
Software packages used.
Enhancement of the time-domain data. This may include digital filtering, solvent filtering, convolution
with a window function, zero filling, or linear prediction.
• Time-domain to frequency-domain conversion. The Fourier transform is the most common method, butalternatives may be employed, such as maximum entropy, maximum likelihood, or Bayesian analysis.
The final data size and digital resolution in each dimension are part of a complete documentation.
• Enhancement of frequency-domain data. Common methods include symmetrization, baseline flattening,
and ridge or solvent suppression.
• Methods used to extract spectral parameters such as resonance frequencies, peak volumes, and coupling
constants.
••

5.3. Referencing of chemical shifts
According to general convention, NMR spectra are displayed with positive frequencies to the left andnegative frequencies to the right of the zero-frequency reference (ref. 6, 7). The IUPAC Commission on
Molecular Structure and Spectroscopy (ref. 6) has recommended that the 1H signal of tetramethylsilane(TMS) be used as the primary reference for the resonance frequencies (and hence chemical shifts) of
protons, and they propose to use the same signal as an indirect reference for other nuclei (ref. 41). The
traditional primary chemical shift standard, however, for aqueous solutions has been the methyl signal of awater-soluble derivative of TMS. Following recent literature (ref. 42), it is recommended that the primarychemical shift standard for all nuclei in aqueous biological investigations be the methyl signal of internal 2,2dimethylsilapentane-5-sulfonic acid (DSS) at low concentration. When it is inappropriate to use DSS as aninternal standard, e.g., in cases where DSS binds to the system under investigation, it is still desirable to use
the position of DSS as the 0 ppm point. In such cases, reference 42 lists alternative reference compounds
and corresponding referencing protocols. In such cases, precise details should be given for the reference
used and the conversion factors employed. In the interest of clearly differentiating such chemical shifts fromthe default IUPAC recommendation for TMS as the reference (ref. 41), it is recommended that the notation
δDSS be used. The chemical shifts of the methyl peaks in the two references are quite similar (ref. 43).
5.3.1. 1H chemical shifts
The methyl resonance of internal DSS at low concentration directly provides the preferred reference signal
for 0 ppm: notation, δ DSS or δ (DSS).
5.3.2. Chemical shifts of other nuclei such as 2H, 13C, 15N, and 31P.
Whereas a number of direct and indi- TABLE 3. Relative Frequencies (Ξ ) of Nuclei of Biomolecular
rect approaches have been described Interest to be Used for Indirect Referencing.a
for the referencing of chemical shifts
NucleusSecondary
ReferenceΞ /MHzof nuclei other than 1H, the indirectreferencereferencing method (ref. 44) has besample
come the preferred approach. Non1
H
100.000000 by definition
proton chemical shifts are referenced
2
H
DSS(internal)
15.3506088
ref. 45
indirectly to the 1H standard using
13
C
DSS (internal)
25.1449530
ref. 42conversion factors derived from ra15
Nliquid NH3 (external)
10.1329118
ref. 42tios of NMR frequencies. The rela31
P(CH
40.4808636
ref. 46
3O)3PO (internal)
tive frequencies are designated by
a These Ξ values were derived originally (as indicated in the table) fromthe symbol Ξ , with 1H conventionthe ratio of the signal frequency of a secondary reference to that of interally at exactly 100 MHz. Ξ values
nal DSS in D2O as the primary reference.
for the nuclei most commonly utilized in studies of proteins and nucleic acids are listed in Table 3. Thus, for example, the zero frequency for130

13C chemical shifts is obtained from the experimentally determined 1H frequency for DSS by multiplying thelatter frequency by the 13C/1H Ξ ratio, 25.1449530 /100.000000 = 0.251449530. In order to tie in with thechemical shift scales used in much of the previous literature, these conversion factors were derived from the
1H/2H frequency ratio of water, 13C frequency of the methyl groups of internal DSS, the 15N frequency of
external liquid ammonia, and the 31P frequency of internal trimethylphosphate, respectively (see reference
42 for details). It is recommended here that the relative frequencies (Table 3) be used as fixed constants not
subject to further change and that they be used for data collected at all temperatures (with no applied temperature correction) and with the primary reference as either internal TMS (δ) or DSS (δDSS). It is anticipated that standardized Ξ values for additional nuclei of interest to biomolecular spectroscopists will be developed and published as recommendations (ref. 41).
6.

Resonance assignments

Resonance assignments constitute a prerequisite for the commonly used structure determination procedures.
Because the final quality of the structure depends to a large extent on the completeness of the underlying
resonance assignments, their full description is important. Deposition of chemical shift assignments is imperative (see 10. below). Quantitative information on the completeness of assignments obtained, including
a listing of the missing assignments, is of prime interest. If, as part of automated assignment procedures,parameters are available that provide a measure for the reliability of a particular assignment, it is desirable
that such parameters be presented along with the assignments themselves.
6.1. Sequence-specific assignment of polypeptide chains
The assignment process for polypeptides consists of sequence-specific assignments of backbone and side
chain resonances.

6.1.1. Sequential backbone assignment
In most cases, a brief description should suffice, since present assignment strategies employ one or both of
the two following approaches.
Assignments derived from sequential NOEs and 1H- 1H J-correlation. If samples with solution conditions(e.g., pH, temperature, ionic strength) different from those reported in the assignment table were used indetermining or validating assignments, this information needs to be noted. It is useful to report other information relevant to the assignment process, such as improvement of the spectral resolution by isotopic
labeling, including 1H/2H exchange of amide groups. It is recommended that this include description of thetypes of J-correlation experiments used in the sequential assignment process.
Sequential assignments derived from homonuclear and heteronuclear J-correlations across peptide bonds.
Although uniform double labeling with 15N and 13C is most commonly employed with this approach, otherlabeling patterns may be used. If non-uniform labeling was employed, important information includes a description of the labeling strategy and a list of the labeled proteins used. In cases where NOEs were used inparallel to establish or validate assignments, this information is of interest.
6.1.2. Amino acid side-chain assignment
Side chain assignments, although conceptually simple, are often complicated by spectral overlap, strong
coupling, and rapid transverse relaxation. Various isotope labeling strategies and NMR techniques havebeen devised to overcome these problems. Unless novel methods have been employed, it should suffice toreference the approaches used. It is recommended that the report contain a list of the experiments used, thefraction of the side chain resonances that have been correlated through J-couplings to the backbone, and theextent to which NOEs have been used in the assignment or validation process.

