Comparison of two crystal polymorphs of NowGFP reveals a new conformational state trapped by crystal packing

Structures of NowGFP obtained from two distinct crystal forms were compared and a new conformational state captured by crystal packing was identified.


Introduction
Proteins can be crystallized in multiple forms, a phenomenon known as crystal polymorphism.A well-known example of protein crystal polymorphism is lysozyme, which exhibits six crystal polymorphs (Vaney et al., 2001).The primary interest in discovering crystal polymorphs of proteins lies in finding a superior crystal form that enables the acquisition of detailed structural information through X-ray crystallography.This crystal form may either have high crystallinity for improved diffraction (Yamada et al., 2017) or possess enhanced thermal and chemical durability (Gerlits et al., 2019).Additionally, different crystal forms can reveal different conformation states of protein molecules which are trapped by crystal lattice packing (Jiang et al., 2013).The availability of different crystal forms provides insight into the conformational flexibility of protein molecules and a detailed understanding of individual conformational states (Zhang et al., 1995).In this study, we demonstrate a new case of protein crystal polymorphism with NowGFP, a variant of green fluorescent protein (GFP).
Fluorescent proteins have become essential tools in cell biology and biomedicine, serving as non-invasive methods for visualizing and tracking cellular and organism-wide processes (Chudakov et al., 2010).Most of these fluorescent proteins are characterized by the tyrosine (Tyr) at the centre of the three residues responsible for forming the chromophore, resulting in fluorescence that spans from green to far-red wavelengths.Recently, a fluorescent protein based on an anionic tryptophan (Trp) was developed and was named WasCFP (Sarkisyan et al., 2012).Derived from the cyan-fluorescent mCerulean (Rizzo et al., 2004), it was engineered by introducing a critical Val61Lys substitution and four additional mutations.WasCFP was further improved to enhance its stability across a broad range of pH levels and temperatures, incorporating 13 additional mutations.This improved variant, which exhibits strong green fluorescence under physiological conditions, has been named NowGFP (Sarkisyan et al., 2015).
Spectral and structural studies of NowGFP consistently reveal pH-dependent changes.Spectrally, NowGFP exhibits green fluorescence (� ex /� em = 493/502 nm) at physiological and higher pH levels, contrasting with cyan fluorescence (� ex / � em = 429/475 nm) under acidic conditions (pH < 6).The intensity ratio between green and cyan fluorescence is highly sensitive to pH conditions.Structurally, NowGFP adopts two distinct pH-dependent conformations at pH 4.8 and pH 9.0.These conformations involve the chromophore and the key residue Lys61, which is believed to play a central role in chromophore ionization and the resultant shifts in the fluorescence spectrum (Pletnev et al., 2015).
We present three crystal structures of NowGFP obtained under various pH conditions from two distinct crystal forms: a monoclinic crystal structure at pH 4.8 (PDB entry 8xh0, 1.45 A resolution) and orthorhombic crystal structures at pH 9.0 (PDB entry 8xh1, 1.7 A ˚resolution) and pH 6.0 (PDB entry 8xh2, 1.8 A ˚resolution).Our study begins with a detailed comparative analysis of crystal contacts and stacking interactions between these two crystal forms.Following this, we identify major differences in the crystal contacts across the two crystal forms and determine how the crystal packing may alter the NowGFP structure.Lastly, we investigate the alternative conformations trapped by the orthorhombic crystal form by examining five NowGFP molecules: one from the monoclinic form at pH 4.8, two from the orthorhombic form at pH 9.0 and two from the orthorhombic form at pH 6.0.This comparison aims to deepen our understanding of how crystal packing influences the pH-dependent conformational changes in NowGFP.