1316.2. Sequence-specific assignment of nucleic acids
The assignment process for nucleic acids consists of two phases. In one part, the sugar-phosphate backbone resonances are assigned, together with the purine H8 and the pyrimidine H5 and H6 resonances. This
assignment usually is based on combined use of NOEs, 1H-1H J-correlation, and 1H- 31P J-correlation, and,in cases where isotopic labeling is used, on 1H- 13C, 13C- 13C, 13C- 15N, and 13C- 31P J-correlations. In thesecond part, the remaining base protons (adenine H2 and all exchange labile protons) are assigned, usually
by NOE methods, but possibly also by isotopic labeling and J-correlation. For both parts, it is important toreport how the resonance assignments were established and what assumptions regarding the conformationof the oligonucleotide were used in making assignments that involve NOEs. Further pertinent information
includes temperature and solvent conditions and, if applicable, the level and pattern of isotopic enrichment.
6.3. Stereospecific assignment of diastereotopic substituents
It is useful to indicate in the assignment table which method was used to make each stereospecific assignment. Unless a novel approach was used, the description may consist of a brief reference to one of themethods in common use.
Analysis of J-couplings and short-range NOEs. It is useful to report the J-couplings and the experiments
used for their measurement along with any NOEs utilized in making the stereospecific assignments. If
computational methods, such as systematic grid searches, were used, they should be referenced or describedbriefly.
Stereoassignment by isotopic labeling. It is customary to report how the labeling was achieved (e.g., isotopic composition of cell growth medium) and what the observed levels of enrichment were for the pertinent sites in the biomolecule.
Stereospecific assignments by reference to preliminary structures. Indicate the software package and any
statistical criteria (confidence level) employed.
6.4. Conformational equilibria
The criteria used to deduce the existence of multiple states (as may result, for example, from partial folding,oligomeric heterogeneity, cofactor or ligand binding heterogeneity, peptide bond cis/trans isomerization, or
hairpin-duplex equilibrium) are of prime interest. A separate designator is supplied for each individual state
and associated with its NMR parameters. Fractional occupancies and interconversion rates for the states(or bounds on these) may be included if known.
7.

Conformational features derived from diagnostic NMR parameters7.1. Polypeptide secondary structure

It is a special feature of protein structure determination by NMR that the secondary polypeptide structure,including the connections between individual segments of regular secondary structure, can be determined
early on in connection with the resonance assignments, before the complete structure calculation is evenstarted. Reports on such identification of regular secondary structures and tight turns will be of interest
also in the foreseeable future, for example, as a preliminary structural characterization of a novel protein or
in connection with studies on protein folding. The information used for secondary structure identification isconcisely documented in a survey diagram of the type shown in Fig. 8. The data in Fig. 8 include spin-spin
coupling constants 3JHNHα, sequential NOEs dαN, dNN, and dβN, medium-range NOEs dαN(i,i+3),dαβ(i,i+3), dαN(i,i+4), dNN(i,i+2), and dαN(i,i+2) (ref. 47), and conformation-dependent chemical shifts forthe α-carbons, ∆δ (13Cα). The diagram can be expanded readily for inclusion of additional parameters ofdiagnostic value for secondary structure determination. Figure 8 gives complete data for identification ofhelical structure (ref. 33) and N-caps in helices (ref. 49). Although segments of extended chain also can be132

Fig. 8. Example showing methods recommended for presenting NMR data supporting the identification ofregular secondary structure in proteins. The 40-residue protein, pheromone Er-2, is used as an illustration(ref. 51). Above the amino acid sequence, black squares identify residues with observably slow hydrogen exchange rates, kex, at the backbone amide (the conditions of the exchange experiment should be specified). Below the amino acid sequence, filled circles identify residues with 3JHNHα < 6.0 Hz, indicative of local α-type
conformation; open circles correspond to 3JHNHα > 8.0 Hz, indicative of residues in extended chain conformation; crosses identify residues with 3JHNHα values 6.08.0 Hz. For the sequential proton-proton NOE connectivities, dαN, dNN, and dβN (dαδ, dNδ, and dβδ for XxxPro dipeptides, dαN, dδN, and dβN for ProXxx dipeptides), thick and thin bars indicate strong and weak NOE intensities, respectively. The observed medium-range
NOEs dαN(i,i+3), dαβ(i,i+3), dαN(i,i+4), dNN(i,i+2), and dαN(i,i+2) are indicated by lines connecting the two
residues that are related by the NOE. 13Cα chemical shifts relative to the random coil values, ∆δ (13Cα), areplotted at the bottom of the figure, where positive values are shifts to lower field. The sequence locations of
three helices are indicated at the bottom; broken lines are used to indicate that the identification of helix 2 fromthese data is uncertain.

recognized from presentations of this type (Fig. 8), additional information on long-range NOEs is needed
for the identification of β -sheets. A two-dimensional drawing of the β -sheets enables a concise presentationof these data, which may even include some details on the experiments used (Fig. 9).

Fig. 9. Presentation of NMR evidence for the presence of β -sheet in a protein (the example shown is Tendamistat (ref. 33).
Slowly exchanging amide protons are shown in bold. Interstrand, long-range 1H1H NOEs dαα(i,j), dαN(i,j), and dNN(i,j)
are indicated by double-headed arrows. Similar representations can be drawn for parallel β -sheets.

1337.2. Polynucleotides
7.2.1. Mononucleosides
The conformation of a mononucleoside within a nucleic acid structure is determined by the sugar conformation and the glycosidic torsion angle. Diagnostic characterization of these structural features often ispossible from simple inspection of the NMR spectra.
• In a ribose ring, 3JH1′H2′ ≈ 1 Hz is diagnostic for the N-puckered conformation (P = 0° to 18°), and3J
H1′H2′ ≈ 7.9 Hz is diagnostic for the S-puckered conformation (P = 144° to 162°).
• In a deoxyribose ring, 3JH1′H2′ ≈ 1.8 Hz is diagnostic for the N-puckered conformation (P = 0° to 18°),
and 3JH1′H2′ ≈ 10 Hz is diagnostic for the S-puckered conformation (P = 144° to 162°).
• The distance between H6 (in pyrimidines) or H8 (in purines) and the sugar ring proton H1′ varies from~ 2.5 C (0.25 nm) in the syn (χ ≈ 60°) orientation to ~ 3.7 C (0.37 nm) in the anti (χ ≈ 240°)orientation. The anti orientation, which is found in A- and B-type helices, therefore, is characterized bycross-relaxation cross peaks of low intensity. The syn conformation, which is found, for example, in the
G-residues in Z-DNA, is characterized by cross-relaxation cross peaks of high intensity. Pyrimidine
residues are rarely found in the syn orientation.
• The distance between H6 (in pyrimidines) or H8 (in purines) and the sugar ring proton H2′ varies from~ 2 C (0.2 nm) in the anti orientation to ~ 4 C (0.4 nm) in the syn orientation. Because these distances
are nearly independent of the sugar pucker, the magnitudes of their cross-relaxation cross peaks are
particularly useful for estimating the magnitude of the glycosidic angle χ.
• Until a specific format for the presentation of these diagnostic data has been agreed upon, description in
the text is recommended.
7.2.2. Backbone angles
In favorable situations, heteronuclear 1H - 31P coupling constants can be used to recognize preferred
backbone conformations. The preferred orientation of the backbone angle β (PO5′C5′C4′) in helixtype structures is in the trans range, which is characterized by relatively small values for 3JH5′P and 3JH5′′P(about 2 to 5 Hz). Unusual folding patterns (e.g., in loop structures) can lead to β values in the g+ range,
characterized by 3JH5′′P ≈ 24 Hz, or the g− range, characterized by 3JH5′P ≈ 24 Hz. In B-type DNA
helices, the torsion angles β and γ are in the preferred β t and γ + conformations, and the fragment
PO5′C5′C4′H4′ forms an all-trans, planar, W-type geometry. This results in a long-range 4JH4′P
coupling constant of 2-3 Hz, which is large enough to lead to discernible 1H - 31P hetero-COSY crosspeaks. In A-type RNA helices, β and γ adopt similar preferred conformations, but the resulting geometry is
not of the ‘ideal’, all-trans, planar, W-type, and thus no 1H - 31P cross peaks are visible for the H4′ - P
combination. The backbone torsion angle ε, which has the preferred orientation εt, is characterized by asimilar effect: when ε switches to the ε − orientation (ε + is forbidden), the fragment H2′C2′C3′
O3′P can form a planar, W-type geometry, which is characterized by the presence of 1H2′ 31P heteroCOSY cross peaks. Until a specific format for the presentation of these data has been proposed, it isrecommended that they be presented either in a table or as part of the text.
7.2.3. Secondary structure
The observation of imino proton resonances between 11.5 and 14.5 ppm is indicative of the presence ofbase-paired helical regions in a nucleic acid. The resonance intensity in this spectral range provides a lower
limit to the number of helical base pairs. The occurrence of imino proton resonances at δDSS > 14.5 ppm isindicative of the presence of protonated cytosine involved in base-pair hydrogen bonding.

134

8.

Collection of input constraints and structure calculation

The collection of constraints used in calculating the structure of a protein or nucleic acid usually is an
iterative process, where an initial fold, determined on the basis of a small number of constraints, supports
the identification of additional constraints, which supplement the original constraints in the next round ofrefinement. Thus, the collection of input constraints can be considered an integral part of the structure
calculation process, and both aspects are dealt with in this section.
8.1. Conformational constraints
8.1.1. Distance constraints from NOE data
It is customary to describe the methods used to assign NOE cross peaks and those employed to convert
NOE cross peak intensities into interproton distance constraints. It is essential that the procedure followed
in determining the constraints be described in sufficient detail that a worker could repeat their derivation
given equivalent NOE data. It is important to specify whether all types of protons were treated equally, or
whether special corrections were made, for example, to account for motional averaging or interactions
involving methyl groups. For oligomeric proteins, it is important to describe if and how intermolecular
NOEs were distinguished from intramolecular interactions.
8.1.2. Torsion angle constraints from J-coupling data
It is usual to specify the methods used to relate J-couplings to structural constraints. This includes the
specific Karplus-type relationships employed, their representation by an energetic penalty function during
the structure calculation (see for example, ref. 50), and possibly additional criteria used to resolve the up to
four-fold degeneracy of the Karplus relation between torsion angle and J-coupling.
8.1.3. Torsion angle constraints from chemical shifts
If chemical-shift-derived torsion angle constraints are used in the structure refinement procedure, it isimportant to describe the energetic penalty function used, the method by which the predicted shift wascalculated, and the refinement stages in which chemical shift information was included.
8.1.4. Constraints representing disulfide bonds, hydrogen bonding, and metal
coordination
Standard upper and lower distance constraints are available to represent disulfide bonds that have been
identified either by chemical methods or from NMR results (ref. 51). Similarly, standard upper and lower
distance constraints are available to represent hydrogen bonds inferred from diagnostic NMR data (see 7.)(ref. 51). Hydrogen bonding distances for the Watson-Crick and Hoogsteen base pairs are usually takenfrom Saenger (ref. 52) with error estimates of ± 0.2 Å (0.02 nm). Justification is needed, in all cases, forthe selection of the ligating atoms and, in the case of metal coordination, for the coordination geometry
used in the structure calculation. In all cases, documentation of how these constraints were incorporated in
the energetic penalty function, and at what stage of the analysis, is of interest.
8.1.5. Supplementary constraints for structure refinement
Structural information may be derived from a variety of additional sources, including evidence of hydration,effects of paramagnetic shift and relaxation reagents, isotope shifts, nuclear quadrupole couplings,
photochemical chemically induced dynamic nuclear polarization (photo CIDNP) experiments, fielddependence of J-couplings, anisotropy of relaxation times, etc. It is important to describe in some detail theway in which these data were collected and the means by which they were applied to the structure
calculation. If an energetic penalty function was used, its functional form should be specified. It is
important to document all assumptions and additional sources of information used in calculating thestructure from the experimental data.