Macromolecule production
The fragment encoding NowGFP with an N-terminal His tag and TEV protease recognition sequence site was cloned into the pET-24a(+) vector and transformed into Escherichia coli strain BL21(DE3) (Invitrogen, USA).Bacterial cultures were grown overnight at 16 � C. Isopropyl �-d-1-thiogalactopyranoside (IPTG) induction was necessary for effective protein expression.The cells were pelleted by centrifugation, resuspended in phosphate-buffered saline (PBS) pH 7.4, 1 mM PMSF, 1 mM TCEP and lysed by an EmulsiFlex-C3 at 103-117 MPa.NowGFP was purified by immobilized metalion affinity chromatography using His60 Ni Superflow Resin (Clontech, USA) and then buffer-exchanged for cleavage by TEV protease on a Cytiva PD-10 DG column (1� TEV protease buffer: 25 mM Tris-HCl, 150 mM NaCl, 1 mM TCEP).TEV protease was applied (10 U ml À 1 , 1:100 ratio) and incubated at 30 � C for 1 h.After incubation, further purification was achieved by size-exclusion chromatography using FPLC (A ¨KTApure 25) with a HiLoad 16/600 Superdex 75 pg column (Cytiva, USA).The purity of the sample was then confirmed by SDS-PAGE analysis (Supplementary Fig. S1).The purified NowGFP protein was concentrated using an Amicon Ultra-15 centrifugal filter unit.The amino-acid sequence and other details are given in Table 1.

Crystallization
Crystallization screening was conducted to identify optimal conditions for the formation of NowGFP crystals.Single crystals in a monoclinic space group (C2) were obtained using KH 2 PO 4 and PEG 3350.Additionally, single crystals in an orthorhombic space group (P2 1 2 1 2 1 ) were obtained using sodium citrate and PEG 4000.Details of the crystallization screening, including other conditions that did not yield diffraction-quality single crystals, are provided in Supplementary Table S1.
To obtain monoclinic crystals, NowGFP was transferred to 20 mM Tris pH 8.0, 200 mM NaCl buffer and concentrated to 12 mg ml À 1 .Crystals suitable for data collection were obtained by the hanging-drop vapour-diffusion method (McPherson, 1982).Typically, 2 ml protein solution was mixed with an equal amount of reservoir solution and incubated at 4 � C for a week.The best crystal was obtained from 16 mM KH 2 PO 4 pH 4.8, 20%(w/v) PEG 3350.
To obtain orthorhombic crystals, NowGFP was transferred to 20 mM Tris pH 8.0, 200 mM NaCl buffer and concentrated to 17 mg ml À 1 .Crystals suitable for data collection were obtained by the hanging-drop vapour-diffusion method.Typically, 2 ml protein solution was mixed with an equal amount of reservoir solution and incubated at 4 � C for a week.The best crystal was obtained from 100 mM sodium citrate pH 6.0, 25%(w/v) PEG 4000.To obtain structures of NowGFP at  2 and Supplementary Fig. S2, respectively.

Data collection and processing
X-ray diffraction data were collected on Pohang Light Source-II (PLS-II) beamline 7A at the Pohang Accelerator Laboratory, Pohang, Republic of Korea.Prior to data collection, the monoclinic crystals were briefly soaked in a cryoprotectant solution consisting of 30%(v/v) glycerol and 70%(v/v) reservoir, while the orthorhombic crystals were soaked in a cryoprotectant solution consisting of 20%(v/v) glycerol and 80%(v/v) reservoir, and then flash-cooled in a 100 K nitrogen stream.A total of 360 images with 1 � oscillation angles were collected with sample-to-detector distances of 150 and 175 mm for monoclinic and orthorhombic crystals, respectively.All diffraction images were processed with HKL-2000 (Otwinowski & Minor, 1997).The absorbed X-ray dose for a single data set was less than 5 � 10 5 Gy, which is much lower than the Henderson dose limit of 1.45 � 10 7 Gy (Henderson, 1990).Data-processing statistics and diffraction images are given in Table 3 and Supplementary Fig. S3, respectively.