1358.1.6. Input for the final structure calculation
Data bank deposition of the input for the final structure calculation is considered to be as important as thedeposition of the final atomic coordinates (10.). Standard formats for the deposition of these constraints(8.1.1.  8.1.5.) are available from the data banks: BMRB (ref. 2) and PDB (ref. 1).
In publications of structures of proteins and nucleic acids, it is customary to provide the following as aconcise overview of the types of input constraints determined. (Prior to this count, NOEs that cannot resultin a constraint due to the distance limits imposed by the covalent geometry, for example, upper limit
distance constraints > 3.3 C (0.33 nm) for vicinal protons, should be eliminated. Upper and lower limits
belonging to the same NOE should be counted as one constraint.):••••

The number and type of NOE (or rotating-frame Overhauser effect (ROE)) constraints, classified as,
intraresidue NOEs (i − j = 0), sequential NOEs (|i − j| = 1), medium-range NOEs (|i − j| <5), long-range
NOEs (all other intramolecular), and intermolecular constraints (if applicable).
The number of torsion angle or J-coupling constraints used, classified by the type of angle (φ, ψ , χ1,
etc., in polypeptides; β , γ, δ, ε, χ, the sugar pucker constraints, and, if used, the staggered
conformations of the α and ζ angles in nucleic acids).
The number of hydrogen bond constraints (with a distinction made between the number of constrainedhydrogen bonds and the number of constraints used to describe these bonds).
If applicable, other types of constraints.
8.2. Methods used for structure calculation and refinement

The computational methods used for structure determination and refinement (including names and versionsof software packages used) need to be specified in adequate detail, and it is important to provide references
to parameter sets, potential functions and values of force constants used, or to include these values
explicitly in the report if they have not been published elsewhere.
8.2.1. Structure calculation
It is desirable that the process for deriving the three-dimensional structure from the NMR data be described
clearly, including the starting conformation(s), numbers and types of constraints used, and the criteria used
to reject unfavorable folds. It is important to describe the protocol used in minimizing the energetic penalty
function during the various stages of refinement (for example, the initial and final temperatures together
with the number of steps, the step size, and the force constants used in simulated annealing protocols).
8.2.2. Preliminary structures to assign additional constraints
Currently, most structure determination protocols remove ambiguities from NOE cross-peak assignmentsby reference to an initial fold calculated with an incomplete set of (ideally unambiguous) constraints. It is
important to document any ambiguity in the initial set of constraints along with the methods used for
assigning additional constraints, for example, by reference to the software used and by reporting adequate
details on the procedures (number of iterations, criteria and cut-off values) employed for the rejection of
NOEs at various stages in the refinement.
8.2.3. Structure refinement
Structure refinement procedures which rely on computationally intensive programs for relaxation matrix
refinement or chemical shift calculations are frequently only incorporated during the final stages of thestructure determination. It is important to provide sufficient details regarding the use of these procedures,
including their effects on the average structure used as input for the refinement, and their effect on the
root-mean-square deviation of the ensemble of structures.

136

9.

Reporting three-dimensional structures
9.1. Presentation of structures

In reporting three-dimensional NMR structures it is common practice to display the entire ensemble ofconformers used for statistical analysis. In addition, a representative conformer, suitable for detailed structural interpretation, often is presented. The representative conformer has been identified variously as the
structure that best satisfies the NMR constraints, as the energy-minimized structure derived from the averaged coordinates of the ensemble, or as the structure that is closest to the averaged coordinates of the ensemble (ref. 53). Subtleties in the averaging of NMR properties (ref. 54), which differ from averaging in Xray crystallography, need to be considered. It is important to specify the total number of conformers calculated and the criteria for selection of the subset of conformers, including the representative conformer used
for display and analysis.
9.2. Agreement of structures with constraints•••

It is important to report complete statistics as to how well the structures satisfy the constraints. The
maximum violations of distance bounds, torsion angle constraints, coupling constants, chemical shifts,
or other experimental constraints used in the structure calculation, are normally reported, together withthe average violation per constraint (± standard deviation).
When computational methods are used that provide a measure of the agreement of the structures withthe constraints or with direct spectroscopic data such as NOE intensities, the final value of the targetfunction or figure of merit (R-factor) normally is reported. In the particular case of relaxation matrix
calculation of NOE intensities, it is appropriate to specify the functional form of the R-factor (e.g., direct, r 6 weighted, etc.).
It is usual to specify the deviations from idealized covalent geometry for bond lengths, bond angles, andimpropers and to indicate which idealized geometry was used. Since covalent bond lengths and angles
are restrained to “ideal” values in all methods currently used to compute NMR structures, deviations
from ideality do not provide an independent assessment of the overall quality of the structures but canidentify problem regions where constraints might be too tight or where constraints may be in conflict asa result of conformational averaging.
9.3. Precision of structures

An ensemble of conformers calculated from the same set of input data is widely used to represent an
NMR-derived structure. The precision of the structure determination commonly is expressed in terms of astatistical analysis of the variation of the atomic coordinates and torsion angles among these conformers.
The precision may then be reported either as average pairwise root mean square (rms.) deviation or the rms.
deviation relative to the mean coordinates. A meaningful description of the precision of local structure isprovided by the circular variance (ref. 55) or the angular order parameter (ref. 56) for torsion angles. It isuseful to specify the method of superposition used in obtaining the mean coordinates. For proteins, the average rms. deviation for Cartesian coordinates usually is reported for a set of backbone heavy atoms, i.e.,
sets of (Cα), (N, Cα, C), or (N, Cα, C, O) atoms, all side-chain heavy atoms, and all heavy atoms (backbone
plus side chain). It is important to indicate whether the reported rms. deviations apply to all amino acidresidues or only to a selected subset. For nucleic acids, the atoms used for calculations of rms. deviations
should be specified.
Useful supplementary information includes evaluation of the rms deviations along the sequence in light ofthe density of constraints per residue, in order to better evaluate the significance of the apparent precision.
9.4. Validation of structures
In common with other methods for three-dimensional structure analysis, for example X-ray diffraction insingle crystals, there is no direct method for absolute validation of the result of an NMR structure determination. Nonetheless, a number of criteria are available for investigating whether the result of an NMR

137

structure determination is “reasonable.” Such approaches have been developed only recently and are being
used to evaluate the results of both NMR and X-ray structure determinations. Additional techniques are
currently in development in different laboratories. These commonly are based on the database of currentlyavailable three-dimensional structures (ref. 5561). Criteria used for structure validation include the following:
•
•
•

10.

Conformational energy (either Lennard-Jones or total energy) with associated force field used.
For proteins, a Ramachandran (φ,ψ ) plot for the backbone torsion angles in the family of conformers.
For more detailed assessment of the stereochemical quality of a protein structure, several computer
programs are available (reviewed in reference 60). These may identify regions of the structure in which
potential problems require further evaluation. Most of these programs have been written primarily forchecking X-ray coordinates, rather than an ensemble of conformers obtained by NMR, but a recently
published suite of programs (ref. 61) has been designed for the validation of NMR structures.
Data bank deposition of NMR structures and supporting data

Deposition in public data banks of the quantitative and semi-quantitative data and references to specificprocedures used to manipulate the data, as described in these recommendations, is strongly encouraged,
even in cases where this duplicates information tabulated in journal articles. The key elements in a data
deposition for an NMR structure include:
•
•
•
•

•
•
•

Representation of the covalent structure(s) of the molecule(s) in the system reported (generally a sequence plus indications of cross links and special modifications) plus information on bound cofactors orother ligands and oligomeric structure.
Tabulation of assigned chemical shifts.
Tabulation of assigned coupling constants.
Tabulation of constraints used in the structure calculation: distance constraints from NOEs (or the
spectroscopic data from which the constraints were derived), torsion angle constraints from J-couplingsand chemical shifts, H-bond constraints, disulfide bridge constraints, and supplementary constraints: it
is important to indicate how the constraints were calibrated and the nature of any pseudoatom or othercorrections (e.g., to account for spin diffusion or spin multiplicity) included in the constraint values andto specify what type of averaging protocol (e.g., center averaging, r-6 averaging, etc.) has been used(ref. 54, 62).
Cartesian coordinates for the family of conformers that represent the result of the structure determination as well as for a single representative conformer: it is important to include all available atoms in thedeposition, including the protons.
Concise description of the solution conditions used for the structure determination (temperature, pH,
ionic composition, etc.).
Literature citations for the studies that originated the deposited data.

Compilers of databases are encouraged to accommodate the deposition of supplementary information of thekind described in the preceding sections either as free text or in a more organized format. This may include
a brief description of the computational techniques (including specification of the software) used to process
the input data, derive and refine the structures, and validate the results.
Additional information usefully available from a database includes tables of NMR relaxation rates, hydrogenexchange rates or protection factors, and thermodynamic parameters characterizing conformational equilibria and ligand binding. Some authors may wish to deposit electronic files containing the free induction decays from a representative cross-relaxation experiment and/or lists of peaks and intensities of the primarydata sets.
In analogy with the reporting of structures determined by X-ray crystallography, it is desirable that journal
editors require, as a condition for acceptance of a publication, database deposition of at least (1) the atomic138

coordinates, (2) the assigned chemical shifts, (3) the assigned J-couplings, and (4) a compilation of all input
constraints used for the structure determination.

Conclusion
The recommendations presented here are designed to support easier communication of NMR data and
NMR structures for proteins and nucleic acids through unified nomenclature and reporting standards.
Much of this document pertains to the reporting of data in journal articles. However, in the best interest of
the future development of structural biology, it is desirable that the bulk of the reported information be
stored in computer-accessible form and be freely accessible to the scientific community. For such purpose,the macromolecular crystallographic community is advocating use of the mmCIF format (ref. 63, 64), whichis compatible with the STAR/CIF format (ref. 65, 66); mmCIF has been implemented in the Nucleic Acid
Database. In recognition of the desirability of developing full compatibility between machine-readable
crystal diffraction and NMR data, it is recommended that a compatible format be used for NMR data fromproteins and nucleic acids. It may prove desirable, in addition, for the databases to develop full compatibility with the ASN.1 data exchange format (ref. 6769), which has been adopted by the U.S. National Center for Biotechnology Information and is being used in the chemical exchange format developed by the
Chemical Abstracts Service (ref. 70). With these goals in mind, international committees, in association
with the Protein Data Bank and BioMagResBank, are in the process of using the present document to develop data dictionaries and author-input protocols for the deposition of macromolecular NMR data
(ref. 71).

References and Notes
1.

2.

3.

4.
5.
6.
7.

8.
9.
10.
11.
12.
13.

Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Jr., Brice, M. D., Rogers, J. R., Kennard,
O., Shimanouchi, & T., Tasumi, M. (1977) The Protein Data Bank: A Computer-Based Archival File for
Macromolecular Structures, J. Mol. Biol. 112, 535542. The Protein Data Bank can be accessed on theInternet at http://www.pdb.bnl.gov/.
Seavey, B. R., Farr, E. A., Westler, W. M., & Markley, J. L. (1991) A Relational Database for SequenceSpecific Protein NMR Data, J. Biomol. NMR 1, 217230; Markley, J. L., Farr, E. A., & Seavey, B. R.
(1991) Appendix: Check List for Protein NMR Data to be Submitted for Publication or for Database Inclusion, J. Biomol. NMR 1, 231236. The Biomolecular NMR Data Bank (BioMagResBank or BMRB) canbe reached on the Internet at http://www.bmrb.wisc.edu/.
Berman, H. M., Olson, W. K., Beveridge, D. L., Westbrook, J., Gelbin, A., Demeny, T., Hsieh, S.-H., Srinivasan, A. R., & Schneider, B. (1992) The Nucleic Acid Database: A Comprehensive Relational Database ofThree-Dimensional Structures of Nucleic Acids, Biophys. J. 63, 751759. The Nucleic Acid Data Bankcan be reached on the Internet at http//www.ndbserver.rutgers.edu:80/.
Hendrickson, W. A., & Wüthrich, K. (1991 ) Macromolecular Structures, Current Biology, London, UK.
Nomenclature Committee of IUBMB (NC-IUBMB) and IUPAC-IUBMB Joint Commission on Biochemical
Nomenclature (JCBN) (1992) Newsletter 1992, Biochem. Int. 26, 567575; and references therein.
Commission on Molecular Structure and Spectroscopy (1972) Recommendations for Presentation of NMR
Data for Publication in Chemical Journals, Pure Appl. Chem. 29, 627628.
Commission on Molecular Structure and Spectroscopy (1976) Presentation of NMR Data for Publication in
Chemical Journals--B. Conventions Relating to Spectra from Nuclei other than Protons (Recommendations1975), Pure Appl. Chem. 45, 219.
Harris, R. K., Kowalewski, J., and Cabral de Menezes, S. (1997) Parameters and Symbols for use in Nuclear Magnetic Resonance, Pure App. Chem. 69, 2489-2495.
Liébecq, C. (ed.) (1992) Biochemical Nomenclature and Related Documents - A Compendium, 2nd ed.,Portland Press on behalf of IUBMB. London.
Bonnett, R. (1978) in The Porphyrins (D. Dolphin, Ed.), Vol. 1, Part A., Academic Press, New York, pp.
127.
IUPAC-IUB Commission on Biochemical Nomenclature (CBN) (1975) Nomenclature of Iron-Sulfur Proteins. Recommendations 1973, Eur. J. Biochem. 35, 12.
IUPAC-IUB Commission on Biochemical Nomenclature (1975) The Nomenclature of Peptide Hormones.
Recommendations 1974, Biochemistry 14, 25592560; reprinted in the Compendium (ref. 9).
IUPAC-IUB Commission on Biochemical Nomenclature (1972) Abbreviated Nomenclature of Synthetic
Polypeptides (Polymerized Amino Acids). Revised Recommendations 1971, Biochemistry 11, 942944;
reprinted in the Compendium (ref. 9).

139

14. Nomenclature Committee of the International Union of Biochemistry (NC-IUB) Nomenclature for Incompletely Specified Bases in Nucleic Acids (1985), Eur. J. Biochem. 150, 15; reprinted in the Compendium(ref. 9).
15. Access to various databases (e.g., GenBank, DDBJ, EBI (EMBL), EC Enzyme Data Bank, PIR, SWISSPROT, PRF, PDB, NDB, BMRB) is provided over the Internet. Useful compilations of Internet addresses
are found, for example, on the home pages of the Protein Data Bank (http://www.pdb.bnl.gov/), BioMagResBank (http://www.bmrb.wisc.edu), the NCSA Biology Workbench (http://biology.ncsa.uiuc.edu), andPedro’s Biomolecular Research Tools (http://www.public.iastate.edu/~pedro/research_tools.html).
16. Protein Identification Resource, Georgetown University, Washington, D.C., USA. The Protein Identification Resource can be searched with tools located at: http://www.gdb.org/Dan/proteins/pir.html.
17. SWISS-PROT Annotated Protein Sequence Database (http://www.expasy.hcuge.ch/sprot/sprot-top.html).
18. International Union of Biochemistry and Molecular Biology (1992) Enzyme Nomenclature. Recommendations 1992, Academic Press, Orlando, Florida.
19. Chemical Abstracts Service, Columbus, OH, USA (http://info.cas.org/).
20. IUPAC-IUB Commission on Biochemical Nomenclature (CBN) (1970) Abbreviations and Symbols for the
Description of the Conformation of Polypeptide Chains. Tentative Rules 1969, Biochemistry 9,34713479; reprinted in the Compendium (ref. 9).
21. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) (1984) Nomenclature and Symbolism for Amino Acids and Peptides. Recommendations 1983, Eur. J. Biochem. 138, 937; reprinted in theCompendium (ref. 9).
22. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) (1983) Abbreviations and Symbols
for the Description of Conformations of Polynucleotide Chains. Recommendations 1982, Eur. J. Biochem.
131, 915; reprinted in the Compendium (ref. 9).
23. Hanson, K. R. (1966) Applications of the Sequence Rule. I. Naming the Paired Ligands g,g at a Tetrahedral Atom Xggij. II. Naming the Two Faces of a Trigonal Atom Yghi, J. Am. Chem. Soc. 88, 27312742.
24. The 1983 recommendations (ref. 21) specified the use of numbers to designate atoms and the use of the R/Srules of Cahn, Ingold, & Prelog (ref. 25 and 26, see also ref. 2729) to define stereochemistry. According
to the R/S rules, a tetrahedral carbon X with prochiral substituents A > B > C = C′ (where the priority is according to decreasing atomic mass and C and C′ have equivalent mass and are labeled arbitrarily) is labeled
as follows: one sights down the AX axis and assumes that C′ takes precedence over C (as it would if itwere replaced, for example, with a heavier isotope). Then, if B, C′, and C (projecting away from theviewer) are clockwise, C′ is labeled as pro-R and C is labeled as pro-S; if they are counterclockwise, C′ islabeled as pro-S and C is labeled as pro-R.
25. Cahn, R. S., Ingold, C. K., & Prelog, V. (1966) Specification of Molecular Chirality, Angew. Chem. Int. Ed.
Engl. 5, 385415; 511.
26. Prelog, V., & Helmchen, G. (1982) Basic Principles of the CIP-System and Proposals for a Revision, Angew.
Chem. Int. Ed. Engl. 21, 567583.
27. Blackwood, J. E., Gladys, C. L., Loening, K. L., Petrarca, A. E., & Rush, J. E. (1968) Unambiguous Specification of Stereoisomerism about a Double Bond, J. Am. Chem. Soc. 90, 509510.
28. Blackwood, J. E., Gladys, C. L., Petrarca, A. E., Powell, W. H., & Rush, J. E. (1968) Unique and Unambiguous Specification of Stereoisomerism about a Double Bond in Nomenclature and Other Notation Systems,
J. Chem. Doc. 8, 30-32.
29. IUPAC Commission on Nomenclature of Organic Chemistry (CNOC) (1976) Nomenclature of Organic
Chemistry, Section E: Stereochemistry. Recommendations 1974, Pure Appl. Chem. 45, 1130 (1976); reprinted in the Compendium (ref. 9).
30. Wüthrich, K., Billeter, M., & Braun, W. (1983) Pseudo-Structures for the 20 Common Amino Acids for Use
in Studies of Protein Conformations by Measurements of Intramolecular ProtonProton Distance Constraints with Nuclear Magnetic Resonance, J. Mol. Biol. 169, 949961.
31. Since the experimental NOEs involve distances to the individual protons rather than to the reference pointof the pseudoatom, NOE distance constraints involving pseudoatoms must contain a correction factor. A
conservative and straightforward approach is to set these corrections equal to the distance between thereference point of the pseudoatom and the protons that it replaces. Tables listing these corrections for thecommon amino acid residues have been published (ref. 30, 33) as well as more refined corrections (forexample, see ref. 32, 34).
32. Koning, T. M. G., Boelens, R., & Kaptein, R. (1990) Calculation of the Nuclear Overhauser Effect and Determination of ProtonProton Distances in the Presence of Internal Motions, J. Magn. Reson. 90, 111-123.
33. Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids, Wiley Interscience, New York.
34. Güntert, P., Braun, W., & Wüthrich, K. (1991) Efficient Computation of Three-Dimensional Protein Structures in Solution from Nuclear Magnetic Resonance Data Using the Program DIANA and the Supporting
Programs CALIBA, HABAS and GLOMSA, J. Mol. Biol. 217, 517530.
35. Historical changes in the definition of these angles have contributed to confusion. Their use in numerous
textbooks does not conform to the current IUPAC-IUBMB nomenclature: Clauwaert, J., & Xia, J.-Z. (1993)
Straightening out the Dihedral Angles, Trends Biochem. Sci. 18, 317318; 464.