Structure solution and model refinement
The structures of NowGFP from both crystal forms were determined using the CCP4 suite (Agirre et al., 2023).The crystal structures were solved by the molecular-replacement method with MOLREP (Vagin & Teplyakov, 2010) using the previously solved structure of NowGFP (PDB entry 4rtc; Pletnev et al., 2015) as a model.Crystallographic refinement was performed with REFMAC5 (Murshudov et al., 2011), alternating with manual revision of the model with Coot (Emsley et al., 2010).The location of water molecules and structure validation were performed with Coot.The monoclinic crystal structure (C2) of NowGFP at pH 4.8 was determined at 1.45 A ˚resolution and orthorhombic crystal structures (P2 1 2 1 2 1 ) of NowGFP at pH 9.0 and 6.0 were solved   55, 51.04, 55.52 51.24, 52.28, 195.30 51.17, 51.71, 196 0.072 0.079 0.071 at 1.7 and 1.8 A ˚resolution, respectively.The coordinates and structure factors for NowGFP in the monoclinic form at pH 4.8, the orthorhombic form at pH 9.0 and the orthorhombic form at pH 6.0 were deposited in the Protein Data Bank under accession codes 8xh0, 8xh1 and 8xh2, respectively.Structuresolution and data-refinement statistics are given in Table 4.All structural figures were rendered with PyMOL (Schro ¨dinger).

Crystal structures of two crystal polymorphs
The assemblies of protein molecules within the unit cells of both orthorhombic and monoclinic crystals are illustrated in Fig. 1.For the comparative study of the two crystal forms, we chose the orthorhombic crystal structure obtained at pH 9.0 for comparison with the monoclinic structure since it has a higher resolution (1.7 A ˚).Comparison between the lower resolution orthorhombic crystal structure obtained at pH 6.0 (1.8A ˚) and the monoclinic structure revealed nearly identical results to the higher pH (pH 9.0) counterpart.This comparison is detailed in Supplementary Figs.S4-S7 and Supplementary Tables S2 and S3.
For simplicity, we refer to chain A of the NowGFP structure in the monoclinic form as Mono.Similarly, we refer to chains A and B in the orthorhombic form as Orth(A) and Orth(B), respectively.Comparative analysis of Mono, Orth(A) and Orth(B) indicates that there are no significant differences in the overall protein structures, with the C � -C � r.m.s.d.being less than 0.4 A ˚.However, partial differences are observed in the r.m.s.d.from ideal angles, B factors and solvation free energy (Tables 4 and 5).These differences will be discussed in detail in Section 3.3.

Crystal contacts and related crystal packing
The term 'crystal contacts' denotes the interactions between protein molecules that emerge through the crystallization process (Janin & Rodier, 1995;Dasgupta et al., 1997).In this section, the crystal contacts between NowGFP molecules are considered using PISA analysis (Krissinel & Henrick, 2005, 2007) and the protein crystal packing is discussed.All crystal contacts from each molecule are listed in Table 6.
In the monoclinic (C2) crystal form, the crystal contacts are categorized into three types, denoted as contacts I, II and III.Contact I is between molecules 1 and 4 (or 2 and 3) (Figs.2a  and 2b), which has the largest contact area for Mono.More specifically, contact I comes from two different symmetryrelated molecules (Mono) at (À x + 1/2, y À 1/2, À z À 1) and (À x + 1/2, y + 1/2, À z À 1) and connects molecules 1 and 4 (or 2 and 3) to form a zigzag linear assembly along the b axis (Fig. 2d).Contact II is between molecules 1 and 2 (or 3 and 4) (Figs.2a and 2c).More specifically, contact II comes from symmetry-related molecules at (À x, y, À z À 1) and connects two types of linear assemblies (1-4 and 2-3) to form layers in the ab plane (Fig. 2d).The symmetry operation between two neighbouring linear assemblies is (À x, y, À z), which is the same as the symmetry operation between molecules 1 and 2 or 3 and 4 (represented as opposite white arrows in Fig. 2d).Contact III is between molecules 1 and 3 (or 2 and 4), which has the smallest contact area (Figs.2a and 2b).More specifically, contact III comes from two different symmetry-related molecules at (À x + 1/2, y À 1/2, À z) and (À x + 1/2, y + 1/2, À z) and results from the packing of sheets along the c axis (Fig. 2d).

The structural alteration of NowGFP results from crystal contacts
Comparison of the crystal contacts of three types of molecules [Mono, Orth(A) and Orth(B)] from two different crystal forms shows that contacts I, I A and I B are similar in structure and sequence (blue and yellow in Fig. 4).Notably, linear assemblies derived from contacts I, I A and I B are also almost identical (Figs. 2a,.However, there is a significant difference between contacts II and II AB (red in Fig. 4).This difference is responsible for the packing mode of the linear assemblies: parallel for the monoclinic form and perpendicular for the orthorhombic form.There are minor differences in the other contacts, including contacts III, III AB , IV AB and V B , but those have a much smaller contact area compared with contacts I, I A , I B , II and II AB (Table 6).In addition, the histogram of the distances between atom pairs for each crystal contact indicates that contacts I, I A , I B , II and II AB are more closely interacting compared with others (Figs.4e and 4f ).We thus focused on analysing the differences between contacts II and II AB as represented in Fig. 5.
For contact II in the monoclinic crystal form, there are no atom pairs closer than 3.0 A ˚(Table 6).Phe99, Tyr182, Gln157 and Lys156 from two molecules contact each other without any water molecule in between.Gln157 also contacts the main chain of Arg96 and Gln183.Additionally, residues corresponding to contact II show no significant deviation from Mono to Orth(A) and Orth(B) (Fig. 5a).This indicates that contact II does not significantly influence the overall protein structure.
For contact II AB in the orthorhombic crystal form, there are four atom pairs closer than 3.0 A ˚(Table 6).Two of them come from the arginine-aspartic acid salt bridge between Asp180 of Orth(A) and Arg168 of Orth(B).The other two are from Tyr182 and Tyr164 of Orth(A), which are connected to the main chain of Val176 and Asp173 of Orth(B) through hydrogen bonding.Compared with Mono, a significant shift of the main chain is observed for residues 170-176 of Orth(B), which mainly involves contact II AB and the loop between the �8 and �9 strands (Figs.5b and 5c).This indicates that contact Protein molecules from the monoclinic crystal form and the orthorhombic crystal form.
The total numbers of atoms and residues in the refined NowGFP molecule (residues 2-231) are 1809 and 228, respectively.H atoms are not considered.All values are from PISA analysis (Krissinel & Henrick, 2005, 2007).

Crystal form
Name of molecule No. of atoms on the surface † No. of residues on the surface † Surface area ‡ (A ˚2) Solvation energy ‡ (kcal mol

Table 6
Crystal contacts of the monoclinic crystal form and the orthorhombic crystal form.
Symmetry operations for symmetry-related neighbouring molecules are given in parentheses.H atoms are not considered.All values are from PISA analysis (Krissinel & Henrick, 2005, 2007).

Name of contact
No. of atoms in the contact No. of residues in the contact

Contact area † (A ˚2)
Solvation energy ‡ (kcal mol À 1 ) No. of atom pairs closer than 3.0/3.5A Mono V B 6 2 76.5 -1.2 0/1 † Surface area buried by the intermolecular interface.‡ Solvation energy gain upon formation of the interface.This value does not include the effect of satisfied hydrogen bonds and salt bridges across the interface.II AB might be responsible for the structural shifts in Orth(B).Additionally, this shift causes residues 141-147, which are part of the �7 strand, to move away from the �10 strand (Fig. 5).Specifically, the C � -C � distances between Asn144 in the �7 strand and Gln207 in the �10 strand are 6.3, 6.1 and 6.7 A ˚for Mono, Orth(A) and Orth(B), respectively.
The evidence that Orth(B) has a more unstable structure compared with Orth(A) or Mono is that the r.m.s.d.from ideal angles, average B factor and solvation free energy all show the highest values for Orth(B).In detail, the r.m.s.d.s from ideal angles for Mono, Orth(A) and Orth(B) are �2.0 � , �1.9 � and �2.1 � , respectively, the average B factors of the main chain from Mono, Orth(A) and Orth(B) are �22.7,�26.8 and 31.0A ˚2, respectively, and the solvation free energy from the isolated structure of each molecule is �À 215 kcal mol À 1 for Orth(A) and Mono and �À 209 kcal mol À 1 for Orth(B) (Tables 4 and 5).These results indicate that Orth(A) exhibits similar conformational behaviour to Mono, while Orth(B) represents a relatively unstable conformational state.
Moreover, protein crystals are highly hydrated, with those with lower solvent content typically having better diffraction quality (Matthews, 1968;Zhang et al., 1995).However, solvent-content estimation reveals that the orthorhombic Table 7 Two alternative conformations of the key residue Lys61.