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36. Wilmott, C. M., & Thornton, J. M. (1990) β-turns and their Distortions: A Proposed New Nomenclature,
Protein Eng. 3, 479493.
37. Richardson, J. S. (1981) The Anatomy and Taxonomy of Protein Structure, Adv. Protein Chem. 34,
167339.
38. Milner-White, E. J., Bell, L. H., & Maccallum, P. H. (1992) Pyrrolidine Ring Puckering in cis- andtrans-Proline Residues in Proteins and Polypeptides. Different Puckers are Favoured in Certain Situations,J. Mol. Biol. 228, 725734.
39. de Leeuw, H. P. M., Haasnoot, C. A. G., & Altona, C. (1980) Empirical Correlations between
Conformational Parameters in β-D-Furanoside Fragments Derived from a Statistical Survey of Crystal
Structures of Nucleic acid Constituents, Isr. J. Chem. 20, 108126.
40. Haasnoot, C. A. G., de Leeuw, F. A. A. M., de Leeuw, H. P. M., & Altona, C. (1981) The Relationshipbetween Proton-Proton NMR Coupling Constants and Substituent Electronegativities Org. Magn. Reson.
15, 4352.
41. Harris, R. K., Becker, E. D., Bremser, W., Cabral de Menezes, S., Goodfellow, R., & Granger, P. IUPAC(Commission on Molecular Structure and Spectroscopy, I.5) Recommendations for NMR Nomenclature. A.
Nuclear Spin Properties and Conventions for Chemical Shifts, (in preparation).
42. Wishart, D. S., Bigam, C. G., Yao, J., Abildgaard, F., Dyson, H. J., Oldfield, E., Markley, J. L., & Sykes, B.
D. (1995) 1H, 13C and 15N Chemical Shift Referencing in Biomolecular NMR, J. Biomol. NMR. 6,
135140.
43. In a dilute aqueous solution at 298 K (0.3 mM DSS and 0.03 mM TMS in D2O at pH* 7), the signal of
TMS is at δDSS = -0.0173 ppm (i.e., is at low frequency, historically referred to as “upfield”, from that of
DSS); in a dilute organic solution at 298 K (0.03 mM TMS and 0.01 mM DSS in perdeuterated dimethyl
sulfoxide), the signal of TMS is at δDSS = 0.0246 ppm (i.e., to high frequency from that of DSS) from that ofDSS.
44. Live, D. H., Davis, D. G., Agosta, W. C., & Cowburn, D. (1984) Long Range Hydrogen Bond MediatedEffects in Peptides: 15N NMR Study of Gramicidin S in Water and Organic Solvents, J. Am. Chem. Soc.
106, 19391943.
45. The Ξ value recommended here for 2H was determined from the ratios of 1H and 2H signals of a water sample at 298 K as measured by Dr. Frits Abildgaard and David M. LeMaster at the National Magnetic Resonance Facility at Madison. This value is a surrogate for the isotopomers of DSS, and its derivation assumes
that there is no primary isotope effect on the ratio.
46. The Ξ value recommended here for 31P is the average of values determined independently, one at the National Magnetic Resonance Facility at Madison by F. Abildgaard (40.4808651), and the other at the National Institutes of Health by A. Bax (40.480862). Measurements were made as 298 K in D2O solution containing 10% trimethylphosphate as the indirect reference and DSS as the direct reference.
47. NOE distances may be represented rigorously by notation analogous to that used for J-couplings. However,since protons are always implied, a shorthand notation is widely used: for example, dαN for dHαHN, dNN fordHNHN, dβN for dHβHN, and dαβ for dHαHβ.
48. Williamson, M. P., Havel, T. F., & Wüthrich, K. (1985) Solution Conformation of Proteinase Inhibitor IIA
from Bull Seminal Plasma by 1H NMR Spectroscopy and Distance Geometry, J. Mol. Biol. 182, 295315.
49. Gronenborn, A. M., & Clore, G. M. (1994) Identification of N-Terminal Helix Capping Boxes by Means of
13
C Chemical Shifts, J. Biomol. NMR 4, 455458.
50. Eberstadt, M., Gemmecker, G., Mierke, D. F., Kessler, H. (1995) Scalar Coupling ConstantsTheir Analysis and their Application for the Elucidation of Structures, Angew. Chem. Int. Ed. Engl. 34, 16711695.
51. Ottiger, M., Szyperski, T., Luginbühl, L., Ortenzi, C., Luporini, P., Bradshaw, R. A., & Wüthrich, K.
(1994) The NMR Solution Structure of the Pheromone Er-2 from the Ciliated Protozoan Euplotes raikovi,
Protein Science 3, 15151526.
52. Saenger, W., (1983) Principles of Nucleic Acid Structure, Springer Verlag, New York, pp. 123124.
53. Sutcliffe, M. J. (1993) Representing an Ensemble of NMR-Derived Protein Structures by a Single Structure,
Protein Sci. 2, 936944.
54. Fletcher, C. M., Jones, D. N. M., Diamond, R., Neuhaus, D. (1996) Treatment of NOE Constraints Involving Equivalent or Nonstereoassigned Protons in Calculations of Biomacromolecular Structures, J. Biomol.
NMR 8, 292310.
55. MacArthur, M. W., & Thornton, J. M. (1993) Conformational Analysis of Protein Structures Derived fromNMR Data, Proteins 17, 232251.
56. Hyberts, S. G., Goldberg, M. S., Havel, T. F., Wagner, G. (1992) The Solution Structure of Eglin c Based on
Measurements of Many NOEs and Coupling Constants and its Comparison with X-ray Structures, Protein
Sci. 1, 736751.
57. Colovos, C., & Yeates, T. O. (1993) Verification of Protein Structures: Patterns of Nonbonded Atomic Interactions, Protein Sci. 2, 15111519.
58. Sippl, M. J. (1993) Recognition of Errors in 3-Dimensional Structures of Proteins, Proteins 17, 355362.
59. Vriend, G., & Sander, C. (1993) Quality Control of Protein Models: Directional Atomic Contact Analysis,
J. Appl. Cryst. 26, 4760.