New conformational state of NowGFP trapped by crystal lattice packing
In a previous study by Pletnev and coworkers, it was revealed that the key residue Lys61 plays a central role in the ionization process of the chromophore, showing significant pH-dependent conformational changes.Specifically, at high pH the N � atom of Lys61 makes two hydrogen bonds: one to the N " atom of Trp66 and the other to the O "1 atom of Glu222 (k1 conformation).This dual bonding helps to form a non'; covalent connection and promotes the deprotonation of Trp66.In contrast, at lower pH the orientation of Lys61 shifts away from Trp66, instead forming a hydrogen bond to the N "2 atom of Gln207 (k2 conformation; Pletnev et al., 2015).
To elucidate the alternative conformation state trapped by the orthorhombic crystal lattice, a total of five NowGFP molecules are compared (one molecule from the monoclinic crystal structure at pH 4.8 and four molecules from the orthorhombic crystal structures at pH 6.0 and pH 9.0).
In the case of the Orth(A) and Mono structures, the key residue Lys61 has �80% k1 conformation and �20% k2 conformation under a high-pH condition (pH 9.0).This shifts to �50% k1 conformation and �50% k2 conformation as the pH decreases to 6.0, and finally to �20% k1 conformation and �80% k2 conformation under an acidic pH condition (pH 4.8) (Figs.6a, 6c and 6e).This trend of conformational shifts from k1 to k2 as the pH decreases is consistent with previously published structures: the monoclinic structure at pH 9.0 (PDB entry 4rtc, 1.18 A ˚resolution) and pH 4.8 (PDB entry 4rys, 1.35 A ˚resolution) (Supplementary Fig. S8; Pletnev et al., 2015).Even though the high-resolution structure of NowGFP shows a partial trans conformation of the chromophore at pH 4.8, this is difficult to observe in our moderate-resolution structure (1.45A ˚).
In the case of the Orth(B) structure, however, the conformation of Lys61 is almost 100% k1 at both pH 9.0 and pH 6.0, and the distance between the N " atoms of the Trp66 indole and the N � atom of Lys61 in the k1 conformation is �0.3A ˚closer compared with the Orth(A) structure (Figs.6a-6d).It appears that strong hydrogen bonding between the Trp66 indole and the k1 conformation of Lys61 is responsible for the high occupancies of the k1 conformation in the Orth(B) structure.Considering that Orth(B) has an unstable structure compared with Orth(A) and Mono, and this instability is linked to crystal contact II AB as detailed in Section 3.3, it is evident that the alteration of the Lys61 conformation in the Orth(B) structure is a result of orthorhombic crystal packing.Table 7 shows the � 1 and � 2 angles of two alternative conformations of the key residue Lys61.
While this alternative conformational state could be dismissed as merely an artefact of crystal packing, a previous study of crystal polymorphs of photoactive yellow protein indicates that the crystal lattice does not simply impose arbitrary conformational changes on the protein molecule (van Aalten et al., 2000).Instead, the protein shifts along essential eigenvectors to adapt to different lattice environments.Therefore, different protein structures from crystal polymorphs may represent the inherent conformational flexibility of the molecule (van Aalten et al., 1997).
Previous studies, including the unfolding and backbone dynamics of GFP, reveal that �-strands 7, 8, 9 and 10 exhibit greater flexibility (Huang et al., 2007;Seifert et al., 2003).Additionally, the parental WasCFP shows conformational flexibility in the �7 and �10 strands, which opens the solvent channel between these two �-strands.This flexibility is expected to play a crucial role in managing solvent access to the chromophore (Laricheva et al., 2015).These observations are consistent with our finding that the r.m.s.d. of the mainchain atoms between two structures from crystal polymorphs shows higher values at residues corresponding to �-strands 7, 8, 9 and 10.The most significant deviations are noted in the loop between the �8 and �9 strands, resulting in opening of the solvent channel between the �7 and �10 strands (Fig. 5).Based on these correspondences, the new conformational state of NowGFP captured by crystal packing in this study is likely to represent one of the potential conformational states inherent to this protein.

Conclusions
In this study, we report the discovery of a novel orthorhombic crystal form of NowGFP and conduct a detailed comparison with the known monoclinic crystal form.Our investigations primarily focused on the crystal contacts, revealing that both forms exhibit similar zigzag linear assemblies of protein molecules resulting from crystal contact I.The key distinction between the two forms lies in their stacking modes: parallel stacking for the monoclinic form and perpendicular stacking for the orthorhombic form.This difference in packing correlates with a specific crystal contact, referred to as crystal contact II (or II AB ), and results in an alteration of one molecule in the symmetry unit of the orthorhombic crystal form, designated as Orth(B).Given that these structural shifts are predominantly concentrated between �-strands 7-10, which are known for their partial flexibility, we propose that this altered molecule represents an alternative conformational state of NowGFP.In contrast, the other molecule in the orthorhombic form, Orth(A), remains unchanged and is similar to that found in the monoclinic form.
Significantly, this new conformational state of NowGFP captured in the orthorhombic crystal packing exhibits a different functional behaviour: the key residue Lys61, which is known for its pH-dependent shift from the k1 to the k2 conformation, appears to be locked in the k1 configuration regardless of pH conditions.This contrasts with the unaltered molecule, in which Lys61 exhibits pH-dependent movement as expected.These observations provide valuable insights into how crystal lattice packing influences the conformational states of protein molecules, enhancing our understanding of the conformational flexibility of protein structures.

Figure 1
Figure 1Assembly of the protein molecules in the monoclinic (a-c) and orthorhombic (d-f ) crystal forms.Crystal structures are viewed along the c axis (a, d), a axis (b, e) and b axis (c, f ).The secondary structures of NowGFP are represented as ribbons, while the unit cell of each crystal form is outlined in a black box.For the monoclinic form, molecules 1, 2, 3 and 4 are coloured blue, purple, light purple and light blue, respectively.For the orthorhombic form, molecules 1A, 2A, 3A, 4A, 1B, 2B, 3B and 4B are coloured blue, light blue, light purple, purple, yellow, light orange, light yellow and orange, respectively.

Figure 2
Figure 2 Crystal contacts and packing of NowGFP molecules in the monoclinic crystal structure.The secondary structures of NowGFP are represented as ribbons, and atoms belonging to crystal contacts are represented as surfaces.(a) Contacts I and II are viewed along the c axis.(b) Contacts I and III are viewed along the a axis.(c) Contacts II and III are viewed along the b axis.Contacts I, II and III are coloured blue, red and grey, respectively.(d) Schematic diagram of crystal packing.Crystal contacts are represented by coloured arrows, and linear assemblies connected by contact I are represented as blue-or purple-coloured boxes.Molecules connected by the blue-coloured contact I are linked along the b axis.Linear assemblies are stacked parallel by contacts II and III along the a axis and c axis, respectively.The symmetry operation between assemblies 1-4 and 2-3, indicated by opposite white arrows, is (À x, y, À z).