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60. MacArthur, M. W., Laskowski, R. A, Thornton, J. M. (1994) Knowledge-Based Validation of ProteinStructure Coordinates Derived by X-ray Crystallography and NMR Spectroscopy, Current Opinion Struct.
Biol. 4, 731737.
61. Laskowski, R. A., Rullmann, J. A. C., MacArthur, M. W., Kaptein, R., & Thornton, J. M. (1996) AQUA
and PROCHECKNMR: Programs for Checking the Quality of Protein Structures Solved by NMR. J.
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62. Jardetzky, O., Roberts, G. C. K. (1981) NMR in Molecular Biology, Academic Press, New York, pp.
126142.
63. Watenpaugh, K. D., Berman, H. M., Bourne, P. E., & Fitzgerald, P. M. D. (1993) The Macromolecular CIF
Dictionary, Acta Crystallogr. Ser. A 49 (Supplement), IVI International Congress (IUCr), Abstract
OCM18.02.07.
64. Fitzgerald, P. M. D., Berman, H., Bourne, P., & Watenpaugh, K (1993) The Macromolecular CIF Dictionary, Abstract No. D008, American Crystallographic Association Annual Meeting, Albuquerque, NM, May
2328 (available from: http://ndbserver.rutgers.edu:80/mmcif/).
65. Hall, S. R., & Spadaccini, N. (1994) The STAR File: Detailed Specifications, J. Chem. Inf. Comput. Sci.
34, 505508.
66. Hall, S. R., Allen, F. H., & Brown, I. D., [International Union of Crystallography, Commission on Crystallographic Data, Commission on Journals, Working Party on Crystallographic Information] (1991) The
Crystallographic Information File (CIF): a New Standard Archive File for Crystallography, Acta Crystallogr. Ser. A 47, 655685.
67. International Organization for Standardization (1987) Information Processing SystemsOpen Systems InterconnectionSpecification of Abstract Syntax Notation One (ASN.1), Technical Report ISO8824.
68. International Organization for Standardization (1987) Information Processing SystemsOpen Systems InterconnectionSpecification of Basic Encoding Rules for Abstract Syntax Notation One (ASN.1), Technical Report ISO8825.
69. Ostell, J. (1990) GenInfo ASN.1 Syntax: Sequences, Technical Report 1 & 2, National Center for Biotechnology Information, National Library of Medicine.
70. Mockus, J., & Steckert, T. D. (1994) CXF Chemical eXchange Format Version 1.0, Chemical Abstracts
Service: Columbus, OH, USA.
71. Ulrich, E. L., & Markley, J. L. (1994) A Data Dictionary and Information File for the Exchange and Archiving of NMR Experimental Results, 35th Experimental Nuclear Magnetic Resonance Conference, April
1015, Asilomar, California, Abs. WP 112, p. 264.
The BioMagResBank web pages(http://www.bmrb.wisc.edu/) contain complete information about this effort including user entry protocols
and software.

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