Figure 3
Figure 3 Crystal contacts and packing of NowGFP molecules in the orthorhombic crystal structure.The secondary structures of NowGFP are represented as ribbons, and atoms belonging to crystal contacts are represented as surfaces.(a) Contact I A is viewed along the c axis.(b) Contacts I B and V B are viewed along the c axis.(c) Contacts I A and I B are viewed along the b axis.(d) Contacts II AB , III AB and IV AB are viewed along the a axis.Contacts I A , I B , II AB , III AB , IV AB and V B are coloured blue, yellow, red, green, light blue and grey, respectively.(e) Schematic diagram of protein packing.Crystal contacts are represented by coloured arrows and linear assemblies connected by contact I A are represented as blue-or purple-coloured boxes, while linear assemblies connected by contact I B are represented as yellow-or orange-coloured boxes.The linear assemblies connected by contacts I A and I B are linked along the a axis and the b axis, respectively.Linear assemblies composed of chains A and B are stacked perpendicular by contacts II AB , III AB and IV AB along the c axis.Linear assemblies composed of chain B are stacked parallel by contact V B along the a axis.The symmetry operation between two assemblies composed of the same chains, 1A-2A and 3A-4A (or 1B-3B and 2B-4B), indicated by opposite white arrows, is (À x + 1/2, À y, z + 1/2).

Figure 4
Figure 4 Comparison of crystal contacts for three types of molecules.(a) Chain A of the monoclinic structure, Mono. 1, 2, 3 and 4 are identical.(b) Chain A of the orthorhombic structure, Orth(A).1A, 2A, 3A and 4A are identical.(c) Chain B of the orthorhombic structure, Orth(B).1B, 2B, 3B and 4B are identical.(d) Amino-acid sequence representation of the three molecules.Thr65, Trp66 and Gly67 were replaced by the chromophore.For Mono, atoms belonging to contacts I, II and III are coloured blue, red and grey, respectively.For Orth(A) and Orth(B), atoms belonging to contacts I A , I B , II AB , III AB , IV AB and V B are coloured blue, yellow, red, green, light blue and grey, respectively.Histograms of the distances between atom pairs in each crystal contact from (e) the monoclinic and ( f ) the orthorhombic crystal forms are shown.

Figure 5
Figure 5 Comparison of contacts II and II AB .(a) Two molecules in the monoclinic crystal structure connected by contact II are presented.The Orth(A) and Orth(B) structures are superposed with the Mono structure and coloured grey.For clarity, only the main chains of Arg96 and Gln183 are represented.(b) Two molecules from the orthorhombic structure connected by contact II AB are presented.The Mono structures are superposed with Orth(A) and Orth(B) and coloured grey.For clarity, only the main chains of Val176, Ser175, Gly174 and Asp173 are represented.The r.m.s.d. between the main chains of two superposed molecules is shown as a putty representation, with a blue-white-red colour profile.Crystal contacts less than 3.0 A ˚are represented as red dotted lines.Stick representations of chromophores are coloured green.The electron-density map (2F o À F c map) has a cutoff level of 2.0�.(c) The r.m.s.d.plot of the main-chain atoms of two structures from different crystal forms.Residues close to the N-terminus and C-terminus are ignored, and only the residues numbered from 4 to 229 are considered.Residue numbers 65, 66 and 67 are ignored since these are substituted with the chromophore.The region with the most significant r.m.s.d., spanning residues 170-176, is indicated by a red arrow.

Figure 6
Figure 6The chromophore Thr65-Trp66-Gly67 and key residue Lys61 under various pH conditions.The structures are from (a, b) the orthorhombic crystal at pH 9.0, (c, d) the orthorhombic crystal at pH 6.0 and (e) the monoclinic crystal at pH 4.8.The electron-density map (2F o À F c map) has a cutoff level of 0.8�.Black dotted lines between the N " atom of the Trp66 indole and the N � atom of Lys61 represent hydrogen bonding.Orth(B), which is a new conformational state of NowGFP caused by an orthorhombic crystal lattice, shows the dominant k1 conformation of Lys61, while both Mono and Orth(A) exhibit pH-dependent conformational shifts of Lys61.

Table 2
Crystallization conditions for the two crystal forms of NowGFP.The soaking solution is used to adjust crystals to different pH conditions.

Table 3
Data-collection statistics for NowGFP crystals.
Values in parentheses are for the outer shell.

Table 4
Structure-refinement statistics for NowGFP crystals.Values in parentheses are for the outer shell.